http://2009.igem.org/wiki/index.php?title=Special:Contributions/Brendan1&feed=atom&limit=50&target=Brendan1&year=&month=2009.igem.org - User contributions [en]2024-03-28T23:52:31ZFrom 2009.igem.orgMediaWiki 1.16.5http://2009.igem.org/Team:Wash_U/ProjectTeam:Wash U/Project2009-11-10T00:48:59Z<p>Brendan1: /* Results */</p>
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== '''Introduction''' == <br />
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Our project goal is to maximize the photosynthetic productivity of a series of photobioreactors containing ''Rhodobacter sphaeroides'' under both high and low light intensities by synthetically regulating the size of the light-harvesting antenna complex LH2. We chose to undertake such a project using ''R. sphaeroides'' due to its well-characterized photosynthetic and genetic system. <br />
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The antenna system functions to expand the spectrum of light available for photosynthesis by absorbing different wavelengths than that of the reaction center. Essentially, it is like the dish around the main receiver of any antenna. Marine bacteria, such as ''R. sphaeroides'', evolved very large antenna complexes to absorb light in a natural environment, where there is great competition for photons. As a result, the photosynthetic machinery is saturated at low light intensities in a synthetic non-competitive environment, such as a photobioreactor. This causes up to 95% of incidental photons to be dissipated as heat or fluorescence by bacteria through a process called Non-Photochemical Quenching (NPQ) (Mussgnug et al., 2007). In essence, these photons are being wasted as NPQ reduces light penetration into and across bioreactors and starves the shaded cells for for photons. One method that has been shown to improve photosynthetic productivity in photobioreactors is the reduction of light-harvesting antenna sizes (Polle et al., 2002, Mussgnug et al., 2007). Though, current approaches to this end are difficult to precisely control from the perspective of biological engineering and synthetic biology. <br />
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Our intention is to create a dynamic system that varies antenna size, is dependent on incident light intensity, and can be readily optimized using bioengineering principles. This synthetic regulation of the LH2 complex should result in bacteria grown under high light intensities expressing fewer LH2 complexes than cells that are more shaded, leading to a reduction of wasted incident photons while maintaining a high overall absorbance across subsequent bioreactors. Consequently, we expect to see an increase in the total photosynthetic productivity of our mutant ''Rhodobacter sphaeroides'' when compared to the wild type grown under similar conditions. <br />
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|<font size="2" style="color:black;">'''Wild Type Energy Production in a series of Bioreactors'''<br />
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|<font size="2" style="color:black;">'''Synthetically Regulated Antenna Energy Production in Bioreactors'''<br />
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[[Image:Slide09b.jpg| 450px | left]][[Image:Slide10 a.jpg| 450px | right]]<br />
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[https://2009.igem.org/Team:Wash_U/Project Back To Top]<br />
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== '''Organism''' ==<br />
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''Rhodobacter sphaeroides'' is a purple Alphaproteobacteria. It is metabolically flexible: it can grow heterotrophically, via aerobic and anaerobic respiration, as well as phototrophically under anaerobic conditions with light. <br />
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''R. sphaeroides'' is one of the best understood photosynthetic organisms. The photosystem is located in intracytoplasmic membrane invaginations and the main components are the Light Haresting Complex 2 (LH2), Light Harvesting Complex 1 (LH1), and the Reaction Center (RC). These pigment-protein complexes non-covalently bind bacteriochlorophylls and carotenoids. LH1 and RC make up the core complex and are often found in the ratio of 1:1. LH2 is more peripheral and naturally ranges in the ratio to LH1/RC of 3.0:1 to 6.7:1 under varying light conditions (Scheuring et al., 2005). LH2 absorbs photons maximally at the wavelengths of 850 and 800 nm and funnels its energy to LH1 and the reaction center for photochemistry. (Scheuring et al., 2005). <br />
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The two subunits of LH2 are coded for by the pucB/A genes and are naturally promoted by the puc promoter. <br />
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<br><div style="text-align: center;">'''ICM of ''R. sphaeroides'' '''[[Image:R sphaeroides photosystem.png| 500 px | center]]<center>source:Sener et al.2007<br />
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|<font size="2" style="color:black;">'''Plate of ''R. sphaeroides'' 2.4.1'''<br />
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[[Image:R sphaeroides aborbance.png| 450px | left]][[Image:R sphaeroides 0986.png| 450px | right]]<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br>source: Walz et al. 1998<br><br />
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====Online Resources for ''R. sphaeroides''====<br />
[http://www.kazusa.or.jp/codon/cgi-bin/showcodon.cgi?species=272943 Codon Usage Table for ''R. sphaeroides'' 2.41]<br />
<br> [http://www.ncbi.nlm.nih.gov/sites/entrez?db=genome&cmd=Retrieve&dopt=Overview&list_uids=18843 ''R. sphaeroides'' 2.41 NCBI Genome Tools]<br />
<br> [http://archaea.ucsc.edu/cgi-bin/hgPcr?wp_target=&db=0&org=Rhodobacter+sphaeroides+2+4+1&wp_f=&wp_r=&wp_size=2000&wp_perfect=15&wp_good=15&wp_showPage=true&hgsid=159191 ''In silico'' PCR of ''R. sphaeroides'' 2.41 genome]<br />
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[https://2009.igem.org/Team:Wash_U/Project Back To Top]<br />
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=='''Regulation'''==<br />
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[[Image:pucregulation-fullpanels.png|950px]]<br />
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'''The Natural System (Left)''' Under high oxygen tension, PpsR prevents transcription from the puc promoter of the pucBA genes, and thus prevents the expression of LH2(1st panel). Under low oxygen tension, this repression is removed by activated AppA, thus allowing expression of LH2 from pucBA(2nd panel).<br><br />
'''Synthetic Regulation System (Right)''' Our system takes advantage of constitutive protein expression from the puc promoter at low oxygen tension, and replaces pucBA with Cph8 at this site. Placing pucBA expression under the control of the ompC promoter allows for Cph8/OmpR control of LH2 expression under varying light conditions. Under high intensity of incident light, Cph8 will not be autophosphorylated, preventing expression of pucBA (3rd panel). When light intensity is lower, Cph8 will autophosphorylate, thus phosphorylating and activating OmpR and promoting expression of LH2 from the ompC promoter.<br><br />
This regulation system was designed such that light is able to penetrate deeper into and through photobioreactors where light is plentiful.<br />
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<br>[https://2009.igem.org/Team:Wash_U/Project Back To Top]<br />
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== '''Results''' ==<br />
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For methods, materials, and procedures regarding these results, please see [https://2009.igem.org/Team:Wash_U/Protocol#Tissue_Flask_Experiment Tissue Flask Experiment] or [https://2009.igem.org/Team:Wash_U/Biological_Parts#Modeling Modeling]<br />
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'''Figure 1'''<br />
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|<font size="2" style="color:#black;">'''a''' Tissue Flask Position 1 Optical Density of Wild Type and DCBomega<br />
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|<font size="2" style="color:#black;">'''b''' Tissue Flask Position 2 Optical Density of Wild Type and DCBomega<br />
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[[Image:Tissue Flask 2b.png| 450 px| left]][[Image:Tissue Flask 1b.png| 450px | right]]<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
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|<font size="2" style="color:#black;">'''c''' Tissue Flask Position 3 Optical Density of Wild Type and DCBomega<br />
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|<font size="2" style="color:#black;">'''d''' Tissue Flask Position 4 Optical Density of Wild Type and DCBomega<br />
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[[Image:Tissue Flask 3b.png| 450px | left]][[Image:Tissue Flask 4b.png| 450px | right]]<br />
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'''e''' Tissue Flask Position 5 Optical Density of Wild Type and DBComega<br />
[[Image:Tissue Flask 5b.png| 450px | center]]<br><br />
Figure 1a,b,c,d,e are the optical density of a tissue flask, as assessed at 600 nm, over the course of the 6 days of the experiment.<br />
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'''Figure 2'''<br />
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|<font size="2" style="color:#black;">'''a''' The Change in the Optical Density of the Tissue Flasks for the WT<br />
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|<font size="2" style="color:#black;">'''b''' The Change in the Optical Density of the Tissue Flasks for DBComega<br />
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[[Image:3D 241.png| 450 px | left]][[Image:3D DBCOmega2.png| 450 px | right]]<br />
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Figure 2a and 2b display the growth of the tissue flasks as measured by OD assessed as absorbance at 600 nm. The graph is organized such that those closest to the "days" axis were closest to the light source (i.e. flask 1 is closer than flask 2).<br />
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'''Figure 3'''<br />
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[[Image:Bar 241 b.png| 450px | left]][[Image:Bar DBCOmega b.png| 450px | right]]<br />
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Figure 3a and 3b display the cumulative growth of the tissue flasks as measured by the sum of the optical density of the respective cell type's tissue flasks at a given day. Furthermore, the contribution of a given tissue flask to cumulative growth is displayed.<br><br />
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'''Figure 4'''<br />
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[[image:sphaeroides tissue flask.jpg|450 px|left|]][[image:dbc tissue flask.jpg|450px|right]]<br />
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Figure 4A and 4B display the appearance of the tissue flasks for both strains on day 6. They are arranged from left to right with left being closed to the light source (flask 1) and right furthest (flask 5)<br />
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'''Figure 5'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:#black;">'''a''' R. sphaeroides Spectrum by Flask Distance from Source <br />
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|<font size="2" style="color:#black;">'''b'''DBComega Spectrum by Flask Distance from Source<br />
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[[Image: WT irradiance 3d.png| 450 px | left]][[Image: DBComega irradiance 3d.png| 450 px | right]]<br />
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Contour of Absolute Irradiance by Flash distance from Source<br />
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|<font size="2" style="color:#black;">'''c''' R. Sphaeroides 2.4.1<br />
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[[Image: r sphaeroides day 2 a.jpg| 450px | left]][[Image: dbc day 4b.jpg| 450px | right]]<br />
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'''e''' Wild Type Absolute Irradiance Behind Flask 1: Day 6<br />
[[Image: irradiance WT flask 1 day6.png| 450px | center]]<br />
Figure 5 shows the results of the absolute irradiance data taken using the sprectroradiomter. A and B shows absolute irradiance on a given flask at each wavelength. Figure C and D represent the data for the same days as A and B in two dimensions. Figure E is the absolute irradiance behind only flask 1 on day 6. <br><br />
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|<font size="2" style="color:#black;">'''Figure 7'''<br />
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|<font size="2" style="color:#black;">Nonlinear least-squares estimation of WT LH2 saturation curve<br />
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|<font size="2" style="color:#black;">Simulated Optical Density of Mutant and Wild Type Bioreactors<br>Layers One and Two Layers Three, Four and Five<br />
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[[Image: saturation for WT as inferred.png| 450px | left]][[Image: OD after 3,4, and 5.jpg| 215px |right]][[Image: OD after flask 1 and 2.jpg| 215px | center]]<br />
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|<font size="2" style="color:#black;">Figure 7 shows the results of modeling the WT vs. mutant regulation system<br />
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[https://2009.igem.org/Team:Wash_U/Project Back To Top]<br />
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== '''Analysis''' ==<br />
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Though we were not able to have our mutant functionally express our system, the tissue flask experiment was an interesting way to observe the effects of a full knockout of the Light Harvesting Antenna Complex 2 (LH2) vs the Wild Type on an array of consecutive photobioreactors. A couple of conclusions can be drawn from this data that can be used in modeling our synthetic regulation system for LH2 and in the future design of systems for the optimization of Light Harvesting Antenna size for photobioreactors . <br><br><br />
The most striking difference between the Wild Type Rhodobacter Sphaeroides 2.41 and DBCOmega (LH2 knockout) growth patterns is that the first flask in the WT (closest to the light source) grew less than the second flask, while the converse was true in DBCOmega (figure 2a and 2b). This can be attributed to the photosystem saturation curves for the respective cultures. The Wild Type has an LH2 complex, meaning that their antenna size is inherently larger and that their photosystem will be saturated at lower light intensities than the LH2 deficient mutant. We observed that the incident light intensity used in this experiment led to oversaturation for the first WT tissue flask and resulted in growth-inhibiting photodamage, as is evidenced by its lesser growth relative to the second culture. In contrast, photodamage was not observed in DBCOmega as is evidenced by the first tissue flask that grew at the fastest respective rate. Furthermore, it appears that this photodamage slowed the growth of the first WT tissue flask to the point that the OD of this tissue flask was nearly equal to that of DBCOmega after 5 days of growth(.987 vs. .967) (figure 1a). As such, the contribution of LH2 to the growth of the WT in flask 1 can be assessed to be little after day 3 when the growth curve slows- though, this light (at 800 and 850 nm) was still greatly absorbed by flask 1 (Figure 5e). It is likely that much of this light was then wasted through Non-Photochemical Quenching (NPQ), depleting the flasks behind flask 1 of photons at the LH2 absorbtion peaks and decreasing the cumulative productivity of flasks 1-5. This effect of wasting photons though NPQ under high light intensities is one that we sought to minimize in the design of our synthetic regulation for the pucB/A genes. <br><br><br />
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A further a comparison of the WT and DBCOmega experiments reveals that the 3rd tissue flask in each had almost identical growth rates (figure 1c), while the DBCOmega 4th and 5th tissue flask outperformed that of the WT (figure 1d and 1e). This can simply be attributed to the higher density of cells in the 2nd flask of the WT that led to an overall lesser quantity of photons passing through to the 4th and 5th flasks in the WT experiment and likely resulting in heterotrophic growth (though this will need to be confirmed with a "dark control" experiment that should be completed in time for the Jamboree). This decrease in available photons for the WT can be seen by viewing the absolute irradiance data (incident light intensity at a given wavelength) after the third culture for the WT (figure 5a and 5c). This decrease in irradiance is the most pronounced for the LH2 absorption bands at 800 and 850 nm, as was expected. Our synthetic regulation system for pucB/A was designed with the intention of allowing greater penetration of light through the first couple of flasks at the LH2 wavelengths, as expression of pucB/A is inversely correlated to incident light intensity.<br><br><br />
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Overall, the cumulative culture growth of the WT exceeded that of DBCOmega (figure 3) due to the performance of the second tissue flask of the WT relative to that of DBCOmega (figure 1b) despite the photodamage that occurred in tissue flask 1 for the WT. This cumulative culture growth can be considered photosynthetic productivty and is proportional to the amount of energy/desired product that can be obtained from these cultures. <br><br><br />
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It is an interesting exercise to model how our mutant system would perform under these same conditions. To do so, the LH2 saturation curve for the WT (figure 6) and the relative contribution of LH1/LH2 light absorbance to growth was derived from our empirical data and applied to a model that took into account incident light and gave the relative growth in terms of optical density as an output. In this model, our synthetic regulation system of pucB/A allowed our mutant to outperform the wild type species (figure 7). This was due to the performance of the 3rd, 4th, and 5th tissue flask relative to those of the wild type (see figure 7 layers 1,2 vs 1-5). The reason for this is that the wild type LH2 light saturation curve does not vary with changes in incident light intensity as the wild type expresses pucB/A at a uniform level under low oxygen conditions. As a result, the first two layers of Wild Type bioreactors absorb nearly all the available light and starve the bioreactors behind them for photons. <br><br><br />
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Conversely, our mutant was modeled to have a dynamic expression of pucB/A based on the incident light intensity. This light response curve was modeled as an inverse exponential for the Cph8/OmpR ompC Promoter system and the saturation curve changes for each tissue flask in accordance with the incident light intensity- as light intensity decreases, the expression pucB/A increases. This results in more light passing through the first 2 flasks and facilitating greater growth in flasks 3, 4 and 5 as they express large antenna complexes. Nonetheless, we believe that growth across the bioreactors could be further maximized by increasing antenna size in the first 2 bioreactors and sacrificing growth in the 4th and 5th. This could be achieved with a feedback mechanism that sets a minimum level for LH2 expression. <br />
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='''Conclusion'''=<br />
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Though we were not able to conduct the tissue flask growth experiment on our mutant, the results of this experiment on our controls (WT and DBComega) match the assumptions that we had laid out at the beginning of the project, allowed us to draw further conclusions about the efficiency of light-based growth for a series of bioreactors, and to create a model for our mutant based on empirical data. <br> <br />
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*Based on this model, we have concluded that it would be favorable to have a feedback regulation mechanism that sets a minimum expression level for pucB/A to further improve photosynthetic productivity. <br><br />
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*We intend to conduct the tissue flask w DBComega+prkcbc3 before jamboree and compare/interpret these results using our characterization data for the pucB/A and the puc promoter.<br />
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*We will conduct the tissue flask experiment with our functioning mutant in future and compare the results to our model<br />
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*Finally, we would like to characterize the puc promoter under various light conditions and additional oxygen tensions. <br><br />
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'''Overall- our project has demonstrated the potential for a synthetically regulated light harvesting antenna to improve photosynthetic productivity for a series of photobioreactors vs. the wild type.''' <br />
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[https://2009.igem.org/Team:Wash_U/Project Back To Top]<br />
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== '''References''' ==<br />
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# "2007-2008 Catalog & Technical Reference." <u>New England BioLabs Inc.</u> (2007).<br />
# Alon, Uri. <u>Introduction to systems biology and the design principles of biological networks</u>. Boca Raton, FL: Chapman & Hall, 2006.<br />
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# Bower, James M. <u>Computational Modeling of Genetic and Biochemical Networks (Computational Molecular Biology).</u> New York: M.I.T. PRESS, 2001.<br />
# Braatsch, Stephan et al. "A single Flavoprotein AppA, Integrates Both Redox and Light Signals in ''Rhodobacter sphaeroides.''" <u>Molecular Microbiology.</u> Vol. 45.3 (2002): 827-836.<br />
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# Fowler, Gregory J. S. and C. Neil Hunter. "The Synthesis and Assembly of Functional High and Low Light LH2 Antenna Complexes from ''Rhodopseudomonas palustris'' in ''Rhodobacter sphaeroides.''" <u>The Journal of Biological Chemistry.</u> Vol. 271.23 (1996): 13,356-13,361.<br />
# Gabrielsen, Mads et al. "Peripheral Complexes of Purple Bacteria." <u>The Purple Phototropic Bacteria.</u> (2009): 135-153.<br />
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# Harwood, Caroline S. "Nitrogenase-Catalyzed Hydrogen Production by Purple Nonsulfur Photosynthetic Bacteria." <u>Bioenergy.</u> (2008): 259-271.<br />
# Jager, Andreas et al. "The AppA and PpsR Proteins from ''Rhodobacter sphaeroides'' Can Establish a Redox-Dependent Signal Chain but Fail to Transmit Blue-Light Signals in Other Bacteria." <u>Journal of Bacteriology.</u> Vol. 189.6 (2007): 2,274-2,282.<br />
# Jones, M. R. et al. "Mutants of ''Rhodobacter sphaeroides'' Lacking One or More Pigment-Protein Complexes and Complementation with Reaction-Centre, LH1, and LH2 Genes." <u>Molecular Microbiology.</u> Vol. 6.9 (1992): 1,173-1,184.<br />
# Kondo, Toshihiko et al. "Enhancement of Hydrogen Production by a Photosynthetic Bacterium Mutant with Reduced Pigment." <u>Journal of Bioscience and Bioengineering.</u> Vol. 93.2 (2002): 145-150.<br />
# Lagarias, J. Clark. <u>PCB from Spirulina</u>. Personal Communication. June 2009.<br />
# Laible, Philip D. "Foreign Gene Expression in Photosynthetic Bacteria." <u>The Purple Phototropic Bacteria.</u> (2009): 839-860.<br />
# Lee, Jeong K. and Samuel Kaplan. "Transcriptional Regulation of ''puc'' Operon Expression in ''Rhodobacter sphaeroides.''" <u>Journal of Biological Chemistry.</u> Vol. 270.35 (1995): 20,453-20,458.<br />
# Levskaya, Anselmetal et al. "Engineering ''Escherichia coli'' to See Light." <u>Nature.</u> Vol. 438 (2005): 441-442.<br />
# "Life Sciences Catalog 2009-2010." <u>National Diagnostics.</u> (2009).<br />
# Melis, Anastasios. "Solar Energy Conversion Efficiencies in Photosynthesis: Minimizing the Chlorophyll Antennae to Maximize Efficiency." <u>Plant Science.</u> (2009).<br />
# Mizuno, Takeshi and Shoji Mizushima. "Characterization by Deletion and Localized Mutagenesis in Vitro of the Promoter Region of the ''Escherichia coli'' OmpC Gene and Importance of the Upstream DNA Domain in Positive Regulation by the OmpR Protein." <u>Journal of Bacteriology.</u> Vol. 168.1 (1986): 86-95.<br />
# Moskvin, Oleg V. et al. "Transcriptome Analysis of the ''Rhodobacter sphaeroides'' PspR Regulon: PspR as a Master Regulator of Photosystem Development." <u>Journal of Bacteriology.</u> Vol. 187.6 (2005): 2,148-2,156.<br />
# Mussgnug, Jan H. et al. "Engineering photosynthetic light capture: impacts on improved solar energy to biomass conversion." <u> Plant Biotechnology Journal</u> Vol. 5 Issue 6. (2007) pp. 802-814<br />
# "NEBCutter V2.0." <u>New England BioLabs.</u> 2009. 6 June 2009 <http://tools.neb.com/NEBcutter2/index.php>.<br />
# "OligoAnalyzer 3.1." <u>Integrated DNA Technologies.</u> 2009. 6 June 2009. <http://www.idtdna.com/analyzer/Applications/OligoAnalyzer/><br />
# Sambrook, Joseph and David W. Russell. <u>Molecular Cloning: A Laboratory Manual.</u> Vol. 1-3. New York: Cold Springs Harbor Laboratory Press, 2001.<br />
# Scheuring, Simon and Sturgis, James N. "Chromatic Adaptation of Photosynthetic Membranes" <u>Science</u> Vol. 309 (2005) pp.484-487<br />
# Sener, Melih K. et al. "Atomic-level structural and functional model of a bacterial photosynthetic membrane vesicle" <u>Proceedings of the National Academy of Sciences of the United States of America.</u> Vol. 104, Number 40,pp. 15723-15728<br />
# Shimizu-Sato, Sae et al. "A Light-Switchable Gene Promoter System." <u>Nature Publishing Group.</u> Vol. 20 (2002): 1,041-1044.<br />
# Smith, Harry and M. Geoffrey Holmes. <u>Techniquies in Photomorphogenesis.</u> London: Academic Press, Inc., 1984.<br />
# <u>System modeling in cellular biology from concepts to nuts and bolts.</u> Cambridge, MA: MIT P, 2006.<br />
# Terry, Matthew J. "Biosynthesis and Analysis of Bilins." <u>Heme, Chlorophyll, and Bilins: Methods and Protocols.</u> (2002) 273-291. <br />
# Wang, Wanneng et al. "Heterologous Synthesis and Assembly of Functional LHII Antenna Complexes from ''Rhodovulum sulfidophilum'' in ''Rhodobacter sphaeroides'' Mutant." <u>Molecular Biology Reports.</u> Vol. 12.3 (2008).<br />
# Walz, Thomas et al. "Projection Structures of Three Photosynthetic Complexes from Rhodobacter sphaeroides: LH2 at 6 A, LH1 and RC-LH1 at 25 A" <u>Journal of Molecular Biology</u> Vol. 282 pp. 833-845<br />
# Yakovlev, A. G. et al. "Light Control Over the Size of an Antenna Unit Building Block as an Efficient Strategy for Light Harvesting in Photosynthesis." <u>Federation of European Biochemical Societies.</u> (2002):129-132.<br />
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{{WashUbottom}}</div>Brendan1http://2009.igem.org/Team:Wash_U/ProjectTeam:Wash U/Project2009-11-10T00:47:56Z<p>Brendan1: /* Results */</p>
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__NOTOC__<br />
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== '''Introduction''' == <br />
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Our project goal is to maximize the photosynthetic productivity of a series of photobioreactors containing ''Rhodobacter sphaeroides'' under both high and low light intensities by synthetically regulating the size of the light-harvesting antenna complex LH2. We chose to undertake such a project using ''R. sphaeroides'' due to its well-characterized photosynthetic and genetic system. <br />
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The antenna system functions to expand the spectrum of light available for photosynthesis by absorbing different wavelengths than that of the reaction center. Essentially, it is like the dish around the main receiver of any antenna. Marine bacteria, such as ''R. sphaeroides'', evolved very large antenna complexes to absorb light in a natural environment, where there is great competition for photons. As a result, the photosynthetic machinery is saturated at low light intensities in a synthetic non-competitive environment, such as a photobioreactor. This causes up to 95% of incidental photons to be dissipated as heat or fluorescence by bacteria through a process called Non-Photochemical Quenching (NPQ) (Mussgnug et al., 2007). In essence, these photons are being wasted as NPQ reduces light penetration into and across bioreactors and starves the shaded cells for for photons. One method that has been shown to improve photosynthetic productivity in photobioreactors is the reduction of light-harvesting antenna sizes (Polle et al., 2002, Mussgnug et al., 2007). Though, current approaches to this end are difficult to precisely control from the perspective of biological engineering and synthetic biology. <br />
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Our intention is to create a dynamic system that varies antenna size, is dependent on incident light intensity, and can be readily optimized using bioengineering principles. This synthetic regulation of the LH2 complex should result in bacteria grown under high light intensities expressing fewer LH2 complexes than cells that are more shaded, leading to a reduction of wasted incident photons while maintaining a high overall absorbance across subsequent bioreactors. Consequently, we expect to see an increase in the total photosynthetic productivity of our mutant ''Rhodobacter sphaeroides'' when compared to the wild type grown under similar conditions. <br />
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== '''Organism''' ==<br />
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''Rhodobacter sphaeroides'' is a purple Alphaproteobacteria. It is metabolically flexible: it can grow heterotrophically, via aerobic and anaerobic respiration, as well as phototrophically under anaerobic conditions with light. <br />
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''R. sphaeroides'' is one of the best understood photosynthetic organisms. The photosystem is located in intracytoplasmic membrane invaginations and the main components are the Light Haresting Complex 2 (LH2), Light Harvesting Complex 1 (LH1), and the Reaction Center (RC). These pigment-protein complexes non-covalently bind bacteriochlorophylls and carotenoids. LH1 and RC make up the core complex and are often found in the ratio of 1:1. LH2 is more peripheral and naturally ranges in the ratio to LH1/RC of 3.0:1 to 6.7:1 under varying light conditions (Scheuring et al., 2005). LH2 absorbs photons maximally at the wavelengths of 850 and 800 nm and funnels its energy to LH1 and the reaction center for photochemistry. (Scheuring et al., 2005). <br />
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The two subunits of LH2 are coded for by the pucB/A genes and are naturally promoted by the puc promoter. <br />
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<br><div style="text-align: center;">'''ICM of ''R. sphaeroides'' '''[[Image:R sphaeroides photosystem.png| 500 px | center]]<center>source:Sener et al.2007<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:black;">'''Plate of ''R. sphaeroides'' 2.4.1'''<br />
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[[Image:R sphaeroides aborbance.png| 450px | left]][[Image:R sphaeroides 0986.png| 450px | right]]<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br>source: Walz et al. 1998<br><br />
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====Online Resources for ''R. sphaeroides''====<br />
[http://www.kazusa.or.jp/codon/cgi-bin/showcodon.cgi?species=272943 Codon Usage Table for ''R. sphaeroides'' 2.41]<br />
<br> [http://www.ncbi.nlm.nih.gov/sites/entrez?db=genome&cmd=Retrieve&dopt=Overview&list_uids=18843 ''R. sphaeroides'' 2.41 NCBI Genome Tools]<br />
<br> [http://archaea.ucsc.edu/cgi-bin/hgPcr?wp_target=&db=0&org=Rhodobacter+sphaeroides+2+4+1&wp_f=&wp_r=&wp_size=2000&wp_perfect=15&wp_good=15&wp_showPage=true&hgsid=159191 ''In silico'' PCR of ''R. sphaeroides'' 2.41 genome]<br />
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=='''Regulation'''==<br />
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[[Image:pucregulation-fullpanels.png|950px]]<br />
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'''The Natural System (Left)''' Under high oxygen tension, PpsR prevents transcription from the puc promoter of the pucBA genes, and thus prevents the expression of LH2(1st panel). Under low oxygen tension, this repression is removed by activated AppA, thus allowing expression of LH2 from pucBA(2nd panel).<br><br />
'''Synthetic Regulation System (Right)''' Our system takes advantage of constitutive protein expression from the puc promoter at low oxygen tension, and replaces pucBA with Cph8 at this site. Placing pucBA expression under the control of the ompC promoter allows for Cph8/OmpR control of LH2 expression under varying light conditions. Under high intensity of incident light, Cph8 will not be autophosphorylated, preventing expression of pucBA (3rd panel). When light intensity is lower, Cph8 will autophosphorylate, thus phosphorylating and activating OmpR and promoting expression of LH2 from the ompC promoter.<br><br />
This regulation system was designed such that light is able to penetrate deeper into and through photobioreactors where light is plentiful.<br />
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<br>[https://2009.igem.org/Team:Wash_U/Project Back To Top]<br />
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== '''Results''' ==<br />
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For methods, materials, and procedures regarding these results, please see [https://2009.igem.org/Team:Wash_U/Protocol#Tissue_Flask_Experiment Tissue Flask Experiment] or [https://2009.igem.org/Team:Wash_U/Biological_Parts#Modeling Modeling]<br />
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'''Figure 1'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:#black;">'''b''' Tissue Flask Position 2 Optical Density of Wild Type and DCBomega<br />
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[[Image:Tissue Flask 2b.png| 450 px| left]][[Image:Tissue Flask 1b.png| 450px | right]]<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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[[Image:Tissue Flask 3b.png| 450px | left]][[Image:Tissue Flask 4b.png| 450px | right]]<br />
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'''e''' Tissue Flask Position 5 Optical Density of Wild Type and DBComega<br />
[[Image:Tissue Flask 5b.png| 450px | center]]<br><br />
Figure 1a,b,c,d,e are the optical density of a tissue flask, as assessed at 600 nm, over the course of the 6 days of the experiment.<br />
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'''Figure 2'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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[[Image:3D 241.png| 450 px | left]][[Image:3D DBCOmega2.png| 450 px | right]]<br />
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Figure 2a and 2b display the growth of the tissue flasks as measured by OD assessed as absorbance at 600 nm. The graph is organized such that those closest to the "days" axis were closest to the light source (i.e. flask 1 is closer than flask 2).<br />
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'''Figure 3'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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[[Image:Bar 241 b.png| 450px | left]][[Image:Bar DBCOmega b.png| 450px | right]]<br />
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Figure 3a and 3b display the cumulative growth of the tissue flasks as measured by the sum of the optical density of the respective cell type's tissue flasks at a given day. Furthermore, the contribution of a given tissue flask to cumulative growth is displayed.<br><br />
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'''Figure 4'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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[[image:sphaeroides tissue flask.jpg|450 px|left|]][[image:dbc tissue flask.jpg|450px|right]]<br />
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Figure 4A and 4B display the appearance of the tissue flasks for both strains on day 6. They are arranged from left to right with left being closed to the light source (flask 1) and right furthest (flask 5)<br />
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'''Figure 5'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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[[Image: WT irradiance 3d.png| 450 px | left]][[Image: DBComega irradiance 3d.png| 450 px | right]]<br />
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Contour of Absolute Irradiance by Flash distance from Source<br />
{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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[[Image: r sphaeroides day 2 a.jpg| 450px | left]][[Image: dbc day 4b.jpg| 450px | right]]<br />
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'''e''' Wild Type Absolute Irradiance Behind Flask 1: Day 6<br />
[[Image: irradiance WT flask 1 day6.png| 450px | center]]<br />
Figure 5 shows the results of the absolute irradiance data taken using the sprectroradiomter. A and B shows absolute irradiance on a given flask at each wavelength. Figure C and D represent the data for the same days as A and B in two dimensions. Figure E is the absolute irradiance behind only flask 1 on day 6. <br><br />
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Simulated Optical Density of Mutant and Wild Type Bioreactors<br><br />
Nonlinear least-squares estimation of WT LH2 saturation curve <br><br />
[[Image: saturation for WT as inferred.png| 450px | left]][[Image: OD after 3,4, and 5.jpg| 215px |right]][[Image: OD after flask 1 and 2.jpg| 215px | center]]<br />
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[https://2009.igem.org/Team:Wash_U/Project Back To Top]<br />
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== '''Analysis''' ==<br />
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Though we were not able to have our mutant functionally express our system, the tissue flask experiment was an interesting way to observe the effects of a full knockout of the Light Harvesting Antenna Complex 2 (LH2) vs the Wild Type on an array of consecutive photobioreactors. A couple of conclusions can be drawn from this data that can be used in modeling our synthetic regulation system for LH2 and in the future design of systems for the optimization of Light Harvesting Antenna size for photobioreactors . <br><br><br />
The most striking difference between the Wild Type Rhodobacter Sphaeroides 2.41 and DBCOmega (LH2 knockout) growth patterns is that the first flask in the WT (closest to the light source) grew less than the second flask, while the converse was true in DBCOmega (figure 2a and 2b). This can be attributed to the photosystem saturation curves for the respective cultures. The Wild Type has an LH2 complex, meaning that their antenna size is inherently larger and that their photosystem will be saturated at lower light intensities than the LH2 deficient mutant. We observed that the incident light intensity used in this experiment led to oversaturation for the first WT tissue flask and resulted in growth-inhibiting photodamage, as is evidenced by its lesser growth relative to the second culture. In contrast, photodamage was not observed in DBCOmega as is evidenced by the first tissue flask that grew at the fastest respective rate. Furthermore, it appears that this photodamage slowed the growth of the first WT tissue flask to the point that the OD of this tissue flask was nearly equal to that of DBCOmega after 5 days of growth(.987 vs. .967) (figure 1a). As such, the contribution of LH2 to the growth of the WT in flask 1 can be assessed to be little after day 3 when the growth curve slows- though, this light (at 800 and 850 nm) was still greatly absorbed by flask 1 (Figure 5e). It is likely that much of this light was then wasted through Non-Photochemical Quenching (NPQ), depleting the flasks behind flask 1 of photons at the LH2 absorbtion peaks and decreasing the cumulative productivity of flasks 1-5. This effect of wasting photons though NPQ under high light intensities is one that we sought to minimize in the design of our synthetic regulation for the pucB/A genes. <br><br><br />
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A further a comparison of the WT and DBCOmega experiments reveals that the 3rd tissue flask in each had almost identical growth rates (figure 1c), while the DBCOmega 4th and 5th tissue flask outperformed that of the WT (figure 1d and 1e). This can simply be attributed to the higher density of cells in the 2nd flask of the WT that led to an overall lesser quantity of photons passing through to the 4th and 5th flasks in the WT experiment and likely resulting in heterotrophic growth (though this will need to be confirmed with a "dark control" experiment that should be completed in time for the Jamboree). This decrease in available photons for the WT can be seen by viewing the absolute irradiance data (incident light intensity at a given wavelength) after the third culture for the WT (figure 5a and 5c). This decrease in irradiance is the most pronounced for the LH2 absorption bands at 800 and 850 nm, as was expected. Our synthetic regulation system for pucB/A was designed with the intention of allowing greater penetration of light through the first couple of flasks at the LH2 wavelengths, as expression of pucB/A is inversely correlated to incident light intensity.<br><br><br />
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Overall, the cumulative culture growth of the WT exceeded that of DBCOmega (figure 3) due to the performance of the second tissue flask of the WT relative to that of DBCOmega (figure 1b) despite the photodamage that occurred in tissue flask 1 for the WT. This cumulative culture growth can be considered photosynthetic productivty and is proportional to the amount of energy/desired product that can be obtained from these cultures. <br><br><br />
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It is an interesting exercise to model how our mutant system would perform under these same conditions. To do so, the LH2 saturation curve for the WT (figure 6) and the relative contribution of LH1/LH2 light absorbance to growth was derived from our empirical data and applied to a model that took into account incident light and gave the relative growth in terms of optical density as an output. In this model, our synthetic regulation system of pucB/A allowed our mutant to outperform the wild type species (figure 7). This was due to the performance of the 3rd, 4th, and 5th tissue flask relative to those of the wild type (see figure 7 layers 1,2 vs 1-5). The reason for this is that the wild type LH2 light saturation curve does not vary with changes in incident light intensity as the wild type expresses pucB/A at a uniform level under low oxygen conditions. As a result, the first two layers of Wild Type bioreactors absorb nearly all the available light and starve the bioreactors behind them for photons. <br><br><br />
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Conversely, our mutant was modeled to have a dynamic expression of pucB/A based on the incident light intensity. This light response curve was modeled as an inverse exponential for the Cph8/OmpR ompC Promoter system and the saturation curve changes for each tissue flask in accordance with the incident light intensity- as light intensity decreases, the expression pucB/A increases. This results in more light passing through the first 2 flasks and facilitating greater growth in flasks 3, 4 and 5 as they express large antenna complexes. Nonetheless, we believe that growth across the bioreactors could be further maximized by increasing antenna size in the first 2 bioreactors and sacrificing growth in the 4th and 5th. This could be achieved with a feedback mechanism that sets a minimum level for LH2 expression. <br />
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='''Conclusion'''=<br />
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Though we were not able to conduct the tissue flask growth experiment on our mutant, the results of this experiment on our controls (WT and DBComega) match the assumptions that we had laid out at the beginning of the project, allowed us to draw further conclusions about the efficiency of light-based growth for a series of bioreactors, and to create a model for our mutant based on empirical data. <br> <br />
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*Based on this model, we have concluded that it would be favorable to have a feedback regulation mechanism that sets a minimum expression level for pucB/A to further improve photosynthetic productivity. <br><br />
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*We intend to conduct the tissue flask w DBComega+prkcbc3 before jamboree and compare/interpret these results using our characterization data for the pucB/A and the puc promoter.<br />
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*We will conduct the tissue flask experiment with our functioning mutant in future and compare the results to our model<br />
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*Finally, we would like to characterize the puc promoter under various light conditions and additional oxygen tensions. <br><br />
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'''Overall- our project has demonstrated the potential for a synthetically regulated light harvesting antenna to improve photosynthetic productivity for a series of photobioreactors vs. the wild type.''' <br />
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[https://2009.igem.org/Team:Wash_U/Project Back To Top]<br />
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== '''References''' ==<br />
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# "2007-2008 Catalog & Technical Reference." <u>New England BioLabs Inc.</u> (2007).<br />
# Alon, Uri. <u>Introduction to systems biology and the design principles of biological networks</u>. Boca Raton, FL: Chapman & Hall, 2006.<br />
# Ausubel, Fred et al. <u>Short Protocols in Molecular Biology: Third Edition.</u> Canada: John Wiley and Sons, Inc., 1999.<br />
# "BioBrick Assembly Help." <u>Registry of Standard Biological Parts.</u> 2009. 6 July 2009. <http://partsregistry.org/Main_Page>.<br />
# Bower, James M. <u>Computational Modeling of Genetic and Biochemical Networks (Computational Molecular Biology).</u> New York: M.I.T. PRESS, 2001.<br />
# Braatsch, Stephan et al. "A single Flavoprotein AppA, Integrates Both Redox and Light Signals in ''Rhodobacter sphaeroides.''" <u>Molecular Microbiology.</u> Vol. 45.3 (2002): 827-836.<br />
# Brown, Stanley B. et al. "Bile Pigment Synthesis in Plants: Incorporation of Haem into Phycocyanobilin and Phycocyanobiliproteins in Cyanidium Caldarium." <u>Biochemistry.</u> Vol. 194 (1981): 137-147.<br />
# Dragnea, Vladimira et al. "Time-Resolved Spectroscopic Studies of the AppA Blue-Light Receptor BLUF Domain from ''Rhodobacter sphaeroides'' <u>Biochemistry.</u> Vol. 44 (2005): 15,978-15,985.<br />
# "Entrez Nucleotide Search." <u>National Center for Biotechnology Information.</u> 2009. 6 June 2009 <http://www.ncbi.nlm.nih.gov/nuccore/49175990?from=3533887&to=3534606&report=gbwithparts>.<br />
# Fowler, Gregory J. S. and C. Neil Hunter. "The Synthesis and Assembly of Functional High and Low Light LH2 Antenna Complexes from ''Rhodopseudomonas palustris'' in ''Rhodobacter sphaeroides.''" <u>The Journal of Biological Chemistry.</u> Vol. 271.23 (1996): 13,356-13,361.<br />
# Gabrielsen, Mads et al. "Peripheral Complexes of Purple Bacteria." <u>The Purple Phototropic Bacteria.</u> (2009): 135-153.<br />
# "GenElute HP Plasmid Miniprep Kit: User Guide." <u>Sigma-Aldrich</u>. (2008).<br />
# Harwood, Caroline S. "Nitrogenase-Catalyzed Hydrogen Production by Purple Nonsulfur Photosynthetic Bacteria." <u>Bioenergy.</u> (2008): 259-271.<br />
# Jager, Andreas et al. "The AppA and PpsR Proteins from ''Rhodobacter sphaeroides'' Can Establish a Redox-Dependent Signal Chain but Fail to Transmit Blue-Light Signals in Other Bacteria." <u>Journal of Bacteriology.</u> Vol. 189.6 (2007): 2,274-2,282.<br />
# Jones, M. R. et al. "Mutants of ''Rhodobacter sphaeroides'' Lacking One or More Pigment-Protein Complexes and Complementation with Reaction-Centre, LH1, and LH2 Genes." <u>Molecular Microbiology.</u> Vol. 6.9 (1992): 1,173-1,184.<br />
# Kondo, Toshihiko et al. "Enhancement of Hydrogen Production by a Photosynthetic Bacterium Mutant with Reduced Pigment." <u>Journal of Bioscience and Bioengineering.</u> Vol. 93.2 (2002): 145-150.<br />
# Lagarias, J. Clark. <u>PCB from Spirulina</u>. Personal Communication. June 2009.<br />
# Laible, Philip D. "Foreign Gene Expression in Photosynthetic Bacteria." <u>The Purple Phototropic Bacteria.</u> (2009): 839-860.<br />
# Lee, Jeong K. and Samuel Kaplan. "Transcriptional Regulation of ''puc'' Operon Expression in ''Rhodobacter sphaeroides.''" <u>Journal of Biological Chemistry.</u> Vol. 270.35 (1995): 20,453-20,458.<br />
# Levskaya, Anselmetal et al. "Engineering ''Escherichia coli'' to See Light." <u>Nature.</u> Vol. 438 (2005): 441-442.<br />
# "Life Sciences Catalog 2009-2010." <u>National Diagnostics.</u> (2009).<br />
# Melis, Anastasios. "Solar Energy Conversion Efficiencies in Photosynthesis: Minimizing the Chlorophyll Antennae to Maximize Efficiency." <u>Plant Science.</u> (2009).<br />
# Mizuno, Takeshi and Shoji Mizushima. "Characterization by Deletion and Localized Mutagenesis in Vitro of the Promoter Region of the ''Escherichia coli'' OmpC Gene and Importance of the Upstream DNA Domain in Positive Regulation by the OmpR Protein." <u>Journal of Bacteriology.</u> Vol. 168.1 (1986): 86-95.<br />
# Moskvin, Oleg V. et al. "Transcriptome Analysis of the ''Rhodobacter sphaeroides'' PspR Regulon: PspR as a Master Regulator of Photosystem Development." <u>Journal of Bacteriology.</u> Vol. 187.6 (2005): 2,148-2,156.<br />
# Mussgnug, Jan H. et al. "Engineering photosynthetic light capture: impacts on improved solar energy to biomass conversion." <u> Plant Biotechnology Journal</u> Vol. 5 Issue 6. (2007) pp. 802-814<br />
# "NEBCutter V2.0." <u>New England BioLabs.</u> 2009. 6 June 2009 <http://tools.neb.com/NEBcutter2/index.php>.<br />
# "OligoAnalyzer 3.1." <u>Integrated DNA Technologies.</u> 2009. 6 June 2009. <http://www.idtdna.com/analyzer/Applications/OligoAnalyzer/><br />
# Sambrook, Joseph and David W. Russell. <u>Molecular Cloning: A Laboratory Manual.</u> Vol. 1-3. New York: Cold Springs Harbor Laboratory Press, 2001.<br />
# Scheuring, Simon and Sturgis, James N. "Chromatic Adaptation of Photosynthetic Membranes" <u>Science</u> Vol. 309 (2005) pp.484-487<br />
# Sener, Melih K. et al. "Atomic-level structural and functional model of a bacterial photosynthetic membrane vesicle" <u>Proceedings of the National Academy of Sciences of the United States of America.</u> Vol. 104, Number 40,pp. 15723-15728<br />
# Shimizu-Sato, Sae et al. "A Light-Switchable Gene Promoter System." <u>Nature Publishing Group.</u> Vol. 20 (2002): 1,041-1044.<br />
# Smith, Harry and M. Geoffrey Holmes. <u>Techniquies in Photomorphogenesis.</u> London: Academic Press, Inc., 1984.<br />
# <u>System modeling in cellular biology from concepts to nuts and bolts.</u> Cambridge, MA: MIT P, 2006.<br />
# Terry, Matthew J. "Biosynthesis and Analysis of Bilins." <u>Heme, Chlorophyll, and Bilins: Methods and Protocols.</u> (2002) 273-291. <br />
# Wang, Wanneng et al. "Heterologous Synthesis and Assembly of Functional LHII Antenna Complexes from ''Rhodovulum sulfidophilum'' in ''Rhodobacter sphaeroides'' Mutant." <u>Molecular Biology Reports.</u> Vol. 12.3 (2008).<br />
# Walz, Thomas et al. "Projection Structures of Three Photosynthetic Complexes from Rhodobacter sphaeroides: LH2 at 6 A, LH1 and RC-LH1 at 25 A" <u>Journal of Molecular Biology</u> Vol. 282 pp. 833-845<br />
# Yakovlev, A. G. et al. "Light Control Over the Size of an Antenna Unit Building Block as an Efficient Strategy for Light Harvesting in Photosynthesis." <u>Federation of European Biochemical Societies.</u> (2002):129-132.<br />
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{{WashUbottom}}</div>Brendan1http://2009.igem.org/Team:Wash_U/ProjectTeam:Wash U/Project2009-11-10T00:41:04Z<p>Brendan1: /* Results */</p>
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__NOTOC__<br />
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== '''Introduction''' == <br />
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Our project goal is to maximize the photosynthetic productivity of a series of photobioreactors containing ''Rhodobacter sphaeroides'' under both high and low light intensities by synthetically regulating the size of the light-harvesting antenna complex LH2. We chose to undertake such a project using ''R. sphaeroides'' due to its well-characterized photosynthetic and genetic system. <br />
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The antenna system functions to expand the spectrum of light available for photosynthesis by absorbing different wavelengths than that of the reaction center. Essentially, it is like the dish around the main receiver of any antenna. Marine bacteria, such as ''R. sphaeroides'', evolved very large antenna complexes to absorb light in a natural environment, where there is great competition for photons. As a result, the photosynthetic machinery is saturated at low light intensities in a synthetic non-competitive environment, such as a photobioreactor. This causes up to 95% of incidental photons to be dissipated as heat or fluorescence by bacteria through a process called Non-Photochemical Quenching (NPQ) (Mussgnug et al., 2007). In essence, these photons are being wasted as NPQ reduces light penetration into and across bioreactors and starves the shaded cells for for photons. One method that has been shown to improve photosynthetic productivity in photobioreactors is the reduction of light-harvesting antenna sizes (Polle et al., 2002, Mussgnug et al., 2007). Though, current approaches to this end are difficult to precisely control from the perspective of biological engineering and synthetic biology. <br />
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Our intention is to create a dynamic system that varies antenna size, is dependent on incident light intensity, and can be readily optimized using bioengineering principles. This synthetic regulation of the LH2 complex should result in bacteria grown under high light intensities expressing fewer LH2 complexes than cells that are more shaded, leading to a reduction of wasted incident photons while maintaining a high overall absorbance across subsequent bioreactors. Consequently, we expect to see an increase in the total photosynthetic productivity of our mutant ''Rhodobacter sphaeroides'' when compared to the wild type grown under similar conditions. <br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:black;">'''Wild Type Energy Production in a series of Bioreactors'''<br />
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|<font size="2" style="color:black;">'''Synthetically Regulated Antenna Energy Production in Bioreactors'''<br />
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[[Image:Slide09b.jpg| 450px | left]][[Image:Slide10 a.jpg| 450px | right]]<br />
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== '''Organism''' ==<br />
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''Rhodobacter sphaeroides'' is a purple Alphaproteobacteria. It is metabolically flexible: it can grow heterotrophically, via aerobic and anaerobic respiration, as well as phototrophically under anaerobic conditions with light. <br />
<br> <br><br />
''R. sphaeroides'' is one of the best understood photosynthetic organisms. The photosystem is located in intracytoplasmic membrane invaginations and the main components are the Light Haresting Complex 2 (LH2), Light Harvesting Complex 1 (LH1), and the Reaction Center (RC). These pigment-protein complexes non-covalently bind bacteriochlorophylls and carotenoids. LH1 and RC make up the core complex and are often found in the ratio of 1:1. LH2 is more peripheral and naturally ranges in the ratio to LH1/RC of 3.0:1 to 6.7:1 under varying light conditions (Scheuring et al., 2005). LH2 absorbs photons maximally at the wavelengths of 850 and 800 nm and funnels its energy to LH1 and the reaction center for photochemistry. (Scheuring et al., 2005). <br />
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The two subunits of LH2 are coded for by the pucB/A genes and are naturally promoted by the puc promoter. <br />
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<br><div style="text-align: center;">'''ICM of ''R. sphaeroides'' '''[[Image:R sphaeroides photosystem.png| 500 px | center]]<center>source:Sener et al.2007<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:black;">'''Plate of ''R. sphaeroides'' 2.4.1'''<br />
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[[Image:R sphaeroides aborbance.png| 450px | left]][[Image:R sphaeroides 0986.png| 450px | right]]<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br>source: Walz et al. 1998<br><br />
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====Online Resources for ''R. sphaeroides''====<br />
[http://www.kazusa.or.jp/codon/cgi-bin/showcodon.cgi?species=272943 Codon Usage Table for ''R. sphaeroides'' 2.41]<br />
<br> [http://www.ncbi.nlm.nih.gov/sites/entrez?db=genome&cmd=Retrieve&dopt=Overview&list_uids=18843 ''R. sphaeroides'' 2.41 NCBI Genome Tools]<br />
<br> [http://archaea.ucsc.edu/cgi-bin/hgPcr?wp_target=&db=0&org=Rhodobacter+sphaeroides+2+4+1&wp_f=&wp_r=&wp_size=2000&wp_perfect=15&wp_good=15&wp_showPage=true&hgsid=159191 ''In silico'' PCR of ''R. sphaeroides'' 2.41 genome]<br />
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=='''Regulation'''==<br />
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[[Image:pucregulation-fullpanels.png|950px]]<br />
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'''The Natural System (Left)''' Under high oxygen tension, PpsR prevents transcription from the puc promoter of the pucBA genes, and thus prevents the expression of LH2(1st panel). Under low oxygen tension, this repression is removed by activated AppA, thus allowing expression of LH2 from pucBA(2nd panel).<br><br />
'''Synthetic Regulation System (Right)''' Our system takes advantage of constitutive protein expression from the puc promoter at low oxygen tension, and replaces pucBA with Cph8 at this site. Placing pucBA expression under the control of the ompC promoter allows for Cph8/OmpR control of LH2 expression under varying light conditions. Under high intensity of incident light, Cph8 will not be autophosphorylated, preventing expression of pucBA (3rd panel). When light intensity is lower, Cph8 will autophosphorylate, thus phosphorylating and activating OmpR and promoting expression of LH2 from the ompC promoter.<br><br />
This regulation system was designed such that light is able to penetrate deeper into and through photobioreactors where light is plentiful.<br />
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<br>[https://2009.igem.org/Team:Wash_U/Project Back To Top]<br />
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== '''Results''' ==<br />
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For methods, materials, and procedures regarding these results, please see [https://2009.igem.org/Team:Wash_U/Protocol#Tissue_Flask_Experiment Tissue Flask Experiment] or [https://2009.igem.org/Team:Wash_U/Biological_Parts#Modeling Modeling]<br />
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'''Figure 1'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:#black;">'''a''' Tissue Flask Position 1 Optical Density of Wild Type and DCBomega<br />
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|<font size="2" style="color:#black;">'''b''' Tissue Flask Position 2 Optical Density of Wild Type and DCBomega<br />
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[[Image:Tissue Flask 2b.png| 450 px| left]][[Image:Tissue Flask 1b.png| 450px | right]]<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:#black;">'''d''' Tissue Flask Position 4 Optical Density of Wild Type and DCBomega<br />
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[[Image:Tissue Flask 3b.png| 450px | left]][[Image:Tissue Flask 4b.png| 450px | right]]<br />
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'''e''' Tissue Flask Position 5 Optical Density of Wild Type and DBComega<br />
[[Image:Tissue Flask 5b.png| 450px | center]]<br><br />
Figure 1a,b,c,d,e are the optical density of a tissue flask, as assessed at 600 nm, over the course of the 6 days of the experiment.<br />
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'''Figure 2'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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[[Image:3D 241.png| 450 px | left]][[Image:3D DBCOmega2.png| 450 px | right]]<br />
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Figure 2a and 2b display the growth of the tissue flasks as measured by OD assessed as absorbance at 600 nm. The graph is organized such that those closest to the "days" axis were closest to the light source (i.e. flask 1 is closer than flask 2).<br />
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'''Figure 3'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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[[Image:Bar 241 b.png| 450px | left]][[Image:Bar DBCOmega b.png| 450px | right]]<br />
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Figure 3a and 3b display the cumulative growth of the tissue flasks as measured by the sum of the optical density of the respective cell type's tissue flasks at a given day. Furthermore, the contribution of a given tissue flask to cumulative growth is displayed.<br><br />
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'''Figure 4'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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[[image:sphaeroides tissue flask.jpg|450 px|left|]][[image:dbc tissue flask.jpg|450px|right]]<br />
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Figure 4A and 4B display the appearance of the tissue flasks for both strains on day 6. They are arranged from left to right with left being closed to the light source (flask 1) and right furthest (flask 5)<br />
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'''Figure 5'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:#black;">'''a''' R. sphaeroides Spectrum by Flask Distance from Source <br />
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|<font size="2" style="color:#black;">'''b'''DBComega Spectrum by Flask Distance from Source<br />
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[[Image: WT irradiance 3d.png| 450 px | left]][[Image: DBComega irradiance 3d.png| 450 px | right]]<br />
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Contour of Absolute Irradiance by Flash distance from Source<br />
{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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[[Image: r sphaeroides day 2 a.jpg| 450px | left]][[Image: dbc day 4b.jpg| 450px | right]]<br />
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<div style="text-align: center;"><br />
'''e''' Wild Type Absolute Irradiance Behind Flask 1: Day 6<br />
[[Image: irradiance WT flask 1 day6.png| 450px | center]]<br />
Figure 5 shows the results of the absolute irradiance data taken using the sprectroradiomter. A and B shows absolute irradiance on a given flask at each wavelength. Figure C and D represent the data for the same days as A and B in two dimensions. Figure E is the absolute irradiance behind only flask 1 on day 6. <br><br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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Simulated Optical Density of Mutant and Wild Type Bioreactors<br><br />
Nonlinear least-squares estimation of WT LH2 saturation curve Layers One and Two . Layers Three, Four and Five<br><br />
[[Image: saturation for WT as inferred.png| 450px | left]][[Image: OD after 3,4, and 5.jpg| 215px |right]][[Image: OD after flask 1 and 2.jpg| 215px | center]]<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:#black;">Figure 6 derived from absolute irradiance data from WT tissue flask experiment<br />
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[https://2009.igem.org/Team:Wash_U/Project Back To Top]<br />
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== '''Analysis''' ==<br />
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Though we were not able to have our mutant functionally express our system, the tissue flask experiment was an interesting way to observe the effects of a full knockout of the Light Harvesting Antenna Complex 2 (LH2) vs the Wild Type on an array of consecutive photobioreactors. A couple of conclusions can be drawn from this data that can be used in modeling our synthetic regulation system for LH2 and in the future design of systems for the optimization of Light Harvesting Antenna size for photobioreactors . <br><br><br />
The most striking difference between the Wild Type Rhodobacter Sphaeroides 2.41 and DBCOmega (LH2 knockout) growth patterns is that the first flask in the WT (closest to the light source) grew less than the second flask, while the converse was true in DBCOmega (figure 2a and 2b). This can be attributed to the photosystem saturation curves for the respective cultures. The Wild Type has an LH2 complex, meaning that their antenna size is inherently larger and that their photosystem will be saturated at lower light intensities than the LH2 deficient mutant. We observed that the incident light intensity used in this experiment led to oversaturation for the first WT tissue flask and resulted in growth-inhibiting photodamage, as is evidenced by its lesser growth relative to the second culture. In contrast, photodamage was not observed in DBCOmega as is evidenced by the first tissue flask that grew at the fastest respective rate. Furthermore, it appears that this photodamage slowed the growth of the first WT tissue flask to the point that the OD of this tissue flask was nearly equal to that of DBCOmega after 5 days of growth(.987 vs. .967) (figure 1a). As such, the contribution of LH2 to the growth of the WT in flask 1 can be assessed to be little after day 3 when the growth curve slows- though, this light (at 800 and 850 nm) was still greatly absorbed by flask 1 (Figure 5e). It is likely that much of this light was then wasted through Non-Photochemical Quenching (NPQ), depleting the flasks behind flask 1 of photons at the LH2 absorbtion peaks and decreasing the cumulative productivity of flasks 1-5. This effect of wasting photons though NPQ under high light intensities is one that we sought to minimize in the design of our synthetic regulation for the pucB/A genes. <br><br><br />
<br />
A further a comparison of the WT and DBCOmega experiments reveals that the 3rd tissue flask in each had almost identical growth rates (figure 1c), while the DBCOmega 4th and 5th tissue flask outperformed that of the WT (figure 1d and 1e). This can simply be attributed to the higher density of cells in the 2nd flask of the WT that led to an overall lesser quantity of photons passing through to the 4th and 5th flasks in the WT experiment and likely resulting in heterotrophic growth (though this will need to be confirmed with a "dark control" experiment that should be completed in time for the Jamboree). This decrease in available photons for the WT can be seen by viewing the absolute irradiance data (incident light intensity at a given wavelength) after the third culture for the WT (figure 5a and 5c). This decrease in irradiance is the most pronounced for the LH2 absorption bands at 800 and 850 nm, as was expected. Our synthetic regulation system for pucB/A was designed with the intention of allowing greater penetration of light through the first couple of flasks at the LH2 wavelengths, as expression of pucB/A is inversely correlated to incident light intensity.<br><br><br />
<br />
Overall, the cumulative culture growth of the WT exceeded that of DBCOmega (figure 3) due to the performance of the second tissue flask of the WT relative to that of DBCOmega (figure 1b) despite the photodamage that occurred in tissue flask 1 for the WT. This cumulative culture growth can be considered photosynthetic productivty and is proportional to the amount of energy/desired product that can be obtained from these cultures. <br><br><br />
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It is an interesting exercise to model how our mutant system would perform under these same conditions. To do so, the LH2 saturation curve for the WT (figure 6) and the relative contribution of LH1/LH2 light absorbance to growth was derived from our empirical data and applied to a model that took into account incident light and gave the relative growth in terms of optical density as an output. In this model, our synthetic regulation system of pucB/A allowed our mutant to outperform the wild type species (figure 7). This was due to the performance of the 3rd, 4th, and 5th tissue flask relative to those of the wild type (see figure 7 layers 1,2 vs 1-5). The reason for this is that the wild type LH2 light saturation curve does not vary with changes in incident light intensity as the wild type expresses pucB/A at a uniform level under low oxygen conditions. As a result, the first two layers of Wild Type bioreactors absorb nearly all the available light and starve the bioreactors behind them for photons. <br><br><br />
<br />
Conversely, our mutant was modeled to have a dynamic expression of pucB/A based on the incident light intensity. This light response curve was modeled as an inverse exponential for the Cph8/OmpR ompC Promoter system and the saturation curve changes for each tissue flask in accordance with the incident light intensity- as light intensity decreases, the expression pucB/A increases. This results in more light passing through the first 2 flasks and facilitating greater growth in flasks 3, 4 and 5 as they express large antenna complexes. Nonetheless, we believe that growth across the bioreactors could be further maximized by increasing antenna size in the first 2 bioreactors and sacrificing growth in the 4th and 5th. This could be achieved with a feedback mechanism that sets a minimum level for LH2 expression. <br />
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='''Conclusion'''=<br />
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Though we were not able to conduct the tissue flask growth experiment on our mutant, the results of this experiment on our controls (WT and DBComega) match the assumptions that we had laid out at the beginning of the project, allowed us to draw further conclusions about the efficiency of light-based growth for a series of bioreactors, and to create a model for our mutant based on empirical data. <br> <br />
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*Based on this model, we have concluded that it would be favorable to have a feedback regulation mechanism that sets a minimum expression level for pucB/A to further improve photosynthetic productivity. <br><br />
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*We intend to conduct the tissue flask w DBComega+prkcbc3 before jamboree and compare/interpret these results using our characterization data for the pucB/A and the puc promoter.<br />
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*We will conduct the tissue flask experiment with our functioning mutant in future and compare the results to our model<br />
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*Finally, we would like to characterize the puc promoter under various light conditions and additional oxygen tensions. <br><br />
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'''Overall- our project has demonstrated the potential for a synthetically regulated light harvesting antenna to improve photosynthetic productivity for a series of photobioreactors vs. the wild type.''' <br />
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[https://2009.igem.org/Team:Wash_U/Project Back To Top]<br />
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== '''References''' ==<br />
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# "2007-2008 Catalog & Technical Reference." <u>New England BioLabs Inc.</u> (2007).<br />
# Alon, Uri. <u>Introduction to systems biology and the design principles of biological networks</u>. Boca Raton, FL: Chapman & Hall, 2006.<br />
# Ausubel, Fred et al. <u>Short Protocols in Molecular Biology: Third Edition.</u> Canada: John Wiley and Sons, Inc., 1999.<br />
# "BioBrick Assembly Help." <u>Registry of Standard Biological Parts.</u> 2009. 6 July 2009. <http://partsregistry.org/Main_Page>.<br />
# Bower, James M. <u>Computational Modeling of Genetic and Biochemical Networks (Computational Molecular Biology).</u> New York: M.I.T. PRESS, 2001.<br />
# Braatsch, Stephan et al. "A single Flavoprotein AppA, Integrates Both Redox and Light Signals in ''Rhodobacter sphaeroides.''" <u>Molecular Microbiology.</u> Vol. 45.3 (2002): 827-836.<br />
# Brown, Stanley B. et al. "Bile Pigment Synthesis in Plants: Incorporation of Haem into Phycocyanobilin and Phycocyanobiliproteins in Cyanidium Caldarium." <u>Biochemistry.</u> Vol. 194 (1981): 137-147.<br />
# Dragnea, Vladimira et al. "Time-Resolved Spectroscopic Studies of the AppA Blue-Light Receptor BLUF Domain from ''Rhodobacter sphaeroides'' <u>Biochemistry.</u> Vol. 44 (2005): 15,978-15,985.<br />
# "Entrez Nucleotide Search." <u>National Center for Biotechnology Information.</u> 2009. 6 June 2009 <http://www.ncbi.nlm.nih.gov/nuccore/49175990?from=3533887&to=3534606&report=gbwithparts>.<br />
# Fowler, Gregory J. S. and C. Neil Hunter. "The Synthesis and Assembly of Functional High and Low Light LH2 Antenna Complexes from ''Rhodopseudomonas palustris'' in ''Rhodobacter sphaeroides.''" <u>The Journal of Biological Chemistry.</u> Vol. 271.23 (1996): 13,356-13,361.<br />
# Gabrielsen, Mads et al. "Peripheral Complexes of Purple Bacteria." <u>The Purple Phototropic Bacteria.</u> (2009): 135-153.<br />
# "GenElute HP Plasmid Miniprep Kit: User Guide." <u>Sigma-Aldrich</u>. (2008).<br />
# Harwood, Caroline S. "Nitrogenase-Catalyzed Hydrogen Production by Purple Nonsulfur Photosynthetic Bacteria." <u>Bioenergy.</u> (2008): 259-271.<br />
# Jager, Andreas et al. "The AppA and PpsR Proteins from ''Rhodobacter sphaeroides'' Can Establish a Redox-Dependent Signal Chain but Fail to Transmit Blue-Light Signals in Other Bacteria." <u>Journal of Bacteriology.</u> Vol. 189.6 (2007): 2,274-2,282.<br />
# Jones, M. R. et al. "Mutants of ''Rhodobacter sphaeroides'' Lacking One or More Pigment-Protein Complexes and Complementation with Reaction-Centre, LH1, and LH2 Genes." <u>Molecular Microbiology.</u> Vol. 6.9 (1992): 1,173-1,184.<br />
# Kondo, Toshihiko et al. "Enhancement of Hydrogen Production by a Photosynthetic Bacterium Mutant with Reduced Pigment." <u>Journal of Bioscience and Bioengineering.</u> Vol. 93.2 (2002): 145-150.<br />
# Lagarias, J. Clark. <u>PCB from Spirulina</u>. Personal Communication. June 2009.<br />
# Laible, Philip D. "Foreign Gene Expression in Photosynthetic Bacteria." <u>The Purple Phototropic Bacteria.</u> (2009): 839-860.<br />
# Lee, Jeong K. and Samuel Kaplan. "Transcriptional Regulation of ''puc'' Operon Expression in ''Rhodobacter sphaeroides.''" <u>Journal of Biological Chemistry.</u> Vol. 270.35 (1995): 20,453-20,458.<br />
# Levskaya, Anselmetal et al. "Engineering ''Escherichia coli'' to See Light." <u>Nature.</u> Vol. 438 (2005): 441-442.<br />
# "Life Sciences Catalog 2009-2010." <u>National Diagnostics.</u> (2009).<br />
# Melis, Anastasios. "Solar Energy Conversion Efficiencies in Photosynthesis: Minimizing the Chlorophyll Antennae to Maximize Efficiency." <u>Plant Science.</u> (2009).<br />
# Mizuno, Takeshi and Shoji Mizushima. "Characterization by Deletion and Localized Mutagenesis in Vitro of the Promoter Region of the ''Escherichia coli'' OmpC Gene and Importance of the Upstream DNA Domain in Positive Regulation by the OmpR Protein." <u>Journal of Bacteriology.</u> Vol. 168.1 (1986): 86-95.<br />
# Moskvin, Oleg V. et al. "Transcriptome Analysis of the ''Rhodobacter sphaeroides'' PspR Regulon: PspR as a Master Regulator of Photosystem Development." <u>Journal of Bacteriology.</u> Vol. 187.6 (2005): 2,148-2,156.<br />
# Mussgnug, Jan H. et al. "Engineering photosynthetic light capture: impacts on improved solar energy to biomass conversion." <u> Plant Biotechnology Journal</u> Vol. 5 Issue 6. (2007) pp. 802-814<br />
# "NEBCutter V2.0." <u>New England BioLabs.</u> 2009. 6 June 2009 <http://tools.neb.com/NEBcutter2/index.php>.<br />
# "OligoAnalyzer 3.1." <u>Integrated DNA Technologies.</u> 2009. 6 June 2009. <http://www.idtdna.com/analyzer/Applications/OligoAnalyzer/><br />
# Sambrook, Joseph and David W. Russell. <u>Molecular Cloning: A Laboratory Manual.</u> Vol. 1-3. New York: Cold Springs Harbor Laboratory Press, 2001.<br />
# Scheuring, Simon and Sturgis, James N. "Chromatic Adaptation of Photosynthetic Membranes" <u>Science</u> Vol. 309 (2005) pp.484-487<br />
# Sener, Melih K. et al. "Atomic-level structural and functional model of a bacterial photosynthetic membrane vesicle" <u>Proceedings of the National Academy of Sciences of the United States of America.</u> Vol. 104, Number 40,pp. 15723-15728<br />
# Shimizu-Sato, Sae et al. "A Light-Switchable Gene Promoter System." <u>Nature Publishing Group.</u> Vol. 20 (2002): 1,041-1044.<br />
# Smith, Harry and M. Geoffrey Holmes. <u>Techniquies in Photomorphogenesis.</u> London: Academic Press, Inc., 1984.<br />
# <u>System modeling in cellular biology from concepts to nuts and bolts.</u> Cambridge, MA: MIT P, 2006.<br />
# Terry, Matthew J. "Biosynthesis and Analysis of Bilins." <u>Heme, Chlorophyll, and Bilins: Methods and Protocols.</u> (2002) 273-291. <br />
# Wang, Wanneng et al. "Heterologous Synthesis and Assembly of Functional LHII Antenna Complexes from ''Rhodovulum sulfidophilum'' in ''Rhodobacter sphaeroides'' Mutant." <u>Molecular Biology Reports.</u> Vol. 12.3 (2008).<br />
# Walz, Thomas et al. "Projection Structures of Three Photosynthetic Complexes from Rhodobacter sphaeroides: LH2 at 6 A, LH1 and RC-LH1 at 25 A" <u>Journal of Molecular Biology</u> Vol. 282 pp. 833-845<br />
# Yakovlev, A. G. et al. "Light Control Over the Size of an Antenna Unit Building Block as an Efficient Strategy for Light Harvesting in Photosynthesis." <u>Federation of European Biochemical Societies.</u> (2002):129-132.<br />
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{{WashUbottom}}</div>Brendan1http://2009.igem.org/Team:Wash_U/ProjectTeam:Wash U/Project2009-11-10T00:40:18Z<p>Brendan1: /* Results */</p>
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__NOTOC__<br />
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== '''Introduction''' == <br />
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Our project goal is to maximize the photosynthetic productivity of a series of photobioreactors containing ''Rhodobacter sphaeroides'' under both high and low light intensities by synthetically regulating the size of the light-harvesting antenna complex LH2. We chose to undertake such a project using ''R. sphaeroides'' due to its well-characterized photosynthetic and genetic system. <br />
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The antenna system functions to expand the spectrum of light available for photosynthesis by absorbing different wavelengths than that of the reaction center. Essentially, it is like the dish around the main receiver of any antenna. Marine bacteria, such as ''R. sphaeroides'', evolved very large antenna complexes to absorb light in a natural environment, where there is great competition for photons. As a result, the photosynthetic machinery is saturated at low light intensities in a synthetic non-competitive environment, such as a photobioreactor. This causes up to 95% of incidental photons to be dissipated as heat or fluorescence by bacteria through a process called Non-Photochemical Quenching (NPQ) (Mussgnug et al., 2007). In essence, these photons are being wasted as NPQ reduces light penetration into and across bioreactors and starves the shaded cells for for photons. One method that has been shown to improve photosynthetic productivity in photobioreactors is the reduction of light-harvesting antenna sizes (Polle et al., 2002, Mussgnug et al., 2007). Though, current approaches to this end are difficult to precisely control from the perspective of biological engineering and synthetic biology. <br />
<br />
Our intention is to create a dynamic system that varies antenna size, is dependent on incident light intensity, and can be readily optimized using bioengineering principles. This synthetic regulation of the LH2 complex should result in bacteria grown under high light intensities expressing fewer LH2 complexes than cells that are more shaded, leading to a reduction of wasted incident photons while maintaining a high overall absorbance across subsequent bioreactors. Consequently, we expect to see an increase in the total photosynthetic productivity of our mutant ''Rhodobacter sphaeroides'' when compared to the wild type grown under similar conditions. <br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:black;">'''Wild Type Energy Production in a series of Bioreactors'''<br />
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[[Image:Slide09b.jpg| 450px | left]][[Image:Slide10 a.jpg| 450px | right]]<br />
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[https://2009.igem.org/Team:Wash_U/Project Back To Top]<br />
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== '''Organism''' ==<br />
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''Rhodobacter sphaeroides'' is a purple Alphaproteobacteria. It is metabolically flexible: it can grow heterotrophically, via aerobic and anaerobic respiration, as well as phototrophically under anaerobic conditions with light. <br />
<br> <br><br />
''R. sphaeroides'' is one of the best understood photosynthetic organisms. The photosystem is located in intracytoplasmic membrane invaginations and the main components are the Light Haresting Complex 2 (LH2), Light Harvesting Complex 1 (LH1), and the Reaction Center (RC). These pigment-protein complexes non-covalently bind bacteriochlorophylls and carotenoids. LH1 and RC make up the core complex and are often found in the ratio of 1:1. LH2 is more peripheral and naturally ranges in the ratio to LH1/RC of 3.0:1 to 6.7:1 under varying light conditions (Scheuring et al., 2005). LH2 absorbs photons maximally at the wavelengths of 850 and 800 nm and funnels its energy to LH1 and the reaction center for photochemistry. (Scheuring et al., 2005). <br />
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The two subunits of LH2 are coded for by the pucB/A genes and are naturally promoted by the puc promoter. <br />
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<br><div style="text-align: center;">'''ICM of ''R. sphaeroides'' '''[[Image:R sphaeroides photosystem.png| 500 px | center]]<center>source:Sener et al.2007<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:black;">'''Plate of ''R. sphaeroides'' 2.4.1'''<br />
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[[Image:R sphaeroides aborbance.png| 450px | left]][[Image:R sphaeroides 0986.png| 450px | right]]<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br>source: Walz et al. 1998<br><br />
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====Online Resources for ''R. sphaeroides''====<br />
[http://www.kazusa.or.jp/codon/cgi-bin/showcodon.cgi?species=272943 Codon Usage Table for ''R. sphaeroides'' 2.41]<br />
<br> [http://www.ncbi.nlm.nih.gov/sites/entrez?db=genome&cmd=Retrieve&dopt=Overview&list_uids=18843 ''R. sphaeroides'' 2.41 NCBI Genome Tools]<br />
<br> [http://archaea.ucsc.edu/cgi-bin/hgPcr?wp_target=&db=0&org=Rhodobacter+sphaeroides+2+4+1&wp_f=&wp_r=&wp_size=2000&wp_perfect=15&wp_good=15&wp_showPage=true&hgsid=159191 ''In silico'' PCR of ''R. sphaeroides'' 2.41 genome]<br />
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[https://2009.igem.org/Team:Wash_U/Project Back To Top]<br />
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=='''Regulation'''==<br />
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[[Image:pucregulation-fullpanels.png|950px]]<br />
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'''The Natural System (Left)''' Under high oxygen tension, PpsR prevents transcription from the puc promoter of the pucBA genes, and thus prevents the expression of LH2(1st panel). Under low oxygen tension, this repression is removed by activated AppA, thus allowing expression of LH2 from pucBA(2nd panel).<br><br />
'''Synthetic Regulation System (Right)''' Our system takes advantage of constitutive protein expression from the puc promoter at low oxygen tension, and replaces pucBA with Cph8 at this site. Placing pucBA expression under the control of the ompC promoter allows for Cph8/OmpR control of LH2 expression under varying light conditions. Under high intensity of incident light, Cph8 will not be autophosphorylated, preventing expression of pucBA (3rd panel). When light intensity is lower, Cph8 will autophosphorylate, thus phosphorylating and activating OmpR and promoting expression of LH2 from the ompC promoter.<br><br />
This regulation system was designed such that light is able to penetrate deeper into and through photobioreactors where light is plentiful.<br />
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<br>[https://2009.igem.org/Team:Wash_U/Project Back To Top]<br />
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== '''Results''' ==<br />
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For methods, materials, and procedures regarding these results, please see [https://2009.igem.org/Team:Wash_U/Protocol#Tissue_Flask_Experiment Tissue Flask Experiment] or [https://2009.igem.org/Team:Wash_U/Biological_Parts#Modeling Modeling]<br />
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<div style="text-align: center;"> <br />
'''Figure 1'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:#black;">'''a''' Tissue Flask Position 1 Optical Density of Wild Type and DCBomega<br />
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[[Image:Tissue Flask 2b.png| 450 px| left]][[Image:Tissue Flask 1b.png| 450px | right]]<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:#black;">'''c''' Tissue Flask Position 3 Optical Density of Wild Type and DCBomega<br />
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|<font size="2" style="color:#black;">'''d''' Tissue Flask Position 4 Optical Density of Wild Type and DCBomega<br />
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[[Image:Tissue Flask 3b.png| 450px | left]][[Image:Tissue Flask 4b.png| 450px | right]]<br />
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'''e''' Tissue Flask Position 5 Optical Density of Wild Type and DBComega<br />
[[Image:Tissue Flask 5b.png| 450px | center]]<br><br />
Figure 1a,b,c,d,e are the optical density of a tissue flask, as assessed at 600 nm, over the course of the 6 days of the experiment.<br />
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'''Figure 2'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:#black;">'''a''' The Change in the Optical Density of the Tissue Flasks for the WT<br />
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|<font size="2" style="color:#black;">'''b''' The Change in the Optical Density of the Tissue Flasks for DBComega<br />
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[[Image:3D 241.png| 450 px | left]][[Image:3D DBCOmega2.png| 450 px | right]]<br />
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Figure 2a and 2b display the growth of the tissue flasks as measured by OD assessed as absorbance at 600 nm. The graph is organized such that those closest to the "days" axis were closest to the light source (i.e. flask 1 is closer than flask 2).<br />
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'''Figure 3'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:#black;">'''a''' Cumulative Growth of Wild Type Tissue Flasks<br />
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[[Image:Bar 241 b.png| 450px | left]][[Image:Bar DBCOmega b.png| 450px | right]]<br />
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Figure 3a and 3b display the cumulative growth of the tissue flasks as measured by the sum of the optical density of the respective cell type's tissue flasks at a given day. Furthermore, the contribution of a given tissue flask to cumulative growth is displayed.<br><br />
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'''Figure 4'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:#black;">'''a''' Wild Type Tissue Flasks<br />
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[[image:sphaeroides tissue flask.jpg|450 px|left|]][[image:dbc tissue flask.jpg|450px|right]]<br />
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Figure 4A and 4B display the appearance of the tissue flasks for both strains on day 6. They are arranged from left to right with left being closed to the light source (flask 1) and right furthest (flask 5)<br />
<div style="text-align: center;"> <br />
'''Figure 5'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:#black;">'''a''' R. sphaeroides Spectrum by Flask Distance from Source <br />
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|<font size="2" style="color:#black;">'''b'''DBComega Spectrum by Flask Distance from Source<br />
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[[Image: WT irradiance 3d.png| 450 px | left]][[Image: DBComega irradiance 3d.png| 450 px | right]]<br />
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Contour of Absolute Irradiance by Flash distance from Source<br />
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|<font size="2" style="color:#black;">'''c''' R. Sphaeroides 2.4.1<br />
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|<font size="2" style="color:#black;">'''d''' DBComega<br />
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[[Image: r sphaeroides day 2 a.jpg| 450px | left]][[Image: dbc day 4b.jpg| 450px | right]]<br />
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<div style="text-align: center;"><br />
'''e''' Wild Type Absolute Irradiance Behind Flask 1: Day 6<br />
[[Image: irradiance WT flask 1 day6.png| 450px | center]]<br />
Figure 5 shows the results of the absolute irradiance data taken using the sprectroradiomter. A and B shows absolute irradiance on a given flask at each wavelength. Figure C and D represent the data for the same days as A and B in two dimensions. Figure E is the absolute irradiance behind only flask 1 on day 6. <br><br />
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Simulated Optical Density of Mutant and Wild Type Bioreactors<br><br />
Nonlinear least-squares estimation of WT LH2 saturation curve Layers One and Two . Layers Three, Four and Five<br><br />
[[Image: saturation for WT as inferred.png| 450px | left]][[Image: OD after 3,4, and 5.jpg| 215px |right]][[Image: OD after flask 1 and 2.jpg| 215px | center]]<br />
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|<font size="2" style="color:#black;">Figure 6 derived from absolute irradiance data from WT tissue flask experiment<br />
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|<font size="2" style="color:#black;">Figure 7 shows the results of modeling the WT vs. mutant regulation system<br />
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[https://2009.igem.org/Team:Wash_U/Project Back To Top]<br />
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== '''Analysis''' ==<br />
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<br />
Though we were not able to have our mutant functionally express our system, the tissue flask experiment was an interesting way to observe the effects of a full knockout of the Light Harvesting Antenna Complex 2 (LH2) vs the Wild Type on an array of consecutive photobioreactors. A couple of conclusions can be drawn from this data that can be used in modeling our synthetic regulation system for LH2 and in the future design of systems for the optimization of Light Harvesting Antenna size for photobioreactors . <br><br><br />
The most striking difference between the Wild Type Rhodobacter Sphaeroides 2.41 and DBCOmega (LH2 knockout) growth patterns is that the first flask in the WT (closest to the light source) grew less than the second flask, while the converse was true in DBCOmega (figure 2a and 2b). This can be attributed to the photosystem saturation curves for the respective cultures. The Wild Type has an LH2 complex, meaning that their antenna size is inherently larger and that their photosystem will be saturated at lower light intensities than the LH2 deficient mutant. We observed that the incident light intensity used in this experiment led to oversaturation for the first WT tissue flask and resulted in growth-inhibiting photodamage, as is evidenced by its lesser growth relative to the second culture. In contrast, photodamage was not observed in DBCOmega as is evidenced by the first tissue flask that grew at the fastest respective rate. Furthermore, it appears that this photodamage slowed the growth of the first WT tissue flask to the point that the OD of this tissue flask was nearly equal to that of DBCOmega after 5 days of growth(.987 vs. .967) (figure 1a). As such, the contribution of LH2 to the growth of the WT in flask 1 can be assessed to be little after day 3 when the growth curve slows- though, this light (at 800 and 850 nm) was still greatly absorbed by flask 1 (Figure 5e). It is likely that much of this light was then wasted through Non-Photochemical Quenching (NPQ), depleting the flasks behind flask 1 of photons at the LH2 absorbtion peaks and decreasing the cumulative productivity of flasks 1-5. This effect of wasting photons though NPQ under high light intensities is one that we sought to minimize in the design of our synthetic regulation for the pucB/A genes. <br><br><br />
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A further a comparison of the WT and DBCOmega experiments reveals that the 3rd tissue flask in each had almost identical growth rates (figure 1c), while the DBCOmega 4th and 5th tissue flask outperformed that of the WT (figure 1d and 1e). This can simply be attributed to the higher density of cells in the 2nd flask of the WT that led to an overall lesser quantity of photons passing through to the 4th and 5th flasks in the WT experiment and likely resulting in heterotrophic growth (though this will need to be confirmed with a "dark control" experiment that should be completed in time for the Jamboree). This decrease in available photons for the WT can be seen by viewing the absolute irradiance data (incident light intensity at a given wavelength) after the third culture for the WT (figure 5a and 5c). This decrease in irradiance is the most pronounced for the LH2 absorption bands at 800 and 850 nm, as was expected. Our synthetic regulation system for pucB/A was designed with the intention of allowing greater penetration of light through the first couple of flasks at the LH2 wavelengths, as expression of pucB/A is inversely correlated to incident light intensity.<br><br><br />
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Overall, the cumulative culture growth of the WT exceeded that of DBCOmega (figure 3) due to the performance of the second tissue flask of the WT relative to that of DBCOmega (figure 1b) despite the photodamage that occurred in tissue flask 1 for the WT. This cumulative culture growth can be considered photosynthetic productivty and is proportional to the amount of energy/desired product that can be obtained from these cultures. <br><br><br />
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It is an interesting exercise to model how our mutant system would perform under these same conditions. To do so, the LH2 saturation curve for the WT (figure 6) and the relative contribution of LH1/LH2 light absorbance to growth was derived from our empirical data and applied to a model that took into account incident light and gave the relative growth in terms of optical density as an output. In this model, our synthetic regulation system of pucB/A allowed our mutant to outperform the wild type species (figure 7). This was due to the performance of the 3rd, 4th, and 5th tissue flask relative to those of the wild type (see figure 7 layers 1,2 vs 1-5). The reason for this is that the wild type LH2 light saturation curve does not vary with changes in incident light intensity as the wild type expresses pucB/A at a uniform level under low oxygen conditions. As a result, the first two layers of Wild Type bioreactors absorb nearly all the available light and starve the bioreactors behind them for photons. <br><br><br />
<br />
Conversely, our mutant was modeled to have a dynamic expression of pucB/A based on the incident light intensity. This light response curve was modeled as an inverse exponential for the Cph8/OmpR ompC Promoter system and the saturation curve changes for each tissue flask in accordance with the incident light intensity- as light intensity decreases, the expression pucB/A increases. This results in more light passing through the first 2 flasks and facilitating greater growth in flasks 3, 4 and 5 as they express large antenna complexes. Nonetheless, we believe that growth across the bioreactors could be further maximized by increasing antenna size in the first 2 bioreactors and sacrificing growth in the 4th and 5th. This could be achieved with a feedback mechanism that sets a minimum level for LH2 expression. <br />
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='''Conclusion'''=<br />
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Though we were not able to conduct the tissue flask growth experiment on our mutant, the results of this experiment on our controls (WT and DBComega) match the assumptions that we had laid out at the beginning of the project, allowed us to draw further conclusions about the efficiency of light-based growth for a series of bioreactors, and to create a model for our mutant based on empirical data. <br> <br />
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*Based on this model, we have concluded that it would be favorable to have a feedback regulation mechanism that sets a minimum expression level for pucB/A to further improve photosynthetic productivity. <br><br />
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*We intend to conduct the tissue flask w DBComega+prkcbc3 before jamboree and compare/interpret these results using our characterization data for the pucB/A and the puc promoter.<br />
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*We will conduct the tissue flask experiment with our functioning mutant in future and compare the results to our model<br />
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*Finally, we would like to characterize the puc promoter under various light conditions and additional oxygen tensions. <br><br />
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'''Overall- our project has demonstrated the potential for a synthetically regulated light harvesting antenna to improve photosynthetic productivity for a series of photobioreactors vs. the wild type.''' <br />
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[https://2009.igem.org/Team:Wash_U/Project Back To Top]<br />
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== '''References''' ==<br />
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# "2007-2008 Catalog & Technical Reference." <u>New England BioLabs Inc.</u> (2007).<br />
# Alon, Uri. <u>Introduction to systems biology and the design principles of biological networks</u>. Boca Raton, FL: Chapman & Hall, 2006.<br />
# Ausubel, Fred et al. <u>Short Protocols in Molecular Biology: Third Edition.</u> Canada: John Wiley and Sons, Inc., 1999.<br />
# "BioBrick Assembly Help." <u>Registry of Standard Biological Parts.</u> 2009. 6 July 2009. <http://partsregistry.org/Main_Page>.<br />
# Bower, James M. <u>Computational Modeling of Genetic and Biochemical Networks (Computational Molecular Biology).</u> New York: M.I.T. PRESS, 2001.<br />
# Braatsch, Stephan et al. "A single Flavoprotein AppA, Integrates Both Redox and Light Signals in ''Rhodobacter sphaeroides.''" <u>Molecular Microbiology.</u> Vol. 45.3 (2002): 827-836.<br />
# Brown, Stanley B. et al. "Bile Pigment Synthesis in Plants: Incorporation of Haem into Phycocyanobilin and Phycocyanobiliproteins in Cyanidium Caldarium." <u>Biochemistry.</u> Vol. 194 (1981): 137-147.<br />
# Dragnea, Vladimira et al. "Time-Resolved Spectroscopic Studies of the AppA Blue-Light Receptor BLUF Domain from ''Rhodobacter sphaeroides'' <u>Biochemistry.</u> Vol. 44 (2005): 15,978-15,985.<br />
# "Entrez Nucleotide Search." <u>National Center for Biotechnology Information.</u> 2009. 6 June 2009 <http://www.ncbi.nlm.nih.gov/nuccore/49175990?from=3533887&to=3534606&report=gbwithparts>.<br />
# Fowler, Gregory J. S. and C. Neil Hunter. "The Synthesis and Assembly of Functional High and Low Light LH2 Antenna Complexes from ''Rhodopseudomonas palustris'' in ''Rhodobacter sphaeroides.''" <u>The Journal of Biological Chemistry.</u> Vol. 271.23 (1996): 13,356-13,361.<br />
# Gabrielsen, Mads et al. "Peripheral Complexes of Purple Bacteria." <u>The Purple Phototropic Bacteria.</u> (2009): 135-153.<br />
# "GenElute HP Plasmid Miniprep Kit: User Guide." <u>Sigma-Aldrich</u>. (2008).<br />
# Harwood, Caroline S. "Nitrogenase-Catalyzed Hydrogen Production by Purple Nonsulfur Photosynthetic Bacteria." <u>Bioenergy.</u> (2008): 259-271.<br />
# Jager, Andreas et al. "The AppA and PpsR Proteins from ''Rhodobacter sphaeroides'' Can Establish a Redox-Dependent Signal Chain but Fail to Transmit Blue-Light Signals in Other Bacteria." <u>Journal of Bacteriology.</u> Vol. 189.6 (2007): 2,274-2,282.<br />
# Jones, M. R. et al. "Mutants of ''Rhodobacter sphaeroides'' Lacking One or More Pigment-Protein Complexes and Complementation with Reaction-Centre, LH1, and LH2 Genes." <u>Molecular Microbiology.</u> Vol. 6.9 (1992): 1,173-1,184.<br />
# Kondo, Toshihiko et al. "Enhancement of Hydrogen Production by a Photosynthetic Bacterium Mutant with Reduced Pigment." <u>Journal of Bioscience and Bioengineering.</u> Vol. 93.2 (2002): 145-150.<br />
# Lagarias, J. Clark. <u>PCB from Spirulina</u>. Personal Communication. June 2009.<br />
# Laible, Philip D. "Foreign Gene Expression in Photosynthetic Bacteria." <u>The Purple Phototropic Bacteria.</u> (2009): 839-860.<br />
# Lee, Jeong K. and Samuel Kaplan. "Transcriptional Regulation of ''puc'' Operon Expression in ''Rhodobacter sphaeroides.''" <u>Journal of Biological Chemistry.</u> Vol. 270.35 (1995): 20,453-20,458.<br />
# Levskaya, Anselmetal et al. "Engineering ''Escherichia coli'' to See Light." <u>Nature.</u> Vol. 438 (2005): 441-442.<br />
# "Life Sciences Catalog 2009-2010." <u>National Diagnostics.</u> (2009).<br />
# Melis, Anastasios. "Solar Energy Conversion Efficiencies in Photosynthesis: Minimizing the Chlorophyll Antennae to Maximize Efficiency." <u>Plant Science.</u> (2009).<br />
# Mizuno, Takeshi and Shoji Mizushima. "Characterization by Deletion and Localized Mutagenesis in Vitro of the Promoter Region of the ''Escherichia coli'' OmpC Gene and Importance of the Upstream DNA Domain in Positive Regulation by the OmpR Protein." <u>Journal of Bacteriology.</u> Vol. 168.1 (1986): 86-95.<br />
# Moskvin, Oleg V. et al. "Transcriptome Analysis of the ''Rhodobacter sphaeroides'' PspR Regulon: PspR as a Master Regulator of Photosystem Development." <u>Journal of Bacteriology.</u> Vol. 187.6 (2005): 2,148-2,156.<br />
# Mussgnug, Jan H. et al. "Engineering photosynthetic light capture: impacts on improved solar energy to biomass conversion." <u> Plant Biotechnology Journal</u> Vol. 5 Issue 6. (2007) pp. 802-814<br />
# "NEBCutter V2.0." <u>New England BioLabs.</u> 2009. 6 June 2009 <http://tools.neb.com/NEBcutter2/index.php>.<br />
# "OligoAnalyzer 3.1." <u>Integrated DNA Technologies.</u> 2009. 6 June 2009. <http://www.idtdna.com/analyzer/Applications/OligoAnalyzer/><br />
# Sambrook, Joseph and David W. Russell. <u>Molecular Cloning: A Laboratory Manual.</u> Vol. 1-3. New York: Cold Springs Harbor Laboratory Press, 2001.<br />
# Scheuring, Simon and Sturgis, James N. "Chromatic Adaptation of Photosynthetic Membranes" <u>Science</u> Vol. 309 (2005) pp.484-487<br />
# Sener, Melih K. et al. "Atomic-level structural and functional model of a bacterial photosynthetic membrane vesicle" <u>Proceedings of the National Academy of Sciences of the United States of America.</u> Vol. 104, Number 40,pp. 15723-15728<br />
# Shimizu-Sato, Sae et al. "A Light-Switchable Gene Promoter System." <u>Nature Publishing Group.</u> Vol. 20 (2002): 1,041-1044.<br />
# Smith, Harry and M. Geoffrey Holmes. <u>Techniquies in Photomorphogenesis.</u> London: Academic Press, Inc., 1984.<br />
# <u>System modeling in cellular biology from concepts to nuts and bolts.</u> Cambridge, MA: MIT P, 2006.<br />
# Terry, Matthew J. "Biosynthesis and Analysis of Bilins." <u>Heme, Chlorophyll, and Bilins: Methods and Protocols.</u> (2002) 273-291. <br />
# Wang, Wanneng et al. "Heterologous Synthesis and Assembly of Functional LHII Antenna Complexes from ''Rhodovulum sulfidophilum'' in ''Rhodobacter sphaeroides'' Mutant." <u>Molecular Biology Reports.</u> Vol. 12.3 (2008).<br />
# Walz, Thomas et al. "Projection Structures of Three Photosynthetic Complexes from Rhodobacter sphaeroides: LH2 at 6 A, LH1 and RC-LH1 at 25 A" <u>Journal of Molecular Biology</u> Vol. 282 pp. 833-845<br />
# Yakovlev, A. G. et al. "Light Control Over the Size of an Antenna Unit Building Block as an Efficient Strategy for Light Harvesting in Photosynthesis." <u>Federation of European Biochemical Societies.</u> (2002):129-132.<br />
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{{WashUbottom}}</div>Brendan1http://2009.igem.org/Team:Wash_U/ProjectTeam:Wash U/Project2009-11-10T00:39:34Z<p>Brendan1: /* Results */</p>
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__NOTOC__<br />
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== '''Introduction''' == <br />
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Our project goal is to maximize the photosynthetic productivity of a series of photobioreactors containing ''Rhodobacter sphaeroides'' under both high and low light intensities by synthetically regulating the size of the light-harvesting antenna complex LH2. We chose to undertake such a project using ''R. sphaeroides'' due to its well-characterized photosynthetic and genetic system. <br />
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The antenna system functions to expand the spectrum of light available for photosynthesis by absorbing different wavelengths than that of the reaction center. Essentially, it is like the dish around the main receiver of any antenna. Marine bacteria, such as ''R. sphaeroides'', evolved very large antenna complexes to absorb light in a natural environment, where there is great competition for photons. As a result, the photosynthetic machinery is saturated at low light intensities in a synthetic non-competitive environment, such as a photobioreactor. This causes up to 95% of incidental photons to be dissipated as heat or fluorescence by bacteria through a process called Non-Photochemical Quenching (NPQ) (Mussgnug et al., 2007). In essence, these photons are being wasted as NPQ reduces light penetration into and across bioreactors and starves the shaded cells for for photons. One method that has been shown to improve photosynthetic productivity in photobioreactors is the reduction of light-harvesting antenna sizes (Polle et al., 2002, Mussgnug et al., 2007). Though, current approaches to this end are difficult to precisely control from the perspective of biological engineering and synthetic biology. <br />
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Our intention is to create a dynamic system that varies antenna size, is dependent on incident light intensity, and can be readily optimized using bioengineering principles. This synthetic regulation of the LH2 complex should result in bacteria grown under high light intensities expressing fewer LH2 complexes than cells that are more shaded, leading to a reduction of wasted incident photons while maintaining a high overall absorbance across subsequent bioreactors. Consequently, we expect to see an increase in the total photosynthetic productivity of our mutant ''Rhodobacter sphaeroides'' when compared to the wild type grown under similar conditions. <br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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[[Image:Slide09b.jpg| 450px | left]][[Image:Slide10 a.jpg| 450px | right]]<br />
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[https://2009.igem.org/Team:Wash_U/Project Back To Top]<br />
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== '''Organism''' ==<br />
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''Rhodobacter sphaeroides'' is a purple Alphaproteobacteria. It is metabolically flexible: it can grow heterotrophically, via aerobic and anaerobic respiration, as well as phototrophically under anaerobic conditions with light. <br />
<br> <br><br />
''R. sphaeroides'' is one of the best understood photosynthetic organisms. The photosystem is located in intracytoplasmic membrane invaginations and the main components are the Light Haresting Complex 2 (LH2), Light Harvesting Complex 1 (LH1), and the Reaction Center (RC). These pigment-protein complexes non-covalently bind bacteriochlorophylls and carotenoids. LH1 and RC make up the core complex and are often found in the ratio of 1:1. LH2 is more peripheral and naturally ranges in the ratio to LH1/RC of 3.0:1 to 6.7:1 under varying light conditions (Scheuring et al., 2005). LH2 absorbs photons maximally at the wavelengths of 850 and 800 nm and funnels its energy to LH1 and the reaction center for photochemistry. (Scheuring et al., 2005). <br />
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The two subunits of LH2 are coded for by the pucB/A genes and are naturally promoted by the puc promoter. <br />
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<br><div style="text-align: center;">'''ICM of ''R. sphaeroides'' '''[[Image:R sphaeroides photosystem.png| 500 px | center]]<center>source:Sener et al.2007<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:black;">'''Plate of ''R. sphaeroides'' 2.4.1'''<br />
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[[Image:R sphaeroides aborbance.png| 450px | left]][[Image:R sphaeroides 0986.png| 450px | right]]<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br>source: Walz et al. 1998<br><br />
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====Online Resources for ''R. sphaeroides''====<br />
[http://www.kazusa.or.jp/codon/cgi-bin/showcodon.cgi?species=272943 Codon Usage Table for ''R. sphaeroides'' 2.41]<br />
<br> [http://www.ncbi.nlm.nih.gov/sites/entrez?db=genome&cmd=Retrieve&dopt=Overview&list_uids=18843 ''R. sphaeroides'' 2.41 NCBI Genome Tools]<br />
<br> [http://archaea.ucsc.edu/cgi-bin/hgPcr?wp_target=&db=0&org=Rhodobacter+sphaeroides+2+4+1&wp_f=&wp_r=&wp_size=2000&wp_perfect=15&wp_good=15&wp_showPage=true&hgsid=159191 ''In silico'' PCR of ''R. sphaeroides'' 2.41 genome]<br />
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[https://2009.igem.org/Team:Wash_U/Project Back To Top]<br />
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=='''Regulation'''==<br />
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[[Image:pucregulation-fullpanels.png|950px]]<br />
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'''The Natural System (Left)''' Under high oxygen tension, PpsR prevents transcription from the puc promoter of the pucBA genes, and thus prevents the expression of LH2(1st panel). Under low oxygen tension, this repression is removed by activated AppA, thus allowing expression of LH2 from pucBA(2nd panel).<br><br />
'''Synthetic Regulation System (Right)''' Our system takes advantage of constitutive protein expression from the puc promoter at low oxygen tension, and replaces pucBA with Cph8 at this site. Placing pucBA expression under the control of the ompC promoter allows for Cph8/OmpR control of LH2 expression under varying light conditions. Under high intensity of incident light, Cph8 will not be autophosphorylated, preventing expression of pucBA (3rd panel). When light intensity is lower, Cph8 will autophosphorylate, thus phosphorylating and activating OmpR and promoting expression of LH2 from the ompC promoter.<br><br />
This regulation system was designed such that light is able to penetrate deeper into and through photobioreactors where light is plentiful.<br />
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<br>[https://2009.igem.org/Team:Wash_U/Project Back To Top]<br />
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== '''Results''' ==<br />
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For methods, materials, and procedures regarding these results, please see [https://2009.igem.org/Team:Wash_U/Protocol#Tissue_Flask_Experiment Tissue Flask Experiment] or [https://2009.igem.org/Team:Wash_U/Biological_Parts#Modeling Modeling]<br />
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<div style="text-align: center;"> <br />
'''Figure 1'''<br />
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[[Image:Tissue Flask 2b.png| 450 px| left]][[Image:Tissue Flask 1b.png| 450px | right]]<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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[[Image:Tissue Flask 3b.png| 450px | left]][[Image:Tissue Flask 4b.png| 450px | right]]<br />
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'''e''' Tissue Flask Position 5 Optical Density of Wild Type and DBComega<br />
[[Image:Tissue Flask 5b.png| 450px | center]]<br><br />
Figure 1a,b,c,d,e are the optical density of a tissue flask, as assessed at 600 nm, over the course of the 6 days of the experiment.<br />
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'''Figure 2'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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[[Image:3D 241.png| 450 px | left]][[Image:3D DBCOmega2.png| 450 px | right]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
Figure 2a and 2b display the growth of the tissue flasks as measured by OD assessed as absorbance at 600 nm. The graph is organized such that those closest to the "days" axis were closest to the light source (i.e. flask 1 is closer than flask 2).<br />
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'''Figure 3'''<br />
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|<font size="2" style="color:#black;">'''a''' Cumulative Growth of Wild Type Tissue Flasks<br />
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[[Image:Bar 241 b.png| 450px | left]][[Image:Bar DBCOmega b.png| 450px | right]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
Figure 3a and 3b display the cumulative growth of the tissue flasks as measured by the sum of the optical density of the respective cell type's tissue flasks at a given day. Furthermore, the contribution of a given tissue flask to cumulative growth is displayed.<br><br />
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'''Figure 4'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:#black;">'''a''' Wild Type Tissue Flasks<br />
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[[image:sphaeroides tissue flask.jpg|450 px|left|]][[image:dbc tissue flask.jpg|450px|right]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
Figure 4A and 4B display the appearance of the tissue flasks for both strains on day 6. They are arranged from left to right with left being closed to the light source (flask 1) and right furthest (flask 5)<br />
<div style="text-align: center;"> <br />
'''Figure 5'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:#black;">'''a''' R. sphaeroides Spectrum by Flask Distance from Source <br />
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|<font size="2" style="color:#black;">'''b'''DBComega Spectrum by Flask Distance from Source<br />
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[[Image: WT irradiance 3d.png| 450 px | left]][[Image: DBComega irradiance 3d.png| 450 px | right]]<br />
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Contour of Absolute Irradiance by Flash distance from Source<br />
{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:#black;">'''c''' R. Sphaeroides 2.4.1<br />
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[[Image: r sphaeroides day 2 a.jpg| 450px | left]][[Image: dbc day 4b.jpg| 450px | right]]<br />
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<div style="text-align: center;"><br />
'''e''' Wild Type Absolute Irradiance Behind Flask 1: Day 6<br />
[[Image: irradiance WT flask 1 day6.png| 450px | center]]<br />
Figure 5 shows the results of the absolute irradiance data taken using the sprectroradiomter. A and B shows absolute irradiance on a given flask at each wavelength. Figure C and D represent the data for the same days as A and B in two dimensions. Figure E is the absolute irradiance behind only flask 1 on day 6. <br><br />
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Simulated Optical Density of Mutant and Wild Type Bioreactors<br><br />
Nonlinear least-squares estimation of WT LH2 saturation curve Layers One and Two . Layers Three, Four and Five<br><br />
[[Image: saturation for WT as inferred.png| 450px | left]][[Image: OD after 3,4, and 5.jpg| 215px |right]][[Image: OD after flask 1 and 2.jpg| 215px | center]]<br />
<br><br><br />
Figure 6 derived from absolute irradiance data from WT tissue flask experiment<br><br><br />
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Figure 7 shows the results of modeling the WT vs. mutant regulation system<br />
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[https://2009.igem.org/Team:Wash_U/Project Back To Top]<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:#black;">Figure 6 derived from absolute irradiance data from WT tissue flask experiment<br />
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== '''Analysis''' ==<br />
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<br />
Though we were not able to have our mutant functionally express our system, the tissue flask experiment was an interesting way to observe the effects of a full knockout of the Light Harvesting Antenna Complex 2 (LH2) vs the Wild Type on an array of consecutive photobioreactors. A couple of conclusions can be drawn from this data that can be used in modeling our synthetic regulation system for LH2 and in the future design of systems for the optimization of Light Harvesting Antenna size for photobioreactors . <br><br><br />
The most striking difference between the Wild Type Rhodobacter Sphaeroides 2.41 and DBCOmega (LH2 knockout) growth patterns is that the first flask in the WT (closest to the light source) grew less than the second flask, while the converse was true in DBCOmega (figure 2a and 2b). This can be attributed to the photosystem saturation curves for the respective cultures. The Wild Type has an LH2 complex, meaning that their antenna size is inherently larger and that their photosystem will be saturated at lower light intensities than the LH2 deficient mutant. We observed that the incident light intensity used in this experiment led to oversaturation for the first WT tissue flask and resulted in growth-inhibiting photodamage, as is evidenced by its lesser growth relative to the second culture. In contrast, photodamage was not observed in DBCOmega as is evidenced by the first tissue flask that grew at the fastest respective rate. Furthermore, it appears that this photodamage slowed the growth of the first WT tissue flask to the point that the OD of this tissue flask was nearly equal to that of DBCOmega after 5 days of growth(.987 vs. .967) (figure 1a). As such, the contribution of LH2 to the growth of the WT in flask 1 can be assessed to be little after day 3 when the growth curve slows- though, this light (at 800 and 850 nm) was still greatly absorbed by flask 1 (Figure 5e). It is likely that much of this light was then wasted through Non-Photochemical Quenching (NPQ), depleting the flasks behind flask 1 of photons at the LH2 absorbtion peaks and decreasing the cumulative productivity of flasks 1-5. This effect of wasting photons though NPQ under high light intensities is one that we sought to minimize in the design of our synthetic regulation for the pucB/A genes. <br><br><br />
<br />
A further a comparison of the WT and DBCOmega experiments reveals that the 3rd tissue flask in each had almost identical growth rates (figure 1c), while the DBCOmega 4th and 5th tissue flask outperformed that of the WT (figure 1d and 1e). This can simply be attributed to the higher density of cells in the 2nd flask of the WT that led to an overall lesser quantity of photons passing through to the 4th and 5th flasks in the WT experiment and likely resulting in heterotrophic growth (though this will need to be confirmed with a "dark control" experiment that should be completed in time for the Jamboree). This decrease in available photons for the WT can be seen by viewing the absolute irradiance data (incident light intensity at a given wavelength) after the third culture for the WT (figure 5a and 5c). This decrease in irradiance is the most pronounced for the LH2 absorption bands at 800 and 850 nm, as was expected. Our synthetic regulation system for pucB/A was designed with the intention of allowing greater penetration of light through the first couple of flasks at the LH2 wavelengths, as expression of pucB/A is inversely correlated to incident light intensity.<br><br><br />
<br />
Overall, the cumulative culture growth of the WT exceeded that of DBCOmega (figure 3) due to the performance of the second tissue flask of the WT relative to that of DBCOmega (figure 1b) despite the photodamage that occurred in tissue flask 1 for the WT. This cumulative culture growth can be considered photosynthetic productivty and is proportional to the amount of energy/desired product that can be obtained from these cultures. <br><br><br />
<br />
It is an interesting exercise to model how our mutant system would perform under these same conditions. To do so, the LH2 saturation curve for the WT (figure 6) and the relative contribution of LH1/LH2 light absorbance to growth was derived from our empirical data and applied to a model that took into account incident light and gave the relative growth in terms of optical density as an output. In this model, our synthetic regulation system of pucB/A allowed our mutant to outperform the wild type species (figure 7). This was due to the performance of the 3rd, 4th, and 5th tissue flask relative to those of the wild type (see figure 7 layers 1,2 vs 1-5). The reason for this is that the wild type LH2 light saturation curve does not vary with changes in incident light intensity as the wild type expresses pucB/A at a uniform level under low oxygen conditions. As a result, the first two layers of Wild Type bioreactors absorb nearly all the available light and starve the bioreactors behind them for photons. <br><br><br />
<br />
Conversely, our mutant was modeled to have a dynamic expression of pucB/A based on the incident light intensity. This light response curve was modeled as an inverse exponential for the Cph8/OmpR ompC Promoter system and the saturation curve changes for each tissue flask in accordance with the incident light intensity- as light intensity decreases, the expression pucB/A increases. This results in more light passing through the first 2 flasks and facilitating greater growth in flasks 3, 4 and 5 as they express large antenna complexes. Nonetheless, we believe that growth across the bioreactors could be further maximized by increasing antenna size in the first 2 bioreactors and sacrificing growth in the 4th and 5th. This could be achieved with a feedback mechanism that sets a minimum level for LH2 expression. <br />
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='''Conclusion'''=<br />
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Though we were not able to conduct the tissue flask growth experiment on our mutant, the results of this experiment on our controls (WT and DBComega) match the assumptions that we had laid out at the beginning of the project, allowed us to draw further conclusions about the efficiency of light-based growth for a series of bioreactors, and to create a model for our mutant based on empirical data. <br> <br />
<br />
*Based on this model, we have concluded that it would be favorable to have a feedback regulation mechanism that sets a minimum expression level for pucB/A to further improve photosynthetic productivity. <br><br />
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*We intend to conduct the tissue flask w DBComega+prkcbc3 before jamboree and compare/interpret these results using our characterization data for the pucB/A and the puc promoter.<br />
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*We will conduct the tissue flask experiment with our functioning mutant in future and compare the results to our model<br />
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*Finally, we would like to characterize the puc promoter under various light conditions and additional oxygen tensions. <br><br />
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'''Overall- our project has demonstrated the potential for a synthetically regulated light harvesting antenna to improve photosynthetic productivity for a series of photobioreactors vs. the wild type.''' <br />
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[https://2009.igem.org/Team:Wash_U/Project Back To Top]<br />
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== '''References''' ==<br />
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# "2007-2008 Catalog & Technical Reference." <u>New England BioLabs Inc.</u> (2007).<br />
# Alon, Uri. <u>Introduction to systems biology and the design principles of biological networks</u>. Boca Raton, FL: Chapman & Hall, 2006.<br />
# Ausubel, Fred et al. <u>Short Protocols in Molecular Biology: Third Edition.</u> Canada: John Wiley and Sons, Inc., 1999.<br />
# "BioBrick Assembly Help." <u>Registry of Standard Biological Parts.</u> 2009. 6 July 2009. <http://partsregistry.org/Main_Page>.<br />
# Bower, James M. <u>Computational Modeling of Genetic and Biochemical Networks (Computational Molecular Biology).</u> New York: M.I.T. PRESS, 2001.<br />
# Braatsch, Stephan et al. "A single Flavoprotein AppA, Integrates Both Redox and Light Signals in ''Rhodobacter sphaeroides.''" <u>Molecular Microbiology.</u> Vol. 45.3 (2002): 827-836.<br />
# Brown, Stanley B. et al. "Bile Pigment Synthesis in Plants: Incorporation of Haem into Phycocyanobilin and Phycocyanobiliproteins in Cyanidium Caldarium." <u>Biochemistry.</u> Vol. 194 (1981): 137-147.<br />
# Dragnea, Vladimira et al. "Time-Resolved Spectroscopic Studies of the AppA Blue-Light Receptor BLUF Domain from ''Rhodobacter sphaeroides'' <u>Biochemistry.</u> Vol. 44 (2005): 15,978-15,985.<br />
# "Entrez Nucleotide Search." <u>National Center for Biotechnology Information.</u> 2009. 6 June 2009 <http://www.ncbi.nlm.nih.gov/nuccore/49175990?from=3533887&to=3534606&report=gbwithparts>.<br />
# Fowler, Gregory J. S. and C. Neil Hunter. "The Synthesis and Assembly of Functional High and Low Light LH2 Antenna Complexes from ''Rhodopseudomonas palustris'' in ''Rhodobacter sphaeroides.''" <u>The Journal of Biological Chemistry.</u> Vol. 271.23 (1996): 13,356-13,361.<br />
# Gabrielsen, Mads et al. "Peripheral Complexes of Purple Bacteria." <u>The Purple Phototropic Bacteria.</u> (2009): 135-153.<br />
# "GenElute HP Plasmid Miniprep Kit: User Guide." <u>Sigma-Aldrich</u>. (2008).<br />
# Harwood, Caroline S. "Nitrogenase-Catalyzed Hydrogen Production by Purple Nonsulfur Photosynthetic Bacteria." <u>Bioenergy.</u> (2008): 259-271.<br />
# Jager, Andreas et al. "The AppA and PpsR Proteins from ''Rhodobacter sphaeroides'' Can Establish a Redox-Dependent Signal Chain but Fail to Transmit Blue-Light Signals in Other Bacteria." <u>Journal of Bacteriology.</u> Vol. 189.6 (2007): 2,274-2,282.<br />
# Jones, M. R. et al. "Mutants of ''Rhodobacter sphaeroides'' Lacking One or More Pigment-Protein Complexes and Complementation with Reaction-Centre, LH1, and LH2 Genes." <u>Molecular Microbiology.</u> Vol. 6.9 (1992): 1,173-1,184.<br />
# Kondo, Toshihiko et al. "Enhancement of Hydrogen Production by a Photosynthetic Bacterium Mutant with Reduced Pigment." <u>Journal of Bioscience and Bioengineering.</u> Vol. 93.2 (2002): 145-150.<br />
# Lagarias, J. Clark. <u>PCB from Spirulina</u>. Personal Communication. June 2009.<br />
# Laible, Philip D. "Foreign Gene Expression in Photosynthetic Bacteria." <u>The Purple Phototropic Bacteria.</u> (2009): 839-860.<br />
# Lee, Jeong K. and Samuel Kaplan. "Transcriptional Regulation of ''puc'' Operon Expression in ''Rhodobacter sphaeroides.''" <u>Journal of Biological Chemistry.</u> Vol. 270.35 (1995): 20,453-20,458.<br />
# Levskaya, Anselmetal et al. "Engineering ''Escherichia coli'' to See Light." <u>Nature.</u> Vol. 438 (2005): 441-442.<br />
# "Life Sciences Catalog 2009-2010." <u>National Diagnostics.</u> (2009).<br />
# Melis, Anastasios. "Solar Energy Conversion Efficiencies in Photosynthesis: Minimizing the Chlorophyll Antennae to Maximize Efficiency." <u>Plant Science.</u> (2009).<br />
# Mizuno, Takeshi and Shoji Mizushima. "Characterization by Deletion and Localized Mutagenesis in Vitro of the Promoter Region of the ''Escherichia coli'' OmpC Gene and Importance of the Upstream DNA Domain in Positive Regulation by the OmpR Protein." <u>Journal of Bacteriology.</u> Vol. 168.1 (1986): 86-95.<br />
# Moskvin, Oleg V. et al. "Transcriptome Analysis of the ''Rhodobacter sphaeroides'' PspR Regulon: PspR as a Master Regulator of Photosystem Development." <u>Journal of Bacteriology.</u> Vol. 187.6 (2005): 2,148-2,156.<br />
# Mussgnug, Jan H. et al. "Engineering photosynthetic light capture: impacts on improved solar energy to biomass conversion." <u> Plant Biotechnology Journal</u> Vol. 5 Issue 6. (2007) pp. 802-814<br />
# "NEBCutter V2.0." <u>New England BioLabs.</u> 2009. 6 June 2009 <http://tools.neb.com/NEBcutter2/index.php>.<br />
# "OligoAnalyzer 3.1." <u>Integrated DNA Technologies.</u> 2009. 6 June 2009. <http://www.idtdna.com/analyzer/Applications/OligoAnalyzer/><br />
# Sambrook, Joseph and David W. Russell. <u>Molecular Cloning: A Laboratory Manual.</u> Vol. 1-3. New York: Cold Springs Harbor Laboratory Press, 2001.<br />
# Scheuring, Simon and Sturgis, James N. "Chromatic Adaptation of Photosynthetic Membranes" <u>Science</u> Vol. 309 (2005) pp.484-487<br />
# Sener, Melih K. et al. "Atomic-level structural and functional model of a bacterial photosynthetic membrane vesicle" <u>Proceedings of the National Academy of Sciences of the United States of America.</u> Vol. 104, Number 40,pp. 15723-15728<br />
# Shimizu-Sato, Sae et al. "A Light-Switchable Gene Promoter System." <u>Nature Publishing Group.</u> Vol. 20 (2002): 1,041-1044.<br />
# Smith, Harry and M. Geoffrey Holmes. <u>Techniquies in Photomorphogenesis.</u> London: Academic Press, Inc., 1984.<br />
# <u>System modeling in cellular biology from concepts to nuts and bolts.</u> Cambridge, MA: MIT P, 2006.<br />
# Terry, Matthew J. "Biosynthesis and Analysis of Bilins." <u>Heme, Chlorophyll, and Bilins: Methods and Protocols.</u> (2002) 273-291. <br />
# Wang, Wanneng et al. "Heterologous Synthesis and Assembly of Functional LHII Antenna Complexes from ''Rhodovulum sulfidophilum'' in ''Rhodobacter sphaeroides'' Mutant." <u>Molecular Biology Reports.</u> Vol. 12.3 (2008).<br />
# Walz, Thomas et al. "Projection Structures of Three Photosynthetic Complexes from Rhodobacter sphaeroides: LH2 at 6 A, LH1 and RC-LH1 at 25 A" <u>Journal of Molecular Biology</u> Vol. 282 pp. 833-845<br />
# Yakovlev, A. G. et al. "Light Control Over the Size of an Antenna Unit Building Block as an Efficient Strategy for Light Harvesting in Photosynthesis." <u>Federation of European Biochemical Societies.</u> (2002):129-132.<br />
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{{WashUbottom}}</div>Brendan1http://2009.igem.org/Team:Wash_U/ProjectTeam:Wash U/Project2009-11-10T00:35:24Z<p>Brendan1: /* Results */</p>
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<div>{{WashUback}}<br />
__NOTOC__<br />
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<div style="text-align: left;"> <font size="4" style="color:black"><br />
== '''Introduction''' == <br />
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Our project goal is to maximize the photosynthetic productivity of a series of photobioreactors containing ''Rhodobacter sphaeroides'' under both high and low light intensities by synthetically regulating the size of the light-harvesting antenna complex LH2. We chose to undertake such a project using ''R. sphaeroides'' due to its well-characterized photosynthetic and genetic system. <br />
<br />
The antenna system functions to expand the spectrum of light available for photosynthesis by absorbing different wavelengths than that of the reaction center. Essentially, it is like the dish around the main receiver of any antenna. Marine bacteria, such as ''R. sphaeroides'', evolved very large antenna complexes to absorb light in a natural environment, where there is great competition for photons. As a result, the photosynthetic machinery is saturated at low light intensities in a synthetic non-competitive environment, such as a photobioreactor. This causes up to 95% of incidental photons to be dissipated as heat or fluorescence by bacteria through a process called Non-Photochemical Quenching (NPQ) (Mussgnug et al., 2007). In essence, these photons are being wasted as NPQ reduces light penetration into and across bioreactors and starves the shaded cells for for photons. One method that has been shown to improve photosynthetic productivity in photobioreactors is the reduction of light-harvesting antenna sizes (Polle et al., 2002, Mussgnug et al., 2007). Though, current approaches to this end are difficult to precisely control from the perspective of biological engineering and synthetic biology. <br />
<br />
Our intention is to create a dynamic system that varies antenna size, is dependent on incident light intensity, and can be readily optimized using bioengineering principles. This synthetic regulation of the LH2 complex should result in bacteria grown under high light intensities expressing fewer LH2 complexes than cells that are more shaded, leading to a reduction of wasted incident photons while maintaining a high overall absorbance across subsequent bioreactors. Consequently, we expect to see an increase in the total photosynthetic productivity of our mutant ''Rhodobacter sphaeroides'' when compared to the wild type grown under similar conditions. <br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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[[Image:Slide09b.jpg| 450px | left]][[Image:Slide10 a.jpg| 450px | right]]<br />
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[https://2009.igem.org/Team:Wash_U/Project Back To Top]<br />
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== '''Organism''' ==<br />
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''Rhodobacter sphaeroides'' is a purple Alphaproteobacteria. It is metabolically flexible: it can grow heterotrophically, via aerobic and anaerobic respiration, as well as phototrophically under anaerobic conditions with light. <br />
<br> <br><br />
''R. sphaeroides'' is one of the best understood photosynthetic organisms. The photosystem is located in intracytoplasmic membrane invaginations and the main components are the Light Haresting Complex 2 (LH2), Light Harvesting Complex 1 (LH1), and the Reaction Center (RC). These pigment-protein complexes non-covalently bind bacteriochlorophylls and carotenoids. LH1 and RC make up the core complex and are often found in the ratio of 1:1. LH2 is more peripheral and naturally ranges in the ratio to LH1/RC of 3.0:1 to 6.7:1 under varying light conditions (Scheuring et al., 2005). LH2 absorbs photons maximally at the wavelengths of 850 and 800 nm and funnels its energy to LH1 and the reaction center for photochemistry. (Scheuring et al., 2005). <br />
<br><br><br />
The two subunits of LH2 are coded for by the pucB/A genes and are naturally promoted by the puc promoter. <br />
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<br><div style="text-align: center;">'''ICM of ''R. sphaeroides'' '''[[Image:R sphaeroides photosystem.png| 500 px | center]]<center>source:Sener et al.2007<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:black;">'''Plate of ''R. sphaeroides'' 2.4.1'''<br />
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[[Image:R sphaeroides aborbance.png| 450px | left]][[Image:R sphaeroides 0986.png| 450px | right]]<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br>source: Walz et al. 1998<br><br />
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====Online Resources for ''R. sphaeroides''====<br />
[http://www.kazusa.or.jp/codon/cgi-bin/showcodon.cgi?species=272943 Codon Usage Table for ''R. sphaeroides'' 2.41]<br />
<br> [http://www.ncbi.nlm.nih.gov/sites/entrez?db=genome&cmd=Retrieve&dopt=Overview&list_uids=18843 ''R. sphaeroides'' 2.41 NCBI Genome Tools]<br />
<br> [http://archaea.ucsc.edu/cgi-bin/hgPcr?wp_target=&db=0&org=Rhodobacter+sphaeroides+2+4+1&wp_f=&wp_r=&wp_size=2000&wp_perfect=15&wp_good=15&wp_showPage=true&hgsid=159191 ''In silico'' PCR of ''R. sphaeroides'' 2.41 genome]<br />
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=='''Regulation'''==<br />
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[[Image:pucregulation-fullpanels.png|950px]]<br />
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'''The Natural System (Left)''' Under high oxygen tension, PpsR prevents transcription from the puc promoter of the pucBA genes, and thus prevents the expression of LH2(1st panel). Under low oxygen tension, this repression is removed by activated AppA, thus allowing expression of LH2 from pucBA(2nd panel).<br><br />
'''Synthetic Regulation System (Right)''' Our system takes advantage of constitutive protein expression from the puc promoter at low oxygen tension, and replaces pucBA with Cph8 at this site. Placing pucBA expression under the control of the ompC promoter allows for Cph8/OmpR control of LH2 expression under varying light conditions. Under high intensity of incident light, Cph8 will not be autophosphorylated, preventing expression of pucBA (3rd panel). When light intensity is lower, Cph8 will autophosphorylate, thus phosphorylating and activating OmpR and promoting expression of LH2 from the ompC promoter.<br><br />
This regulation system was designed such that light is able to penetrate deeper into and through photobioreactors where light is plentiful.<br />
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<br>[https://2009.igem.org/Team:Wash_U/Project Back To Top]<br />
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== '''Results''' ==<br />
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For methods, materials, and procedures regarding these results, please see [https://2009.igem.org/Team:Wash_U/Protocol#Tissue_Flask_Experiment Tissue Flask Experiment] or [https://2009.igem.org/Team:Wash_U/Biological_Parts#Modeling Modeling]<br />
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'''Figure 1'''<br />
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|<font size="2" style="color:#black;">'''a''' Tissue Flask Position 1 Optical Density of Wild Type and DCBomega<br />
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|<font size="2" style="color:#black;">'''b''' Tissue Flask Position 2 Optical Density of Wild Type and DCBomega<br />
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[[Image:Tissue Flask 2b.png| 450 px| left]][[Image:Tissue Flask 1b.png| 450px | right]]<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:#black;">'''c''' Tissue Flask Position 3 Optical Density of Wild Type and DCBomega<br />
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|<font size="2" style="color:#black;">'''d''' Tissue Flask Position 4 Optical Density of Wild Type and DCBomega<br />
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[[Image:Tissue Flask 3b.png| 450px | left]][[Image:Tissue Flask 4b.png| 450px | right]]<br />
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'''e''' Tissue Flask Position 5 Optical Density of Wild Type and DBComega<br />
[[Image:Tissue Flask 5b.png| 450px | center]]<br><br />
Figure 1a,b,c,d,e are the optical density of a tissue flask, as assessed at 600 nm, over the course of the 6 days of the experiment.<br />
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'''Figure 2'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:#black;">'''a''' The Change in the Optical Density of the Tissue Flasks for the WT<br />
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|<font size="2" style="color:#black;">'''b''' The Change in the Optical Density of the Tissue Flasks for DBComega<br />
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[[Image:3D 241.png| 450 px | left]][[Image:3D DBCOmega2.png| 450 px | right]]<br />
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Figure 2a and 2b display the growth of the tissue flasks as measured by OD assessed as absorbance at 600 nm. The graph is organized such that those closest to the "days" axis were closest to the light source (i.e. flask 1 is closer than flask 2).<br />
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'''Figure 3'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:#black;">'''a''' Cumulative Growth of Wild Type Tissue Flasks<br />
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|<font size="2" style="color:#black;">'''b''' Cumulative Growth of the DBComega Tissue Flasks<br />
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[[Image:Bar 241 b.png| 450px | left]][[Image:Bar DBCOmega b.png| 450px | right]]<br />
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Figure 3a and 3b display the cumulative growth of the tissue flasks as measured by the sum of the optical density of the respective cell type's tissue flasks at a given day. Furthermore, the contribution of a given tissue flask to cumulative growth is displayed.<br><br />
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'''Figure 4'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:#black;">'''a''' Wild Type Tissue Flasks<br />
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|<font size="2" style="color:#black;">'''b''' The DBComega Tissue Flasks<br />
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[[image:sphaeroides tissue flask.jpg|450 px|left|]][[image:dbc tissue flask.jpg|450px|right]]<br />
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Figure 4A and 4B display the appearance of the tissue flasks for both strains on day 6. They are arranged from left to right with left being closed to the light source (flask 1) and right furthest (flask 5)<br />
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'''Figure 5'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:#black;">'''a''' R. sphaeroides Spectrum by Flask Distance from Source <br />
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|<font size="2" style="color:#black;">'''b'''DBComega Spectrum by Flask Distance from Source<br />
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[[Image: WT irradiance 3d.png| 450 px | left]][[Image: DBComega irradiance 3d.png| 450 px | right]]<br />
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Contour of Absolute Irradiance by Flash distance from Source<br />
{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:#black;">'''c''' R. Sphaeroides 2.4.1<br />
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|<font size="2" style="color:#black;">'''d''' DBComega<br />
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[[Image: r sphaeroides day 2 a.jpg| 450px | left]][[Image: dbc day 4b.jpg| 450px | right]]<br />
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'''e''' Wild Type Absolute Irradiance Behind Flask 1: Day 6<br />
[[Image: irradiance WT flask 1 day6.png| 450px | center]]<br />
Figure 5 shows the results of the absolute irradiance data taken using the sprectroradiomter. A and B shows absolute irradiance on a given flask at each wavelength. Figure C and D represent the data for the same days as A and B in two dimensions. Figure E is the absolute irradiance behind only flask 1 on day 6. <br><br />
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|<font size="2" style="color:#black;">'''Figure 6'''<br />
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|<font size="2" style="color:#black;">'''Figure 7'''<br />
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Simulated Optical Density of Mutant and Wild Type Bioreactors<br><br />
Nonlinear least-squares estimation of WT LH2 saturation curve Layers One and Two . Layers Three, Four and Five<br><br />
[[Image: saturation for WT as inferred.png| 450px | left]][[Image: OD after 3,4, and 5.jpg| 215px |right]][[Image: OD after flask 1 and 2.jpg| 215px | center]]<br />
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Figure 6 was derived from absolute irradiance data taken from tissue flask experiment for WT<br><br><br />
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Figure 7 shows the results of modeling the WT vs. mutant regulation system. <br />
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[https://2009.igem.org/Team:Wash_U/Project Back To Top]<br />
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== '''Analysis''' ==<br />
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Though we were not able to have our mutant functionally express our system, the tissue flask experiment was an interesting way to observe the effects of a full knockout of the Light Harvesting Antenna Complex 2 (LH2) vs the Wild Type on an array of consecutive photobioreactors. A couple of conclusions can be drawn from this data that can be used in modeling our synthetic regulation system for LH2 and in the future design of systems for the optimization of Light Harvesting Antenna size for photobioreactors . <br><br><br />
The most striking difference between the Wild Type Rhodobacter Sphaeroides 2.41 and DBCOmega (LH2 knockout) growth patterns is that the first flask in the WT (closest to the light source) grew less than the second flask, while the converse was true in DBCOmega (figure 2a and 2b). This can be attributed to the photosystem saturation curves for the respective cultures. The Wild Type has an LH2 complex, meaning that their antenna size is inherently larger and that their photosystem will be saturated at lower light intensities than the LH2 deficient mutant. We observed that the incident light intensity used in this experiment led to oversaturation for the first WT tissue flask and resulted in growth-inhibiting photodamage, as is evidenced by its lesser growth relative to the second culture. In contrast, photodamage was not observed in DBCOmega as is evidenced by the first tissue flask that grew at the fastest respective rate. Furthermore, it appears that this photodamage slowed the growth of the first WT tissue flask to the point that the OD of this tissue flask was nearly equal to that of DBCOmega after 5 days of growth(.987 vs. .967) (figure 1a). As such, the contribution of LH2 to the growth of the WT in flask 1 can be assessed to be little after day 3 when the growth curve slows- though, this light (at 800 and 850 nm) was still greatly absorbed by flask 1 (Figure 5e). It is likely that much of this light was then wasted through Non-Photochemical Quenching (NPQ), depleting the flasks behind flask 1 of photons at the LH2 absorbtion peaks and decreasing the cumulative productivity of flasks 1-5. This effect of wasting photons though NPQ under high light intensities is one that we sought to minimize in the design of our synthetic regulation for the pucB/A genes. <br><br><br />
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A further a comparison of the WT and DBCOmega experiments reveals that the 3rd tissue flask in each had almost identical growth rates (figure 1c), while the DBCOmega 4th and 5th tissue flask outperformed that of the WT (figure 1d and 1e). This can simply be attributed to the higher density of cells in the 2nd flask of the WT that led to an overall lesser quantity of photons passing through to the 4th and 5th flasks in the WT experiment and likely resulting in heterotrophic growth (though this will need to be confirmed with a "dark control" experiment that should be completed in time for the Jamboree). This decrease in available photons for the WT can be seen by viewing the absolute irradiance data (incident light intensity at a given wavelength) after the third culture for the WT (figure 5a and 5c). This decrease in irradiance is the most pronounced for the LH2 absorption bands at 800 and 850 nm, as was expected. Our synthetic regulation system for pucB/A was designed with the intention of allowing greater penetration of light through the first couple of flasks at the LH2 wavelengths, as expression of pucB/A is inversely correlated to incident light intensity.<br><br><br />
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Overall, the cumulative culture growth of the WT exceeded that of DBCOmega (figure 3) due to the performance of the second tissue flask of the WT relative to that of DBCOmega (figure 1b) despite the photodamage that occurred in tissue flask 1 for the WT. This cumulative culture growth can be considered photosynthetic productivty and is proportional to the amount of energy/desired product that can be obtained from these cultures. <br><br><br />
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It is an interesting exercise to model how our mutant system would perform under these same conditions. To do so, the LH2 saturation curve for the WT (figure 6) and the relative contribution of LH1/LH2 light absorbance to growth was derived from our empirical data and applied to a model that took into account incident light and gave the relative growth in terms of optical density as an output. In this model, our synthetic regulation system of pucB/A allowed our mutant to outperform the wild type species (figure 7). This was due to the performance of the 3rd, 4th, and 5th tissue flask relative to those of the wild type (see figure 7 layers 1,2 vs 1-5). The reason for this is that the wild type LH2 light saturation curve does not vary with changes in incident light intensity as the wild type expresses pucB/A at a uniform level under low oxygen conditions. As a result, the first two layers of Wild Type bioreactors absorb nearly all the available light and starve the bioreactors behind them for photons. <br><br><br />
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Conversely, our mutant was modeled to have a dynamic expression of pucB/A based on the incident light intensity. This light response curve was modeled as an inverse exponential for the Cph8/OmpR ompC Promoter system and the saturation curve changes for each tissue flask in accordance with the incident light intensity- as light intensity decreases, the expression pucB/A increases. This results in more light passing through the first 2 flasks and facilitating greater growth in flasks 3, 4 and 5 as they express large antenna complexes. Nonetheless, we believe that growth across the bioreactors could be further maximized by increasing antenna size in the first 2 bioreactors and sacrificing growth in the 4th and 5th. This could be achieved with a feedback mechanism that sets a minimum level for LH2 expression. <br />
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='''Conclusion'''=<br />
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Though we were not able to conduct the tissue flask growth experiment on our mutant, the results of this experiment on our controls (WT and DBComega) match the assumptions that we had laid out at the beginning of the project, allowed us to draw further conclusions about the efficiency of light-based growth for a series of bioreactors, and to create a model for our mutant based on empirical data. <br> <br />
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*Based on this model, we have concluded that it would be favorable to have a feedback regulation mechanism that sets a minimum expression level for pucB/A to further improve photosynthetic productivity. <br><br />
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*We intend to conduct the tissue flask w DBComega+prkcbc3 before jamboree and compare/interpret these results using our characterization data for the pucB/A and the puc promoter.<br />
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*We will conduct the tissue flask experiment with our functioning mutant in future and compare the results to our model<br />
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*Finally, we would like to characterize the puc promoter under various light conditions and additional oxygen tensions. <br><br />
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'''Overall- our project has demonstrated the potential for a synthetically regulated light harvesting antenna to improve photosynthetic productivity for a series of photobioreactors vs. the wild type.''' <br />
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[https://2009.igem.org/Team:Wash_U/Project Back To Top]<br />
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== '''References''' ==<br />
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# "2007-2008 Catalog & Technical Reference." <u>New England BioLabs Inc.</u> (2007).<br />
# Alon, Uri. <u>Introduction to systems biology and the design principles of biological networks</u>. Boca Raton, FL: Chapman & Hall, 2006.<br />
# Ausubel, Fred et al. <u>Short Protocols in Molecular Biology: Third Edition.</u> Canada: John Wiley and Sons, Inc., 1999.<br />
# "BioBrick Assembly Help." <u>Registry of Standard Biological Parts.</u> 2009. 6 July 2009. <http://partsregistry.org/Main_Page>.<br />
# Bower, James M. <u>Computational Modeling of Genetic and Biochemical Networks (Computational Molecular Biology).</u> New York: M.I.T. PRESS, 2001.<br />
# Braatsch, Stephan et al. "A single Flavoprotein AppA, Integrates Both Redox and Light Signals in ''Rhodobacter sphaeroides.''" <u>Molecular Microbiology.</u> Vol. 45.3 (2002): 827-836.<br />
# Brown, Stanley B. et al. "Bile Pigment Synthesis in Plants: Incorporation of Haem into Phycocyanobilin and Phycocyanobiliproteins in Cyanidium Caldarium." <u>Biochemistry.</u> Vol. 194 (1981): 137-147.<br />
# Dragnea, Vladimira et al. "Time-Resolved Spectroscopic Studies of the AppA Blue-Light Receptor BLUF Domain from ''Rhodobacter sphaeroides'' <u>Biochemistry.</u> Vol. 44 (2005): 15,978-15,985.<br />
# "Entrez Nucleotide Search." <u>National Center for Biotechnology Information.</u> 2009. 6 June 2009 <http://www.ncbi.nlm.nih.gov/nuccore/49175990?from=3533887&to=3534606&report=gbwithparts>.<br />
# Fowler, Gregory J. S. and C. Neil Hunter. "The Synthesis and Assembly of Functional High and Low Light LH2 Antenna Complexes from ''Rhodopseudomonas palustris'' in ''Rhodobacter sphaeroides.''" <u>The Journal of Biological Chemistry.</u> Vol. 271.23 (1996): 13,356-13,361.<br />
# Gabrielsen, Mads et al. "Peripheral Complexes of Purple Bacteria." <u>The Purple Phototropic Bacteria.</u> (2009): 135-153.<br />
# "GenElute HP Plasmid Miniprep Kit: User Guide." <u>Sigma-Aldrich</u>. (2008).<br />
# Harwood, Caroline S. "Nitrogenase-Catalyzed Hydrogen Production by Purple Nonsulfur Photosynthetic Bacteria." <u>Bioenergy.</u> (2008): 259-271.<br />
# Jager, Andreas et al. "The AppA and PpsR Proteins from ''Rhodobacter sphaeroides'' Can Establish a Redox-Dependent Signal Chain but Fail to Transmit Blue-Light Signals in Other Bacteria." <u>Journal of Bacteriology.</u> Vol. 189.6 (2007): 2,274-2,282.<br />
# Jones, M. R. et al. "Mutants of ''Rhodobacter sphaeroides'' Lacking One or More Pigment-Protein Complexes and Complementation with Reaction-Centre, LH1, and LH2 Genes." <u>Molecular Microbiology.</u> Vol. 6.9 (1992): 1,173-1,184.<br />
# Kondo, Toshihiko et al. "Enhancement of Hydrogen Production by a Photosynthetic Bacterium Mutant with Reduced Pigment." <u>Journal of Bioscience and Bioengineering.</u> Vol. 93.2 (2002): 145-150.<br />
# Lagarias, J. Clark. <u>PCB from Spirulina</u>. Personal Communication. June 2009.<br />
# Laible, Philip D. "Foreign Gene Expression in Photosynthetic Bacteria." <u>The Purple Phototropic Bacteria.</u> (2009): 839-860.<br />
# Lee, Jeong K. and Samuel Kaplan. "Transcriptional Regulation of ''puc'' Operon Expression in ''Rhodobacter sphaeroides.''" <u>Journal of Biological Chemistry.</u> Vol. 270.35 (1995): 20,453-20,458.<br />
# Levskaya, Anselmetal et al. "Engineering ''Escherichia coli'' to See Light." <u>Nature.</u> Vol. 438 (2005): 441-442.<br />
# "Life Sciences Catalog 2009-2010." <u>National Diagnostics.</u> (2009).<br />
# Melis, Anastasios. "Solar Energy Conversion Efficiencies in Photosynthesis: Minimizing the Chlorophyll Antennae to Maximize Efficiency." <u>Plant Science.</u> (2009).<br />
# Mizuno, Takeshi and Shoji Mizushima. "Characterization by Deletion and Localized Mutagenesis in Vitro of the Promoter Region of the ''Escherichia coli'' OmpC Gene and Importance of the Upstream DNA Domain in Positive Regulation by the OmpR Protein." <u>Journal of Bacteriology.</u> Vol. 168.1 (1986): 86-95.<br />
# Moskvin, Oleg V. et al. "Transcriptome Analysis of the ''Rhodobacter sphaeroides'' PspR Regulon: PspR as a Master Regulator of Photosystem Development." <u>Journal of Bacteriology.</u> Vol. 187.6 (2005): 2,148-2,156.<br />
# Mussgnug, Jan H. et al. "Engineering photosynthetic light capture: impacts on improved solar energy to biomass conversion." <u> Plant Biotechnology Journal</u> Vol. 5 Issue 6. (2007) pp. 802-814<br />
# "NEBCutter V2.0." <u>New England BioLabs.</u> 2009. 6 June 2009 <http://tools.neb.com/NEBcutter2/index.php>.<br />
# "OligoAnalyzer 3.1." <u>Integrated DNA Technologies.</u> 2009. 6 June 2009. <http://www.idtdna.com/analyzer/Applications/OligoAnalyzer/><br />
# Sambrook, Joseph and David W. Russell. <u>Molecular Cloning: A Laboratory Manual.</u> Vol. 1-3. New York: Cold Springs Harbor Laboratory Press, 2001.<br />
# Scheuring, Simon and Sturgis, James N. "Chromatic Adaptation of Photosynthetic Membranes" <u>Science</u> Vol. 309 (2005) pp.484-487<br />
# Sener, Melih K. et al. "Atomic-level structural and functional model of a bacterial photosynthetic membrane vesicle" <u>Proceedings of the National Academy of Sciences of the United States of America.</u> Vol. 104, Number 40,pp. 15723-15728<br />
# Shimizu-Sato, Sae et al. "A Light-Switchable Gene Promoter System." <u>Nature Publishing Group.</u> Vol. 20 (2002): 1,041-1044.<br />
# Smith, Harry and M. Geoffrey Holmes. <u>Techniquies in Photomorphogenesis.</u> London: Academic Press, Inc., 1984.<br />
# <u>System modeling in cellular biology from concepts to nuts and bolts.</u> Cambridge, MA: MIT P, 2006.<br />
# Terry, Matthew J. "Biosynthesis and Analysis of Bilins." <u>Heme, Chlorophyll, and Bilins: Methods and Protocols.</u> (2002) 273-291. <br />
# Wang, Wanneng et al. "Heterologous Synthesis and Assembly of Functional LHII Antenna Complexes from ''Rhodovulum sulfidophilum'' in ''Rhodobacter sphaeroides'' Mutant." <u>Molecular Biology Reports.</u> Vol. 12.3 (2008).<br />
# Walz, Thomas et al. "Projection Structures of Three Photosynthetic Complexes from Rhodobacter sphaeroides: LH2 at 6 A, LH1 and RC-LH1 at 25 A" <u>Journal of Molecular Biology</u> Vol. 282 pp. 833-845<br />
# Yakovlev, A. G. et al. "Light Control Over the Size of an Antenna Unit Building Block as an Efficient Strategy for Light Harvesting in Photosynthesis." <u>Federation of European Biochemical Societies.</u> (2002):129-132.<br />
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{{WashUbottom}}</div>Brendan1http://2009.igem.org/Team:Wash_U/ProjectTeam:Wash U/Project2009-11-10T00:32:17Z<p>Brendan1: /* Results */</p>
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__NOTOC__<br />
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== '''Introduction''' == <br />
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Our project goal is to maximize the photosynthetic productivity of a series of photobioreactors containing ''Rhodobacter sphaeroides'' under both high and low light intensities by synthetically regulating the size of the light-harvesting antenna complex LH2. We chose to undertake such a project using ''R. sphaeroides'' due to its well-characterized photosynthetic and genetic system. <br />
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The antenna system functions to expand the spectrum of light available for photosynthesis by absorbing different wavelengths than that of the reaction center. Essentially, it is like the dish around the main receiver of any antenna. Marine bacteria, such as ''R. sphaeroides'', evolved very large antenna complexes to absorb light in a natural environment, where there is great competition for photons. As a result, the photosynthetic machinery is saturated at low light intensities in a synthetic non-competitive environment, such as a photobioreactor. This causes up to 95% of incidental photons to be dissipated as heat or fluorescence by bacteria through a process called Non-Photochemical Quenching (NPQ) (Mussgnug et al., 2007). In essence, these photons are being wasted as NPQ reduces light penetration into and across bioreactors and starves the shaded cells for for photons. One method that has been shown to improve photosynthetic productivity in photobioreactors is the reduction of light-harvesting antenna sizes (Polle et al., 2002, Mussgnug et al., 2007). Though, current approaches to this end are difficult to precisely control from the perspective of biological engineering and synthetic biology. <br />
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Our intention is to create a dynamic system that varies antenna size, is dependent on incident light intensity, and can be readily optimized using bioengineering principles. This synthetic regulation of the LH2 complex should result in bacteria grown under high light intensities expressing fewer LH2 complexes than cells that are more shaded, leading to a reduction of wasted incident photons while maintaining a high overall absorbance across subsequent bioreactors. Consequently, we expect to see an increase in the total photosynthetic productivity of our mutant ''Rhodobacter sphaeroides'' when compared to the wild type grown under similar conditions. <br />
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== '''Organism''' ==<br />
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''Rhodobacter sphaeroides'' is a purple Alphaproteobacteria. It is metabolically flexible: it can grow heterotrophically, via aerobic and anaerobic respiration, as well as phototrophically under anaerobic conditions with light. <br />
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''R. sphaeroides'' is one of the best understood photosynthetic organisms. The photosystem is located in intracytoplasmic membrane invaginations and the main components are the Light Haresting Complex 2 (LH2), Light Harvesting Complex 1 (LH1), and the Reaction Center (RC). These pigment-protein complexes non-covalently bind bacteriochlorophylls and carotenoids. LH1 and RC make up the core complex and are often found in the ratio of 1:1. LH2 is more peripheral and naturally ranges in the ratio to LH1/RC of 3.0:1 to 6.7:1 under varying light conditions (Scheuring et al., 2005). LH2 absorbs photons maximally at the wavelengths of 850 and 800 nm and funnels its energy to LH1 and the reaction center for photochemistry. (Scheuring et al., 2005). <br />
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The two subunits of LH2 are coded for by the pucB/A genes and are naturally promoted by the puc promoter. <br />
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<br><div style="text-align: center;">'''ICM of ''R. sphaeroides'' '''[[Image:R sphaeroides photosystem.png| 500 px | center]]<center>source:Sener et al.2007<br />
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|<font size="2" style="color:black;">'''LH2 Absorption Spectra'''<br />
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|<font size="2" style="color:black;">'''Plate of ''R. sphaeroides'' 2.4.1'''<br />
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[[Image:R sphaeroides aborbance.png| 450px | left]][[Image:R sphaeroides 0986.png| 450px | right]]<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br>source: Walz et al. 1998<br><br />
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====Online Resources for ''R. sphaeroides''====<br />
[http://www.kazusa.or.jp/codon/cgi-bin/showcodon.cgi?species=272943 Codon Usage Table for ''R. sphaeroides'' 2.41]<br />
<br> [http://www.ncbi.nlm.nih.gov/sites/entrez?db=genome&cmd=Retrieve&dopt=Overview&list_uids=18843 ''R. sphaeroides'' 2.41 NCBI Genome Tools]<br />
<br> [http://archaea.ucsc.edu/cgi-bin/hgPcr?wp_target=&db=0&org=Rhodobacter+sphaeroides+2+4+1&wp_f=&wp_r=&wp_size=2000&wp_perfect=15&wp_good=15&wp_showPage=true&hgsid=159191 ''In silico'' PCR of ''R. sphaeroides'' 2.41 genome]<br />
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[https://2009.igem.org/Team:Wash_U/Project Back To Top]<br />
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=='''Regulation'''==<br />
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[[Image:pucregulation-fullpanels.png|950px]]<br />
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'''The Natural System (Left)''' Under high oxygen tension, PpsR prevents transcription from the puc promoter of the pucBA genes, and thus prevents the expression of LH2(1st panel). Under low oxygen tension, this repression is removed by activated AppA, thus allowing expression of LH2 from pucBA(2nd panel).<br><br />
'''Synthetic Regulation System (Right)''' Our system takes advantage of constitutive protein expression from the puc promoter at low oxygen tension, and replaces pucBA with Cph8 at this site. Placing pucBA expression under the control of the ompC promoter allows for Cph8/OmpR control of LH2 expression under varying light conditions. Under high intensity of incident light, Cph8 will not be autophosphorylated, preventing expression of pucBA (3rd panel). When light intensity is lower, Cph8 will autophosphorylate, thus phosphorylating and activating OmpR and promoting expression of LH2 from the ompC promoter.<br><br />
This regulation system was designed such that light is able to penetrate deeper into and through photobioreactors where light is plentiful.<br />
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<br>[https://2009.igem.org/Team:Wash_U/Project Back To Top]<br />
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== '''Results''' ==<br />
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For methods, materials, and procedures regarding these results, please see [https://2009.igem.org/Team:Wash_U/Protocol#Tissue_Flask_Experiment Tissue Flask Experiment] or [https://2009.igem.org/Team:Wash_U/Biological_Parts#Modeling Modeling]<br />
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'''Figure 1'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:#black;">'''a''' Tissue Flask Position 1 Optical Density of Wild Type and DCBomega<br />
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|<font size="2" style="color:#black;">'''b''' Tissue Flask Position 2 Optical Density of Wild Type and DCBomega<br />
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[[Image:Tissue Flask 2b.png| 450 px| left]][[Image:Tissue Flask 1b.png| 450px | right]]<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:#black;">'''c''' Tissue Flask Position 3 Optical Density of Wild Type and DCBomega<br />
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|<font size="2" style="color:#black;">'''d''' Tissue Flask Position 4 Optical Density of Wild Type and DCBomega<br />
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[[Image:Tissue Flask 3b.png| 450px | left]][[Image:Tissue Flask 4b.png| 450px | right]]<br />
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<br><br><br><br><br><br><br><br><br><br><br><br><br><br><div style="text-align: center;"><br />
'''e''' Tissue Flask Position 5 Optical Density of Wild Type and DBComega<br />
[[Image:Tissue Flask 5b.png| 450px | center]]<br><br />
Figure 1a,b,c,d,e are the optical density of a tissue flask, as assessed at 600 nm, over the course of the 6 days of the experiment.<br />
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'''Figure 2'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:#black;">'''a''' The Change in the Optical Density of the Tissue Flasks for the WT<br />
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|<font size="2" style="color:#black;">'''b''' The Change in the Optical Density of the Tissue Flasks for DBComega<br />
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[[Image:3D 241.png| 450 px | left]][[Image:3D DBCOmega2.png| 450 px | right]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
Figure 2a and 2b display the growth of the tissue flasks as measured by OD assessed as absorbance at 600 nm. The graph is organized such that those closest to the "days" axis were closest to the light source (i.e. flask 1 is closer than flask 2).<br />
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'''Figure 3'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:#black;">'''a''' Cumulative Growth of Wild Type Tissue Flasks<br />
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|<font size="2" style="color:#black;">'''b''' Cumulative Growth of the DBComega Tissue Flasks<br />
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[[Image:Bar 241 b.png| 450px | left]][[Image:Bar DBCOmega b.png| 450px | right]]<br />
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Figure 3a and 3b display the cumulative growth of the tissue flasks as measured by the sum of the optical density of the respective cell type's tissue flasks at a given day. Furthermore, the contribution of a given tissue flask to cumulative growth is displayed.<br><br />
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'''Figure 4'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:#black;">'''a''' Wild Type Tissue Flasks<br />
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|<font size="2" style="color:#black;">'''b''' The DBComega Tissue Flasks<br />
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[[image:sphaeroides tissue flask.jpg|450 px|left|]][[image:dbc tissue flask.jpg|450px|right]]<br />
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Figure 4A and 4B display the appearance of the tissue flasks for both strains on day 6. They are arranged from left to right with left being closed to the light source (flask 1) and right furthest (flask 5)<br />
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'''Figure 5'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:#black;">'''a''' R. sphaeroides Spectrum by Flask Distance from Source <br />
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|<font size="2" style="color:#black;">'''b'''DBComega Spectrum by Flask Distance from Source<br />
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[[Image: WT irradiance 3d.png| 450 px | left]][[Image: DBComega irradiance 3d.png| 450 px | right]]<br />
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Contour of Absolute Irradiance by Flash distance from Source<br />
{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:#black;">'''c''' R. Sphaeroides 2.4.1<br />
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|<font size="2" style="color:#black;">'''d''' DBComega<br />
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[[Image: r sphaeroides day 2 a.jpg| 450px | left]][[Image: dbc day 4b.jpg| 450px | right]]<br />
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<div style="text-align: center;"><br />
'''e''' Wild Type Absolute Irradiance Behind Flask 1: Day 6<br />
[[Image: irradiance WT flask 1 day6.png| 450px | center]]<br />
Figure 5 shows the results of the absolute irradiance data taken using the sprectroradiomter. A and B shows absolute irradiance on a given flask at each wavelength. Figure C and D represent the data for the same days as A and B in two dimensions. Figure E is the absolute irradiance behind only flask 1 on day 6. <br><br />
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|<font size="2" style="color:#black;">'''Figure 6'''<br />
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|<font size="2" style="color:#black;">'''Figure 7'''<br />
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Simulated Optical Density of Mutant and Wild Type Bioreactors<br><br />
Nonlinear least-squares estimation of WT LH2 saturation curve Layers One and Two . Layers Three, Four and Five<br><br />
[[Image: saturation for WT as inferred.png| 450px | left]][[Image: OD after 3,4, and 5.jpg| 215px |right]][[Image: OD after flask 1 and 2.jpg| 215px | center]]<br />
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Figure 6 was derived from the absolute irradiance data taken from the tissue flask experiment for the Wild Type.<br><br><br />
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Figure 7 shows the results of modelling the wild type vs. our mutant regulation system. <br />
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[https://2009.igem.org/Team:Wash_U/Project Back To Top]<br />
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== '''Analysis''' ==<br />
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Though we were not able to have our mutant functionally express our system, the tissue flask experiment was an interesting way to observe the effects of a full knockout of the Light Harvesting Antenna Complex 2 (LH2) vs the Wild Type on an array of consecutive photobioreactors. A couple of conclusions can be drawn from this data that can be used in modeling our synthetic regulation system for LH2 and in the future design of systems for the optimization of Light Harvesting Antenna size for photobioreactors . <br><br><br />
The most striking difference between the Wild Type Rhodobacter Sphaeroides 2.41 and DBCOmega (LH2 knockout) growth patterns is that the first flask in the WT (closest to the light source) grew less than the second flask, while the converse was true in DBCOmega (figure 2a and 2b). This can be attributed to the photosystem saturation curves for the respective cultures. The Wild Type has an LH2 complex, meaning that their antenna size is inherently larger and that their photosystem will be saturated at lower light intensities than the LH2 deficient mutant. We observed that the incident light intensity used in this experiment led to oversaturation for the first WT tissue flask and resulted in growth-inhibiting photodamage, as is evidenced by its lesser growth relative to the second culture. In contrast, photodamage was not observed in DBCOmega as is evidenced by the first tissue flask that grew at the fastest respective rate. Furthermore, it appears that this photodamage slowed the growth of the first WT tissue flask to the point that the OD of this tissue flask was nearly equal to that of DBCOmega after 5 days of growth(.987 vs. .967) (figure 1a). As such, the contribution of LH2 to the growth of the WT in flask 1 can be assessed to be little after day 3 when the growth curve slows- though, this light (at 800 and 850 nm) was still greatly absorbed by flask 1 (Figure 5e). It is likely that much of this light was then wasted through Non-Photochemical Quenching (NPQ), depleting the flasks behind flask 1 of photons at the LH2 absorbtion peaks and decreasing the cumulative productivity of flasks 1-5. This effect of wasting photons though NPQ under high light intensities is one that we sought to minimize in the design of our synthetic regulation for the pucB/A genes. <br><br><br />
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A further a comparison of the WT and DBCOmega experiments reveals that the 3rd tissue flask in each had almost identical growth rates (figure 1c), while the DBCOmega 4th and 5th tissue flask outperformed that of the WT (figure 1d and 1e). This can simply be attributed to the higher density of cells in the 2nd flask of the WT that led to an overall lesser quantity of photons passing through to the 4th and 5th flasks in the WT experiment and likely resulting in heterotrophic growth (though this will need to be confirmed with a "dark control" experiment that should be completed in time for the Jamboree). This decrease in available photons for the WT can be seen by viewing the absolute irradiance data (incident light intensity at a given wavelength) after the third culture for the WT (figure 5a and 5c). This decrease in irradiance is the most pronounced for the LH2 absorption bands at 800 and 850 nm, as was expected. Our synthetic regulation system for pucB/A was designed with the intention of allowing greater penetration of light through the first couple of flasks at the LH2 wavelengths, as expression of pucB/A is inversely correlated to incident light intensity.<br><br><br />
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Overall, the cumulative culture growth of the WT exceeded that of DBCOmega (figure 3) due to the performance of the second tissue flask of the WT relative to that of DBCOmega (figure 1b) despite the photodamage that occurred in tissue flask 1 for the WT. This cumulative culture growth can be considered photosynthetic productivty and is proportional to the amount of energy/desired product that can be obtained from these cultures. <br><br><br />
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It is an interesting exercise to model how our mutant system would perform under these same conditions. To do so, the LH2 saturation curve for the WT (figure 6) and the relative contribution of LH1/LH2 light absorbance to growth was derived from our empirical data and applied to a model that took into account incident light and gave the relative growth in terms of optical density as an output. In this model, our synthetic regulation system of pucB/A allowed our mutant to outperform the wild type species (figure 7). This was due to the performance of the 3rd, 4th, and 5th tissue flask relative to those of the wild type (see figure 7 layers 1,2 vs 1-5). The reason for this is that the wild type LH2 light saturation curve does not vary with changes in incident light intensity as the wild type expresses pucB/A at a uniform level under low oxygen conditions. As a result, the first two layers of Wild Type bioreactors absorb nearly all the available light and starve the bioreactors behind them for photons. <br><br><br />
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Conversely, our mutant was modeled to have a dynamic expression of pucB/A based on the incident light intensity. This light response curve was modeled as an inverse exponential for the Cph8/OmpR ompC Promoter system and the saturation curve changes for each tissue flask in accordance with the incident light intensity- as light intensity decreases, the expression pucB/A increases. This results in more light passing through the first 2 flasks and facilitating greater growth in flasks 3, 4 and 5 as they express large antenna complexes. Nonetheless, we believe that growth across the bioreactors could be further maximized by increasing antenna size in the first 2 bioreactors and sacrificing growth in the 4th and 5th. This could be achieved with a feedback mechanism that sets a minimum level for LH2 expression. <br />
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='''Conclusion'''=<br />
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Though we were not able to conduct the tissue flask growth experiment on our mutant, the results of this experiment on our controls (WT and DBComega) match the assumptions that we had laid out at the beginning of the project, allowed us to draw further conclusions about the efficiency of light-based growth for a series of bioreactors, and to create a model for our mutant based on empirical data. <br> <br />
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*Based on this model, we have concluded that it would be favorable to have a feedback regulation mechanism that sets a minimum expression level for pucB/A to further improve photosynthetic productivity. <br><br />
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*We intend to conduct the tissue flask w DBComega+prkcbc3 before jamboree and compare/interpret these results using our characterization data for the pucB/A and the puc promoter.<br />
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*We will conduct the tissue flask experiment with our functioning mutant in future and compare the results to our model<br />
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*Finally, we would like to characterize the puc promoter under various light conditions and additional oxygen tensions. <br><br />
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'''Overall- our project has demonstrated the potential for a synthetically regulated light harvesting antenna to improve photosynthetic productivity for a series of photobioreactors vs. the wild type.''' <br />
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[https://2009.igem.org/Team:Wash_U/Project Back To Top]<br />
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== '''References''' ==<br />
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# "2007-2008 Catalog & Technical Reference." <u>New England BioLabs Inc.</u> (2007).<br />
# Alon, Uri. <u>Introduction to systems biology and the design principles of biological networks</u>. Boca Raton, FL: Chapman & Hall, 2006.<br />
# Ausubel, Fred et al. <u>Short Protocols in Molecular Biology: Third Edition.</u> Canada: John Wiley and Sons, Inc., 1999.<br />
# "BioBrick Assembly Help." <u>Registry of Standard Biological Parts.</u> 2009. 6 July 2009. <http://partsregistry.org/Main_Page>.<br />
# Bower, James M. <u>Computational Modeling of Genetic and Biochemical Networks (Computational Molecular Biology).</u> New York: M.I.T. PRESS, 2001.<br />
# Braatsch, Stephan et al. "A single Flavoprotein AppA, Integrates Both Redox and Light Signals in ''Rhodobacter sphaeroides.''" <u>Molecular Microbiology.</u> Vol. 45.3 (2002): 827-836.<br />
# Brown, Stanley B. et al. "Bile Pigment Synthesis in Plants: Incorporation of Haem into Phycocyanobilin and Phycocyanobiliproteins in Cyanidium Caldarium." <u>Biochemistry.</u> Vol. 194 (1981): 137-147.<br />
# Dragnea, Vladimira et al. "Time-Resolved Spectroscopic Studies of the AppA Blue-Light Receptor BLUF Domain from ''Rhodobacter sphaeroides'' <u>Biochemistry.</u> Vol. 44 (2005): 15,978-15,985.<br />
# "Entrez Nucleotide Search." <u>National Center for Biotechnology Information.</u> 2009. 6 June 2009 <http://www.ncbi.nlm.nih.gov/nuccore/49175990?from=3533887&to=3534606&report=gbwithparts>.<br />
# Fowler, Gregory J. S. and C. Neil Hunter. "The Synthesis and Assembly of Functional High and Low Light LH2 Antenna Complexes from ''Rhodopseudomonas palustris'' in ''Rhodobacter sphaeroides.''" <u>The Journal of Biological Chemistry.</u> Vol. 271.23 (1996): 13,356-13,361.<br />
# Gabrielsen, Mads et al. "Peripheral Complexes of Purple Bacteria." <u>The Purple Phototropic Bacteria.</u> (2009): 135-153.<br />
# "GenElute HP Plasmid Miniprep Kit: User Guide." <u>Sigma-Aldrich</u>. (2008).<br />
# Harwood, Caroline S. "Nitrogenase-Catalyzed Hydrogen Production by Purple Nonsulfur Photosynthetic Bacteria." <u>Bioenergy.</u> (2008): 259-271.<br />
# Jager, Andreas et al. "The AppA and PpsR Proteins from ''Rhodobacter sphaeroides'' Can Establish a Redox-Dependent Signal Chain but Fail to Transmit Blue-Light Signals in Other Bacteria." <u>Journal of Bacteriology.</u> Vol. 189.6 (2007): 2,274-2,282.<br />
# Jones, M. R. et al. "Mutants of ''Rhodobacter sphaeroides'' Lacking One or More Pigment-Protein Complexes and Complementation with Reaction-Centre, LH1, and LH2 Genes." <u>Molecular Microbiology.</u> Vol. 6.9 (1992): 1,173-1,184.<br />
# Kondo, Toshihiko et al. "Enhancement of Hydrogen Production by a Photosynthetic Bacterium Mutant with Reduced Pigment." <u>Journal of Bioscience and Bioengineering.</u> Vol. 93.2 (2002): 145-150.<br />
# Lagarias, J. Clark. <u>PCB from Spirulina</u>. Personal Communication. June 2009.<br />
# Laible, Philip D. "Foreign Gene Expression in Photosynthetic Bacteria." <u>The Purple Phototropic Bacteria.</u> (2009): 839-860.<br />
# Lee, Jeong K. and Samuel Kaplan. "Transcriptional Regulation of ''puc'' Operon Expression in ''Rhodobacter sphaeroides.''" <u>Journal of Biological Chemistry.</u> Vol. 270.35 (1995): 20,453-20,458.<br />
# Levskaya, Anselmetal et al. "Engineering ''Escherichia coli'' to See Light." <u>Nature.</u> Vol. 438 (2005): 441-442.<br />
# "Life Sciences Catalog 2009-2010." <u>National Diagnostics.</u> (2009).<br />
# Melis, Anastasios. "Solar Energy Conversion Efficiencies in Photosynthesis: Minimizing the Chlorophyll Antennae to Maximize Efficiency." <u>Plant Science.</u> (2009).<br />
# Mizuno, Takeshi and Shoji Mizushima. "Characterization by Deletion and Localized Mutagenesis in Vitro of the Promoter Region of the ''Escherichia coli'' OmpC Gene and Importance of the Upstream DNA Domain in Positive Regulation by the OmpR Protein." <u>Journal of Bacteriology.</u> Vol. 168.1 (1986): 86-95.<br />
# Moskvin, Oleg V. et al. "Transcriptome Analysis of the ''Rhodobacter sphaeroides'' PspR Regulon: PspR as a Master Regulator of Photosystem Development." <u>Journal of Bacteriology.</u> Vol. 187.6 (2005): 2,148-2,156.<br />
# Mussgnug, Jan H. et al. "Engineering photosynthetic light capture: impacts on improved solar energy to biomass conversion." <u> Plant Biotechnology Journal</u> Vol. 5 Issue 6. (2007) pp. 802-814<br />
# "NEBCutter V2.0." <u>New England BioLabs.</u> 2009. 6 June 2009 <http://tools.neb.com/NEBcutter2/index.php>.<br />
# "OligoAnalyzer 3.1." <u>Integrated DNA Technologies.</u> 2009. 6 June 2009. <http://www.idtdna.com/analyzer/Applications/OligoAnalyzer/><br />
# Sambrook, Joseph and David W. Russell. <u>Molecular Cloning: A Laboratory Manual.</u> Vol. 1-3. New York: Cold Springs Harbor Laboratory Press, 2001.<br />
# Scheuring, Simon and Sturgis, James N. "Chromatic Adaptation of Photosynthetic Membranes" <u>Science</u> Vol. 309 (2005) pp.484-487<br />
# Sener, Melih K. et al. "Atomic-level structural and functional model of a bacterial photosynthetic membrane vesicle" <u>Proceedings of the National Academy of Sciences of the United States of America.</u> Vol. 104, Number 40,pp. 15723-15728<br />
# Shimizu-Sato, Sae et al. "A Light-Switchable Gene Promoter System." <u>Nature Publishing Group.</u> Vol. 20 (2002): 1,041-1044.<br />
# Smith, Harry and M. Geoffrey Holmes. <u>Techniquies in Photomorphogenesis.</u> London: Academic Press, Inc., 1984.<br />
# <u>System modeling in cellular biology from concepts to nuts and bolts.</u> Cambridge, MA: MIT P, 2006.<br />
# Terry, Matthew J. "Biosynthesis and Analysis of Bilins." <u>Heme, Chlorophyll, and Bilins: Methods and Protocols.</u> (2002) 273-291. <br />
# Wang, Wanneng et al. "Heterologous Synthesis and Assembly of Functional LHII Antenna Complexes from ''Rhodovulum sulfidophilum'' in ''Rhodobacter sphaeroides'' Mutant." <u>Molecular Biology Reports.</u> Vol. 12.3 (2008).<br />
# Walz, Thomas et al. "Projection Structures of Three Photosynthetic Complexes from Rhodobacter sphaeroides: LH2 at 6 A, LH1 and RC-LH1 at 25 A" <u>Journal of Molecular Biology</u> Vol. 282 pp. 833-845<br />
# Yakovlev, A. G. et al. "Light Control Over the Size of an Antenna Unit Building Block as an Efficient Strategy for Light Harvesting in Photosynthesis." <u>Federation of European Biochemical Societies.</u> (2002):129-132.<br />
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{{WashUbottom}}</div>Brendan1http://2009.igem.org/Team:Wash_U/ProjectTeam:Wash U/Project2009-11-10T00:29:22Z<p>Brendan1: /* Results */</p>
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__NOTOC__<br />
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== '''Introduction''' == <br />
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Our project goal is to maximize the photosynthetic productivity of a series of photobioreactors containing ''Rhodobacter sphaeroides'' under both high and low light intensities by synthetically regulating the size of the light-harvesting antenna complex LH2. We chose to undertake such a project using ''R. sphaeroides'' due to its well-characterized photosynthetic and genetic system. <br />
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The antenna system functions to expand the spectrum of light available for photosynthesis by absorbing different wavelengths than that of the reaction center. Essentially, it is like the dish around the main receiver of any antenna. Marine bacteria, such as ''R. sphaeroides'', evolved very large antenna complexes to absorb light in a natural environment, where there is great competition for photons. As a result, the photosynthetic machinery is saturated at low light intensities in a synthetic non-competitive environment, such as a photobioreactor. This causes up to 95% of incidental photons to be dissipated as heat or fluorescence by bacteria through a process called Non-Photochemical Quenching (NPQ) (Mussgnug et al., 2007). In essence, these photons are being wasted as NPQ reduces light penetration into and across bioreactors and starves the shaded cells for for photons. One method that has been shown to improve photosynthetic productivity in photobioreactors is the reduction of light-harvesting antenna sizes (Polle et al., 2002, Mussgnug et al., 2007). Though, current approaches to this end are difficult to precisely control from the perspective of biological engineering and synthetic biology. <br />
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Our intention is to create a dynamic system that varies antenna size, is dependent on incident light intensity, and can be readily optimized using bioengineering principles. This synthetic regulation of the LH2 complex should result in bacteria grown under high light intensities expressing fewer LH2 complexes than cells that are more shaded, leading to a reduction of wasted incident photons while maintaining a high overall absorbance across subsequent bioreactors. Consequently, we expect to see an increase in the total photosynthetic productivity of our mutant ''Rhodobacter sphaeroides'' when compared to the wild type grown under similar conditions. <br />
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|<font size="2" style="color:black;">'''Wild Type Energy Production in a series of Bioreactors'''<br />
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|<font size="2" style="color:black;">'''Synthetically Regulated Antenna Energy Production in Bioreactors'''<br />
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[[Image:Slide09b.jpg| 450px | left]][[Image:Slide10 a.jpg| 450px | right]]<br />
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[https://2009.igem.org/Team:Wash_U/Project Back To Top]<br />
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== '''Organism''' ==<br />
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''Rhodobacter sphaeroides'' is a purple Alphaproteobacteria. It is metabolically flexible: it can grow heterotrophically, via aerobic and anaerobic respiration, as well as phototrophically under anaerobic conditions with light. <br />
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''R. sphaeroides'' is one of the best understood photosynthetic organisms. The photosystem is located in intracytoplasmic membrane invaginations and the main components are the Light Haresting Complex 2 (LH2), Light Harvesting Complex 1 (LH1), and the Reaction Center (RC). These pigment-protein complexes non-covalently bind bacteriochlorophylls and carotenoids. LH1 and RC make up the core complex and are often found in the ratio of 1:1. LH2 is more peripheral and naturally ranges in the ratio to LH1/RC of 3.0:1 to 6.7:1 under varying light conditions (Scheuring et al., 2005). LH2 absorbs photons maximally at the wavelengths of 850 and 800 nm and funnels its energy to LH1 and the reaction center for photochemistry. (Scheuring et al., 2005). <br />
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The two subunits of LH2 are coded for by the pucB/A genes and are naturally promoted by the puc promoter. <br />
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<br><div style="text-align: center;">'''ICM of ''R. sphaeroides'' '''[[Image:R sphaeroides photosystem.png| 500 px | center]]<center>source:Sener et al.2007<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:black;">'''Plate of ''R. sphaeroides'' 2.4.1'''<br />
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[[Image:R sphaeroides aborbance.png| 450px | left]][[Image:R sphaeroides 0986.png| 450px | right]]<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br>source: Walz et al. 1998<br><br />
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====Online Resources for ''R. sphaeroides''====<br />
[http://www.kazusa.or.jp/codon/cgi-bin/showcodon.cgi?species=272943 Codon Usage Table for ''R. sphaeroides'' 2.41]<br />
<br> [http://www.ncbi.nlm.nih.gov/sites/entrez?db=genome&cmd=Retrieve&dopt=Overview&list_uids=18843 ''R. sphaeroides'' 2.41 NCBI Genome Tools]<br />
<br> [http://archaea.ucsc.edu/cgi-bin/hgPcr?wp_target=&db=0&org=Rhodobacter+sphaeroides+2+4+1&wp_f=&wp_r=&wp_size=2000&wp_perfect=15&wp_good=15&wp_showPage=true&hgsid=159191 ''In silico'' PCR of ''R. sphaeroides'' 2.41 genome]<br />
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[https://2009.igem.org/Team:Wash_U/Project Back To Top]<br />
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=='''Regulation'''==<br />
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[[Image:pucregulation-fullpanels.png|950px]]<br />
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'''The Natural System (Left)''' Under high oxygen tension, PpsR prevents transcription from the puc promoter of the pucBA genes, and thus prevents the expression of LH2(1st panel). Under low oxygen tension, this repression is removed by activated AppA, thus allowing expression of LH2 from pucBA(2nd panel).<br><br />
'''Synthetic Regulation System (Right)''' Our system takes advantage of constitutive protein expression from the puc promoter at low oxygen tension, and replaces pucBA with Cph8 at this site. Placing pucBA expression under the control of the ompC promoter allows for Cph8/OmpR control of LH2 expression under varying light conditions. Under high intensity of incident light, Cph8 will not be autophosphorylated, preventing expression of pucBA (3rd panel). When light intensity is lower, Cph8 will autophosphorylate, thus phosphorylating and activating OmpR and promoting expression of LH2 from the ompC promoter.<br><br />
This regulation system was designed such that light is able to penetrate deeper into and through photobioreactors where light is plentiful.<br />
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<br>[https://2009.igem.org/Team:Wash_U/Project Back To Top]<br />
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== '''Results''' ==<br />
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For methods, materials, and procedures regarding these results, please see [https://2009.igem.org/Team:Wash_U/Protocol#Tissue_Flask_Experiment Tissue Flask Experiment] or [https://2009.igem.org/Team:Wash_U/Biological_Parts#Modeling Modeling]<br />
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'''Figure 1'''<br />
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|<font size="2" style="color:#black;">'''a''' Tissue Flask Position 1 Optical Density of Wild Type and DCBomega<br />
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|<font size="2" style="color:#black;">'''b''' Tissue Flask Position 2 Optical Density of Wild Type and DCBomega<br />
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[[Image:Tissue Flask 2b.png| 450 px| left]][[Image:Tissue Flask 1b.png| 450px | right]]<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:#black;">'''c''' Tissue Flask Position 3 Optical Density of Wild Type and DCBomega<br />
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|<font size="2" style="color:#black;">'''d''' Tissue Flask Position 4 Optical Density of Wild Type and DCBomega<br />
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[[Image:Tissue Flask 3b.png| 450px | left]][[Image:Tissue Flask 4b.png| 450px | right]]<br />
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'''e''' Tissue Flask Position 5 Optical Density of Wild Type and DBComega<br />
[[Image:Tissue Flask 5b.png| 450px | center]]<br><br />
Figure 1a,b,c,d,e are the optical density of a tissue flask, as assessed at 600 nm, over the course of the 6 days of the experiment.<br />
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'''Figure 2'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:#black;">'''b''' The Change in the Optical Density of the Tissue Flasks for DBComega<br />
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[[Image:3D 241.png| 450 px | left]][[Image:3D DBCOmega2.png| 450 px | right]]<br />
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Figure 2a and 2b display the growth of the tissue flasks as measured by OD assessed as absorbance at 600 nm. The graph is organized such that those closest to the "days" axis were closest to the light source (i.e. flask 1 is closer than flask 2).<br />
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'''Figure 3'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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[[Image:Bar 241 b.png| 450px | left]][[Image:Bar DBCOmega b.png| 450px | right]]<br />
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Figure 3a and 3b display the cumulative growth of the tissue flasks as measured by the sum of the optical density of the respective cell type's tissue flasks at a given day. Furthermore, the contribution of a given tissue flask to cumulative growth is displayed.<br><br />
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'''Figure 4'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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[[image:sphaeroides tissue flask.jpg|450 px|left|]][[image:dbc tissue flask.jpg|450px|right]]<br />
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Figure 4A and 4B display the appearance of the tissue flasks for both strains on day 6. They are arranged from left to right with left being closed to the light source (flask 1) and right furthest (flask 5)<br />
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'''Figure 5'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:#black;">'''a''' R. sphaeroides Spectrum by Flask Distance from Source <br />
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|<font size="2" style="color:#black;">'''b'''DBComega Spectrum by Flask Distance from Source<br />
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[[Image: WT irradiance 3d.png| 450 px | left]][[Image: DBComega irradiance 3d.png| 450 px | right]]<br />
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Contour of Absolute Irradiance by Flash distance from Source<br />
{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:#black;">'''c''' R. Sphaeroides 2.4.1<br />
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[[Image: r sphaeroides day 2 a.jpg| 450px | left]][[Image: dbc day 4b.jpg| 450px | right]]<br />
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'''e''' Wild Type Absolute Irradiance Behind Flask 1: Day 6<br />
[[Image: irradiance WT flask 1 day6.png| 450px | center]]<br />
Figure 5 shows the results of the absolute irradiance data taken using the sprectroradiomter. A and B shows absolute irradiance on a given flask at each wavelength. Figure C and D represent the data for the same days as A and B in two dimensions. Figure E is the absolute irradiance behind only flask 1 on day 6. <br><br />
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|<font size="2" style="color:#black;">'''Figure 7'''<br />
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Nonlinear least-squares estimation of WT LH2 saturation curve<br><br />
[[Image: saturation for WT as inferred.png| 450px | left]][[Image: OD after 3,4, and 5.jpg| 210px |right]][[Image: OD after flask 1 and 2.jpg| 210px | center]]<br />
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Figure 6 was derived from the absolute irradiance data taken from the tissue flask experiment for the Wild Type.<br><br><br />
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Simulated Optical Density of Mutant and Wild Type Bioreactors<br><br />
Layers One and Two . Layers Three, Four and Five<br />
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Figure 7 shows the results of modelling the wild type vs. our mutant regulation system. <br />
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[https://2009.igem.org/Team:Wash_U/Project Back To Top]<br />
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== '''Analysis''' ==<br />
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Though we were not able to have our mutant functionally express our system, the tissue flask experiment was an interesting way to observe the effects of a full knockout of the Light Harvesting Antenna Complex 2 (LH2) vs the Wild Type on an array of consecutive photobioreactors. A couple of conclusions can be drawn from this data that can be used in modeling our synthetic regulation system for LH2 and in the future design of systems for the optimization of Light Harvesting Antenna size for photobioreactors . <br><br><br />
The most striking difference between the Wild Type Rhodobacter Sphaeroides 2.41 and DBCOmega (LH2 knockout) growth patterns is that the first flask in the WT (closest to the light source) grew less than the second flask, while the converse was true in DBCOmega (figure 2a and 2b). This can be attributed to the photosystem saturation curves for the respective cultures. The Wild Type has an LH2 complex, meaning that their antenna size is inherently larger and that their photosystem will be saturated at lower light intensities than the LH2 deficient mutant. We observed that the incident light intensity used in this experiment led to oversaturation for the first WT tissue flask and resulted in growth-inhibiting photodamage, as is evidenced by its lesser growth relative to the second culture. In contrast, photodamage was not observed in DBCOmega as is evidenced by the first tissue flask that grew at the fastest respective rate. Furthermore, it appears that this photodamage slowed the growth of the first WT tissue flask to the point that the OD of this tissue flask was nearly equal to that of DBCOmega after 5 days of growth(.987 vs. .967) (figure 1a). As such, the contribution of LH2 to the growth of the WT in flask 1 can be assessed to be little after day 3 when the growth curve slows- though, this light (at 800 and 850 nm) was still greatly absorbed by flask 1 (Figure 5e). It is likely that much of this light was then wasted through Non-Photochemical Quenching (NPQ), depleting the flasks behind flask 1 of photons at the LH2 absorbtion peaks and decreasing the cumulative productivity of flasks 1-5. This effect of wasting photons though NPQ under high light intensities is one that we sought to minimize in the design of our synthetic regulation for the pucB/A genes. <br><br><br />
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A further a comparison of the WT and DBCOmega experiments reveals that the 3rd tissue flask in each had almost identical growth rates (figure 1c), while the DBCOmega 4th and 5th tissue flask outperformed that of the WT (figure 1d and 1e). This can simply be attributed to the higher density of cells in the 2nd flask of the WT that led to an overall lesser quantity of photons passing through to the 4th and 5th flasks in the WT experiment and likely resulting in heterotrophic growth (though this will need to be confirmed with a "dark control" experiment that should be completed in time for the Jamboree). This decrease in available photons for the WT can be seen by viewing the absolute irradiance data (incident light intensity at a given wavelength) after the third culture for the WT (figure 5a and 5c). This decrease in irradiance is the most pronounced for the LH2 absorption bands at 800 and 850 nm, as was expected. Our synthetic regulation system for pucB/A was designed with the intention of allowing greater penetration of light through the first couple of flasks at the LH2 wavelengths, as expression of pucB/A is inversely correlated to incident light intensity.<br><br><br />
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Overall, the cumulative culture growth of the WT exceeded that of DBCOmega (figure 3) due to the performance of the second tissue flask of the WT relative to that of DBCOmega (figure 1b) despite the photodamage that occurred in tissue flask 1 for the WT. This cumulative culture growth can be considered photosynthetic productivty and is proportional to the amount of energy/desired product that can be obtained from these cultures. <br><br><br />
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It is an interesting exercise to model how our mutant system would perform under these same conditions. To do so, the LH2 saturation curve for the WT (figure 6) and the relative contribution of LH1/LH2 light absorbance to growth was derived from our empirical data and applied to a model that took into account incident light and gave the relative growth in terms of optical density as an output. In this model, our synthetic regulation system of pucB/A allowed our mutant to outperform the wild type species (figure 7). This was due to the performance of the 3rd, 4th, and 5th tissue flask relative to those of the wild type (see figure 7 layers 1,2 vs 1-5). The reason for this is that the wild type LH2 light saturation curve does not vary with changes in incident light intensity as the wild type expresses pucB/A at a uniform level under low oxygen conditions. As a result, the first two layers of Wild Type bioreactors absorb nearly all the available light and starve the bioreactors behind them for photons. <br><br><br />
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Conversely, our mutant was modeled to have a dynamic expression of pucB/A based on the incident light intensity. This light response curve was modeled as an inverse exponential for the Cph8/OmpR ompC Promoter system and the saturation curve changes for each tissue flask in accordance with the incident light intensity- as light intensity decreases, the expression pucB/A increases. This results in more light passing through the first 2 flasks and facilitating greater growth in flasks 3, 4 and 5 as they express large antenna complexes. Nonetheless, we believe that growth across the bioreactors could be further maximized by increasing antenna size in the first 2 bioreactors and sacrificing growth in the 4th and 5th. This could be achieved with a feedback mechanism that sets a minimum level for LH2 expression. <br />
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='''Conclusion'''=<br />
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Though we were not able to conduct the tissue flask growth experiment on our mutant, the results of this experiment on our controls (WT and DBComega) match the assumptions that we had laid out at the beginning of the project, allowed us to draw further conclusions about the efficiency of light-based growth for a series of bioreactors, and to create a model for our mutant based on empirical data. <br> <br />
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*Based on this model, we have concluded that it would be favorable to have a feedback regulation mechanism that sets a minimum expression level for pucB/A to further improve photosynthetic productivity. <br><br />
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*We intend to conduct the tissue flask w DBComega+prkcbc3 before jamboree and compare/interpret these results using our characterization data for the pucB/A and the puc promoter.<br />
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*We will conduct the tissue flask experiment with our functioning mutant in future and compare the results to our model<br />
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*Finally, we would like to characterize the puc promoter under various light conditions and additional oxygen tensions. <br><br />
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'''Overall- our project has demonstrated the potential for a synthetically regulated light harvesting antenna to improve photosynthetic productivity for a series of photobioreactors vs. the wild type.''' <br />
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[https://2009.igem.org/Team:Wash_U/Project Back To Top]<br />
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== '''References''' ==<br />
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# "2007-2008 Catalog & Technical Reference." <u>New England BioLabs Inc.</u> (2007).<br />
# Alon, Uri. <u>Introduction to systems biology and the design principles of biological networks</u>. Boca Raton, FL: Chapman & Hall, 2006.<br />
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# Bower, James M. <u>Computational Modeling of Genetic and Biochemical Networks (Computational Molecular Biology).</u> New York: M.I.T. PRESS, 2001.<br />
# Braatsch, Stephan et al. "A single Flavoprotein AppA, Integrates Both Redox and Light Signals in ''Rhodobacter sphaeroides.''" <u>Molecular Microbiology.</u> Vol. 45.3 (2002): 827-836.<br />
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# Jones, M. R. et al. "Mutants of ''Rhodobacter sphaeroides'' Lacking One or More Pigment-Protein Complexes and Complementation with Reaction-Centre, LH1, and LH2 Genes." <u>Molecular Microbiology.</u> Vol. 6.9 (1992): 1,173-1,184.<br />
# Kondo, Toshihiko et al. "Enhancement of Hydrogen Production by a Photosynthetic Bacterium Mutant with Reduced Pigment." <u>Journal of Bioscience and Bioengineering.</u> Vol. 93.2 (2002): 145-150.<br />
# Lagarias, J. Clark. <u>PCB from Spirulina</u>. Personal Communication. June 2009.<br />
# Laible, Philip D. "Foreign Gene Expression in Photosynthetic Bacteria." <u>The Purple Phototropic Bacteria.</u> (2009): 839-860.<br />
# Lee, Jeong K. and Samuel Kaplan. "Transcriptional Regulation of ''puc'' Operon Expression in ''Rhodobacter sphaeroides.''" <u>Journal of Biological Chemistry.</u> Vol. 270.35 (1995): 20,453-20,458.<br />
# Levskaya, Anselmetal et al. "Engineering ''Escherichia coli'' to See Light." <u>Nature.</u> Vol. 438 (2005): 441-442.<br />
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# Melis, Anastasios. "Solar Energy Conversion Efficiencies in Photosynthesis: Minimizing the Chlorophyll Antennae to Maximize Efficiency." <u>Plant Science.</u> (2009).<br />
# Mizuno, Takeshi and Shoji Mizushima. "Characterization by Deletion and Localized Mutagenesis in Vitro of the Promoter Region of the ''Escherichia coli'' OmpC Gene and Importance of the Upstream DNA Domain in Positive Regulation by the OmpR Protein." <u>Journal of Bacteriology.</u> Vol. 168.1 (1986): 86-95.<br />
# Moskvin, Oleg V. et al. "Transcriptome Analysis of the ''Rhodobacter sphaeroides'' PspR Regulon: PspR as a Master Regulator of Photosystem Development." <u>Journal of Bacteriology.</u> Vol. 187.6 (2005): 2,148-2,156.<br />
# Mussgnug, Jan H. et al. "Engineering photosynthetic light capture: impacts on improved solar energy to biomass conversion." <u> Plant Biotechnology Journal</u> Vol. 5 Issue 6. (2007) pp. 802-814<br />
# "NEBCutter V2.0." <u>New England BioLabs.</u> 2009. 6 June 2009 <http://tools.neb.com/NEBcutter2/index.php>.<br />
# "OligoAnalyzer 3.1." <u>Integrated DNA Technologies.</u> 2009. 6 June 2009. <http://www.idtdna.com/analyzer/Applications/OligoAnalyzer/><br />
# Sambrook, Joseph and David W. Russell. <u>Molecular Cloning: A Laboratory Manual.</u> Vol. 1-3. New York: Cold Springs Harbor Laboratory Press, 2001.<br />
# Scheuring, Simon and Sturgis, James N. "Chromatic Adaptation of Photosynthetic Membranes" <u>Science</u> Vol. 309 (2005) pp.484-487<br />
# Sener, Melih K. et al. "Atomic-level structural and functional model of a bacterial photosynthetic membrane vesicle" <u>Proceedings of the National Academy of Sciences of the United States of America.</u> Vol. 104, Number 40,pp. 15723-15728<br />
# Shimizu-Sato, Sae et al. "A Light-Switchable Gene Promoter System." <u>Nature Publishing Group.</u> Vol. 20 (2002): 1,041-1044.<br />
# Smith, Harry and M. Geoffrey Holmes. <u>Techniquies in Photomorphogenesis.</u> London: Academic Press, Inc., 1984.<br />
# <u>System modeling in cellular biology from concepts to nuts and bolts.</u> Cambridge, MA: MIT P, 2006.<br />
# Terry, Matthew J. "Biosynthesis and Analysis of Bilins." <u>Heme, Chlorophyll, and Bilins: Methods and Protocols.</u> (2002) 273-291. <br />
# Wang, Wanneng et al. "Heterologous Synthesis and Assembly of Functional LHII Antenna Complexes from ''Rhodovulum sulfidophilum'' in ''Rhodobacter sphaeroides'' Mutant." <u>Molecular Biology Reports.</u> Vol. 12.3 (2008).<br />
# Walz, Thomas et al. "Projection Structures of Three Photosynthetic Complexes from Rhodobacter sphaeroides: LH2 at 6 A, LH1 and RC-LH1 at 25 A" <u>Journal of Molecular Biology</u> Vol. 282 pp. 833-845<br />
# Yakovlev, A. G. et al. "Light Control Over the Size of an Antenna Unit Building Block as an Efficient Strategy for Light Harvesting in Photosynthesis." <u>Federation of European Biochemical Societies.</u> (2002):129-132.<br />
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{{WashUbottom}}</div>Brendan1http://2009.igem.org/Team:Wash_U/ProjectTeam:Wash U/Project2009-11-10T00:25:19Z<p>Brendan1: /* Results */</p>
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__NOTOC__<br />
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== '''Introduction''' == <br />
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Our project goal is to maximize the photosynthetic productivity of a series of photobioreactors containing ''Rhodobacter sphaeroides'' under both high and low light intensities by synthetically regulating the size of the light-harvesting antenna complex LH2. We chose to undertake such a project using ''R. sphaeroides'' due to its well-characterized photosynthetic and genetic system. <br />
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The antenna system functions to expand the spectrum of light available for photosynthesis by absorbing different wavelengths than that of the reaction center. Essentially, it is like the dish around the main receiver of any antenna. Marine bacteria, such as ''R. sphaeroides'', evolved very large antenna complexes to absorb light in a natural environment, where there is great competition for photons. As a result, the photosynthetic machinery is saturated at low light intensities in a synthetic non-competitive environment, such as a photobioreactor. This causes up to 95% of incidental photons to be dissipated as heat or fluorescence by bacteria through a process called Non-Photochemical Quenching (NPQ) (Mussgnug et al., 2007). In essence, these photons are being wasted as NPQ reduces light penetration into and across bioreactors and starves the shaded cells for for photons. One method that has been shown to improve photosynthetic productivity in photobioreactors is the reduction of light-harvesting antenna sizes (Polle et al., 2002, Mussgnug et al., 2007). Though, current approaches to this end are difficult to precisely control from the perspective of biological engineering and synthetic biology. <br />
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Our intention is to create a dynamic system that varies antenna size, is dependent on incident light intensity, and can be readily optimized using bioengineering principles. This synthetic regulation of the LH2 complex should result in bacteria grown under high light intensities expressing fewer LH2 complexes than cells that are more shaded, leading to a reduction of wasted incident photons while maintaining a high overall absorbance across subsequent bioreactors. Consequently, we expect to see an increase in the total photosynthetic productivity of our mutant ''Rhodobacter sphaeroides'' when compared to the wild type grown under similar conditions. <br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:black;">'''Wild Type Energy Production in a series of Bioreactors'''<br />
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|<font size="2" style="color:black;">'''Synthetically Regulated Antenna Energy Production in Bioreactors'''<br />
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[[Image:Slide09b.jpg| 450px | left]][[Image:Slide10 a.jpg| 450px | right]]<br />
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== '''Organism''' ==<br />
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''Rhodobacter sphaeroides'' is a purple Alphaproteobacteria. It is metabolically flexible: it can grow heterotrophically, via aerobic and anaerobic respiration, as well as phototrophically under anaerobic conditions with light. <br />
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''R. sphaeroides'' is one of the best understood photosynthetic organisms. The photosystem is located in intracytoplasmic membrane invaginations and the main components are the Light Haresting Complex 2 (LH2), Light Harvesting Complex 1 (LH1), and the Reaction Center (RC). These pigment-protein complexes non-covalently bind bacteriochlorophylls and carotenoids. LH1 and RC make up the core complex and are often found in the ratio of 1:1. LH2 is more peripheral and naturally ranges in the ratio to LH1/RC of 3.0:1 to 6.7:1 under varying light conditions (Scheuring et al., 2005). LH2 absorbs photons maximally at the wavelengths of 850 and 800 nm and funnels its energy to LH1 and the reaction center for photochemistry. (Scheuring et al., 2005). <br />
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The two subunits of LH2 are coded for by the pucB/A genes and are naturally promoted by the puc promoter. <br />
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<br><div style="text-align: center;">'''ICM of ''R. sphaeroides'' '''[[Image:R sphaeroides photosystem.png| 500 px | center]]<center>source:Sener et al.2007<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:black;">'''Plate of ''R. sphaeroides'' 2.4.1'''<br />
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[[Image:R sphaeroides aborbance.png| 450px | left]][[Image:R sphaeroides 0986.png| 450px | right]]<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br>source: Walz et al. 1998<br><br />
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====Online Resources for ''R. sphaeroides''====<br />
[http://www.kazusa.or.jp/codon/cgi-bin/showcodon.cgi?species=272943 Codon Usage Table for ''R. sphaeroides'' 2.41]<br />
<br> [http://www.ncbi.nlm.nih.gov/sites/entrez?db=genome&cmd=Retrieve&dopt=Overview&list_uids=18843 ''R. sphaeroides'' 2.41 NCBI Genome Tools]<br />
<br> [http://archaea.ucsc.edu/cgi-bin/hgPcr?wp_target=&db=0&org=Rhodobacter+sphaeroides+2+4+1&wp_f=&wp_r=&wp_size=2000&wp_perfect=15&wp_good=15&wp_showPage=true&hgsid=159191 ''In silico'' PCR of ''R. sphaeroides'' 2.41 genome]<br />
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=='''Regulation'''==<br />
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[[Image:pucregulation-fullpanels.png|950px]]<br />
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'''The Natural System (Left)''' Under high oxygen tension, PpsR prevents transcription from the puc promoter of the pucBA genes, and thus prevents the expression of LH2(1st panel). Under low oxygen tension, this repression is removed by activated AppA, thus allowing expression of LH2 from pucBA(2nd panel).<br><br />
'''Synthetic Regulation System (Right)''' Our system takes advantage of constitutive protein expression from the puc promoter at low oxygen tension, and replaces pucBA with Cph8 at this site. Placing pucBA expression under the control of the ompC promoter allows for Cph8/OmpR control of LH2 expression under varying light conditions. Under high intensity of incident light, Cph8 will not be autophosphorylated, preventing expression of pucBA (3rd panel). When light intensity is lower, Cph8 will autophosphorylate, thus phosphorylating and activating OmpR and promoting expression of LH2 from the ompC promoter.<br><br />
This regulation system was designed such that light is able to penetrate deeper into and through photobioreactors where light is plentiful.<br />
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== '''Results''' ==<br />
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For methods, materials, and procedures regarding these results, please see [https://2009.igem.org/Team:Wash_U/Protocol#Tissue_Flask_Experiment Tissue Flask Experiment] or [https://2009.igem.org/Team:Wash_U/Biological_Parts#Modeling Modeling]<br />
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'''Figure 1'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:#black;">'''b''' Tissue Flask Position 2 Optical Density of Wild Type and DCBomega<br />
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[[Image:Tissue Flask 2b.png| 450 px| left]][[Image:Tissue Flask 1b.png| 450px | right]]<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:#black;">'''c''' Tissue Flask Position 3 Optical Density of Wild Type and DCBomega<br />
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|<font size="2" style="color:#black;">'''d''' Tissue Flask Position 4 Optical Density of Wild Type and DCBomega<br />
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[[Image:Tissue Flask 3b.png| 450px | left]][[Image:Tissue Flask 4b.png| 450px | right]]<br />
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'''e''' Tissue Flask Position 5 Optical Density of Wild Type and DBComega<br />
[[Image:Tissue Flask 5b.png| 450px | center]]<br><br />
Figure 1a,b,c,d,e are the optical density of a tissue flask, as assessed at 600 nm, over the course of the 6 days of the experiment.<br />
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'''Figure 2'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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[[Image:3D 241.png| 450 px | left]][[Image:3D DBCOmega2.png| 450 px | right]]<br />
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Figure 2a and 2b display the growth of the tissue flasks as measured by OD assessed as absorbance at 600 nm. The graph is organized such that those closest to the "days" axis were closest to the light source (i.e. flask 1 is closer than flask 2).<br />
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'''Figure 3'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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[[Image:Bar 241 b.png| 450px | left]][[Image:Bar DBCOmega b.png| 450px | right]]<br />
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Figure 3a and 3b display the cumulative growth of the tissue flasks as measured by the sum of the optical density of the respective cell type's tissue flasks at a given day. Furthermore, the contribution of a given tissue flask to cumulative growth is displayed.<br><br />
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'''Figure 4'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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[[image:sphaeroides tissue flask.jpg|450 px|left|]][[image:dbc tissue flask.jpg|450px|right]]<br />
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Figure 4A and 4B display the appearance of the tissue flasks for both strains on day 6. They are arranged from left to right with left being closed to the light source (flask 1) and right furthest (flask 5)<br />
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'''Figure 5'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:#black;">'''a''' R. sphaeroides Spectrum by Flask Distance from Source <br />
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|<font size="2" style="color:#black;">'''b'''DBComega Spectrum by Flask Distance from Source<br />
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[[Image: WT irradiance 3d.png| 450 px | left]][[Image: DBComega irradiance 3d.png| 450 px | right]]<br />
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Contour of Absolute Irradiance by Flash distance from Source<br />
{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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[[Image: r sphaeroides day 2 a.jpg| 450px | left]][[Image: dbc day 4b.jpg| 450px | right]]<br />
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'''e''' Wild Type Absolute Irradiance Behind Flask 1: Day 6<br />
[[Image: irradiance WT flask 1 day6.png| 450px | center]]<br />
Figure 5 shows the results of the absolute irradiance data taken using the sprectroradiomter. A and B shows absolute irradiance on a given flask at each wavelength. Figure C and D represent the data for the same days as A and B in two dimensions. Figure E is the absolute irradiance behind only flask 1 on day 6. <br><br />
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Nonlinear least-squares estimation of WT LH2 saturation curve<br><br />
[[Image: saturation for WT as inferred.png| 450px | left]][[Image: OD after 3,4, and 5.jpg| 200px |right]][[Image: OD after flask 1 and 2.jpg| 200px | center]]<br />
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Figure 6 was derived from the absolute irradiance data taken from the tissue flask experiment for the Wild Type.<br><br><br />
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'''Figure 7'''<br><br />
Simulated Optical Density of Mutant and Wild Type Bioreactors<br><br />
Layers One and Two . Layers Three, Four and Five<br />
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Figure 7 shows the results of modelling the wild type vs. our mutant regulation system. <br />
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[https://2009.igem.org/Team:Wash_U/Project Back To Top]<br />
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== '''Analysis''' ==<br />
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Though we were not able to have our mutant functionally express our system, the tissue flask experiment was an interesting way to observe the effects of a full knockout of the Light Harvesting Antenna Complex 2 (LH2) vs the Wild Type on an array of consecutive photobioreactors. A couple of conclusions can be drawn from this data that can be used in modeling our synthetic regulation system for LH2 and in the future design of systems for the optimization of Light Harvesting Antenna size for photobioreactors . <br><br><br />
The most striking difference between the Wild Type Rhodobacter Sphaeroides 2.41 and DBCOmega (LH2 knockout) growth patterns is that the first flask in the WT (closest to the light source) grew less than the second flask, while the converse was true in DBCOmega (figure 2a and 2b). This can be attributed to the photosystem saturation curves for the respective cultures. The Wild Type has an LH2 complex, meaning that their antenna size is inherently larger and that their photosystem will be saturated at lower light intensities than the LH2 deficient mutant. We observed that the incident light intensity used in this experiment led to oversaturation for the first WT tissue flask and resulted in growth-inhibiting photodamage, as is evidenced by its lesser growth relative to the second culture. In contrast, photodamage was not observed in DBCOmega as is evidenced by the first tissue flask that grew at the fastest respective rate. Furthermore, it appears that this photodamage slowed the growth of the first WT tissue flask to the point that the OD of this tissue flask was nearly equal to that of DBCOmega after 5 days of growth(.987 vs. .967) (figure 1a). As such, the contribution of LH2 to the growth of the WT in flask 1 can be assessed to be little after day 3 when the growth curve slows- though, this light (at 800 and 850 nm) was still greatly absorbed by flask 1 (Figure 5e). It is likely that much of this light was then wasted through Non-Photochemical Quenching (NPQ), depleting the flasks behind flask 1 of photons at the LH2 absorbtion peaks and decreasing the cumulative productivity of flasks 1-5. This effect of wasting photons though NPQ under high light intensities is one that we sought to minimize in the design of our synthetic regulation for the pucB/A genes. <br><br><br />
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A further a comparison of the WT and DBCOmega experiments reveals that the 3rd tissue flask in each had almost identical growth rates (figure 1c), while the DBCOmega 4th and 5th tissue flask outperformed that of the WT (figure 1d and 1e). This can simply be attributed to the higher density of cells in the 2nd flask of the WT that led to an overall lesser quantity of photons passing through to the 4th and 5th flasks in the WT experiment and likely resulting in heterotrophic growth (though this will need to be confirmed with a "dark control" experiment that should be completed in time for the Jamboree). This decrease in available photons for the WT can be seen by viewing the absolute irradiance data (incident light intensity at a given wavelength) after the third culture for the WT (figure 5a and 5c). This decrease in irradiance is the most pronounced for the LH2 absorption bands at 800 and 850 nm, as was expected. Our synthetic regulation system for pucB/A was designed with the intention of allowing greater penetration of light through the first couple of flasks at the LH2 wavelengths, as expression of pucB/A is inversely correlated to incident light intensity.<br><br><br />
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Overall, the cumulative culture growth of the WT exceeded that of DBCOmega (figure 3) due to the performance of the second tissue flask of the WT relative to that of DBCOmega (figure 1b) despite the photodamage that occurred in tissue flask 1 for the WT. This cumulative culture growth can be considered photosynthetic productivty and is proportional to the amount of energy/desired product that can be obtained from these cultures. <br><br><br />
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It is an interesting exercise to model how our mutant system would perform under these same conditions. To do so, the LH2 saturation curve for the WT (figure 6) and the relative contribution of LH1/LH2 light absorbance to growth was derived from our empirical data and applied to a model that took into account incident light and gave the relative growth in terms of optical density as an output. In this model, our synthetic regulation system of pucB/A allowed our mutant to outperform the wild type species (figure 7). This was due to the performance of the 3rd, 4th, and 5th tissue flask relative to those of the wild type (see figure 7 layers 1,2 vs 1-5). The reason for this is that the wild type LH2 light saturation curve does not vary with changes in incident light intensity as the wild type expresses pucB/A at a uniform level under low oxygen conditions. As a result, the first two layers of Wild Type bioreactors absorb nearly all the available light and starve the bioreactors behind them for photons. <br><br><br />
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Conversely, our mutant was modeled to have a dynamic expression of pucB/A based on the incident light intensity. This light response curve was modeled as an inverse exponential for the Cph8/OmpR ompC Promoter system and the saturation curve changes for each tissue flask in accordance with the incident light intensity- as light intensity decreases, the expression pucB/A increases. This results in more light passing through the first 2 flasks and facilitating greater growth in flasks 3, 4 and 5 as they express large antenna complexes. Nonetheless, we believe that growth across the bioreactors could be further maximized by increasing antenna size in the first 2 bioreactors and sacrificing growth in the 4th and 5th. This could be achieved with a feedback mechanism that sets a minimum level for LH2 expression. <br />
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='''Conclusion'''=<br />
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Though we were not able to conduct the tissue flask growth experiment on our mutant, the results of this experiment on our controls (WT and DBComega) match the assumptions that we had laid out at the beginning of the project, allowed us to draw further conclusions about the efficiency of light-based growth for a series of bioreactors, and to create a model for our mutant based on empirical data. <br> <br />
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*Based on this model, we have concluded that it would be favorable to have a feedback regulation mechanism that sets a minimum expression level for pucB/A to further improve photosynthetic productivity. <br><br />
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*We intend to conduct the tissue flask w DBComega+prkcbc3 before jamboree and compare/interpret these results using our characterization data for the pucB/A and the puc promoter.<br />
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*We will conduct the tissue flask experiment with our functioning mutant in future and compare the results to our model<br />
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*Finally, we would like to characterize the puc promoter under various light conditions and additional oxygen tensions. <br><br />
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'''Overall- our project has demonstrated the potential for a synthetically regulated light harvesting antenna to improve photosynthetic productivity for a series of photobioreactors vs. the wild type.''' <br />
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[https://2009.igem.org/Team:Wash_U/Project Back To Top]<br />
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== '''References''' ==<br />
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# "2007-2008 Catalog & Technical Reference." <u>New England BioLabs Inc.</u> (2007).<br />
# Alon, Uri. <u>Introduction to systems biology and the design principles of biological networks</u>. Boca Raton, FL: Chapman & Hall, 2006.<br />
# Ausubel, Fred et al. <u>Short Protocols in Molecular Biology: Third Edition.</u> Canada: John Wiley and Sons, Inc., 1999.<br />
# "BioBrick Assembly Help." <u>Registry of Standard Biological Parts.</u> 2009. 6 July 2009. <http://partsregistry.org/Main_Page>.<br />
# Bower, James M. <u>Computational Modeling of Genetic and Biochemical Networks (Computational Molecular Biology).</u> New York: M.I.T. PRESS, 2001.<br />
# Braatsch, Stephan et al. "A single Flavoprotein AppA, Integrates Both Redox and Light Signals in ''Rhodobacter sphaeroides.''" <u>Molecular Microbiology.</u> Vol. 45.3 (2002): 827-836.<br />
# Brown, Stanley B. et al. "Bile Pigment Synthesis in Plants: Incorporation of Haem into Phycocyanobilin and Phycocyanobiliproteins in Cyanidium Caldarium." <u>Biochemistry.</u> Vol. 194 (1981): 137-147.<br />
# Dragnea, Vladimira et al. "Time-Resolved Spectroscopic Studies of the AppA Blue-Light Receptor BLUF Domain from ''Rhodobacter sphaeroides'' <u>Biochemistry.</u> Vol. 44 (2005): 15,978-15,985.<br />
# "Entrez Nucleotide Search." <u>National Center for Biotechnology Information.</u> 2009. 6 June 2009 <http://www.ncbi.nlm.nih.gov/nuccore/49175990?from=3533887&to=3534606&report=gbwithparts>.<br />
# Fowler, Gregory J. S. and C. Neil Hunter. "The Synthesis and Assembly of Functional High and Low Light LH2 Antenna Complexes from ''Rhodopseudomonas palustris'' in ''Rhodobacter sphaeroides.''" <u>The Journal of Biological Chemistry.</u> Vol. 271.23 (1996): 13,356-13,361.<br />
# Gabrielsen, Mads et al. "Peripheral Complexes of Purple Bacteria." <u>The Purple Phototropic Bacteria.</u> (2009): 135-153.<br />
# "GenElute HP Plasmid Miniprep Kit: User Guide." <u>Sigma-Aldrich</u>. (2008).<br />
# Harwood, Caroline S. "Nitrogenase-Catalyzed Hydrogen Production by Purple Nonsulfur Photosynthetic Bacteria." <u>Bioenergy.</u> (2008): 259-271.<br />
# Jager, Andreas et al. "The AppA and PpsR Proteins from ''Rhodobacter sphaeroides'' Can Establish a Redox-Dependent Signal Chain but Fail to Transmit Blue-Light Signals in Other Bacteria." <u>Journal of Bacteriology.</u> Vol. 189.6 (2007): 2,274-2,282.<br />
# Jones, M. R. et al. "Mutants of ''Rhodobacter sphaeroides'' Lacking One or More Pigment-Protein Complexes and Complementation with Reaction-Centre, LH1, and LH2 Genes." <u>Molecular Microbiology.</u> Vol. 6.9 (1992): 1,173-1,184.<br />
# Kondo, Toshihiko et al. "Enhancement of Hydrogen Production by a Photosynthetic Bacterium Mutant with Reduced Pigment." <u>Journal of Bioscience and Bioengineering.</u> Vol. 93.2 (2002): 145-150.<br />
# Lagarias, J. Clark. <u>PCB from Spirulina</u>. Personal Communication. June 2009.<br />
# Laible, Philip D. "Foreign Gene Expression in Photosynthetic Bacteria." <u>The Purple Phototropic Bacteria.</u> (2009): 839-860.<br />
# Lee, Jeong K. and Samuel Kaplan. "Transcriptional Regulation of ''puc'' Operon Expression in ''Rhodobacter sphaeroides.''" <u>Journal of Biological Chemistry.</u> Vol. 270.35 (1995): 20,453-20,458.<br />
# Levskaya, Anselmetal et al. "Engineering ''Escherichia coli'' to See Light." <u>Nature.</u> Vol. 438 (2005): 441-442.<br />
# "Life Sciences Catalog 2009-2010." <u>National Diagnostics.</u> (2009).<br />
# Melis, Anastasios. "Solar Energy Conversion Efficiencies in Photosynthesis: Minimizing the Chlorophyll Antennae to Maximize Efficiency." <u>Plant Science.</u> (2009).<br />
# Mizuno, Takeshi and Shoji Mizushima. "Characterization by Deletion and Localized Mutagenesis in Vitro of the Promoter Region of the ''Escherichia coli'' OmpC Gene and Importance of the Upstream DNA Domain in Positive Regulation by the OmpR Protein." <u>Journal of Bacteriology.</u> Vol. 168.1 (1986): 86-95.<br />
# Moskvin, Oleg V. et al. "Transcriptome Analysis of the ''Rhodobacter sphaeroides'' PspR Regulon: PspR as a Master Regulator of Photosystem Development." <u>Journal of Bacteriology.</u> Vol. 187.6 (2005): 2,148-2,156.<br />
# Mussgnug, Jan H. et al. "Engineering photosynthetic light capture: impacts on improved solar energy to biomass conversion." <u> Plant Biotechnology Journal</u> Vol. 5 Issue 6. (2007) pp. 802-814<br />
# "NEBCutter V2.0." <u>New England BioLabs.</u> 2009. 6 June 2009 <http://tools.neb.com/NEBcutter2/index.php>.<br />
# "OligoAnalyzer 3.1." <u>Integrated DNA Technologies.</u> 2009. 6 June 2009. <http://www.idtdna.com/analyzer/Applications/OligoAnalyzer/><br />
# Sambrook, Joseph and David W. Russell. <u>Molecular Cloning: A Laboratory Manual.</u> Vol. 1-3. New York: Cold Springs Harbor Laboratory Press, 2001.<br />
# Scheuring, Simon and Sturgis, James N. "Chromatic Adaptation of Photosynthetic Membranes" <u>Science</u> Vol. 309 (2005) pp.484-487<br />
# Sener, Melih K. et al. "Atomic-level structural and functional model of a bacterial photosynthetic membrane vesicle" <u>Proceedings of the National Academy of Sciences of the United States of America.</u> Vol. 104, Number 40,pp. 15723-15728<br />
# Shimizu-Sato, Sae et al. "A Light-Switchable Gene Promoter System." <u>Nature Publishing Group.</u> Vol. 20 (2002): 1,041-1044.<br />
# Smith, Harry and M. Geoffrey Holmes. <u>Techniquies in Photomorphogenesis.</u> London: Academic Press, Inc., 1984.<br />
# <u>System modeling in cellular biology from concepts to nuts and bolts.</u> Cambridge, MA: MIT P, 2006.<br />
# Terry, Matthew J. "Biosynthesis and Analysis of Bilins." <u>Heme, Chlorophyll, and Bilins: Methods and Protocols.</u> (2002) 273-291. <br />
# Wang, Wanneng et al. "Heterologous Synthesis and Assembly of Functional LHII Antenna Complexes from ''Rhodovulum sulfidophilum'' in ''Rhodobacter sphaeroides'' Mutant." <u>Molecular Biology Reports.</u> Vol. 12.3 (2008).<br />
# Walz, Thomas et al. "Projection Structures of Three Photosynthetic Complexes from Rhodobacter sphaeroides: LH2 at 6 A, LH1 and RC-LH1 at 25 A" <u>Journal of Molecular Biology</u> Vol. 282 pp. 833-845<br />
# Yakovlev, A. G. et al. "Light Control Over the Size of an Antenna Unit Building Block as an Efficient Strategy for Light Harvesting in Photosynthesis." <u>Federation of European Biochemical Societies.</u> (2002):129-132.<br />
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{{WashUbottom}}</div>Brendan1http://2009.igem.org/Team:Wash_U/ProjectTeam:Wash U/Project2009-11-10T00:22:27Z<p>Brendan1: /* Results */</p>
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== '''Introduction''' == <br />
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Our project goal is to maximize the photosynthetic productivity of a series of photobioreactors containing ''Rhodobacter sphaeroides'' under both high and low light intensities by synthetically regulating the size of the light-harvesting antenna complex LH2. We chose to undertake such a project using ''R. sphaeroides'' due to its well-characterized photosynthetic and genetic system. <br />
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The antenna system functions to expand the spectrum of light available for photosynthesis by absorbing different wavelengths than that of the reaction center. Essentially, it is like the dish around the main receiver of any antenna. Marine bacteria, such as ''R. sphaeroides'', evolved very large antenna complexes to absorb light in a natural environment, where there is great competition for photons. As a result, the photosynthetic machinery is saturated at low light intensities in a synthetic non-competitive environment, such as a photobioreactor. This causes up to 95% of incidental photons to be dissipated as heat or fluorescence by bacteria through a process called Non-Photochemical Quenching (NPQ) (Mussgnug et al., 2007). In essence, these photons are being wasted as NPQ reduces light penetration into and across bioreactors and starves the shaded cells for for photons. One method that has been shown to improve photosynthetic productivity in photobioreactors is the reduction of light-harvesting antenna sizes (Polle et al., 2002, Mussgnug et al., 2007). Though, current approaches to this end are difficult to precisely control from the perspective of biological engineering and synthetic biology. <br />
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Our intention is to create a dynamic system that varies antenna size, is dependent on incident light intensity, and can be readily optimized using bioengineering principles. This synthetic regulation of the LH2 complex should result in bacteria grown under high light intensities expressing fewer LH2 complexes than cells that are more shaded, leading to a reduction of wasted incident photons while maintaining a high overall absorbance across subsequent bioreactors. Consequently, we expect to see an increase in the total photosynthetic productivity of our mutant ''Rhodobacter sphaeroides'' when compared to the wild type grown under similar conditions. <br />
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== '''Organism''' ==<br />
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''Rhodobacter sphaeroides'' is a purple Alphaproteobacteria. It is metabolically flexible: it can grow heterotrophically, via aerobic and anaerobic respiration, as well as phototrophically under anaerobic conditions with light. <br />
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''R. sphaeroides'' is one of the best understood photosynthetic organisms. The photosystem is located in intracytoplasmic membrane invaginations and the main components are the Light Haresting Complex 2 (LH2), Light Harvesting Complex 1 (LH1), and the Reaction Center (RC). These pigment-protein complexes non-covalently bind bacteriochlorophylls and carotenoids. LH1 and RC make up the core complex and are often found in the ratio of 1:1. LH2 is more peripheral and naturally ranges in the ratio to LH1/RC of 3.0:1 to 6.7:1 under varying light conditions (Scheuring et al., 2005). LH2 absorbs photons maximally at the wavelengths of 850 and 800 nm and funnels its energy to LH1 and the reaction center for photochemistry. (Scheuring et al., 2005). <br />
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The two subunits of LH2 are coded for by the pucB/A genes and are naturally promoted by the puc promoter. <br />
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<br><div style="text-align: center;">'''ICM of ''R. sphaeroides'' '''[[Image:R sphaeroides photosystem.png| 500 px | center]]<center>source:Sener et al.2007<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:black;">'''Plate of ''R. sphaeroides'' 2.4.1'''<br />
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[[Image:R sphaeroides aborbance.png| 450px | left]][[Image:R sphaeroides 0986.png| 450px | right]]<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br>source: Walz et al. 1998<br><br />
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====Online Resources for ''R. sphaeroides''====<br />
[http://www.kazusa.or.jp/codon/cgi-bin/showcodon.cgi?species=272943 Codon Usage Table for ''R. sphaeroides'' 2.41]<br />
<br> [http://www.ncbi.nlm.nih.gov/sites/entrez?db=genome&cmd=Retrieve&dopt=Overview&list_uids=18843 ''R. sphaeroides'' 2.41 NCBI Genome Tools]<br />
<br> [http://archaea.ucsc.edu/cgi-bin/hgPcr?wp_target=&db=0&org=Rhodobacter+sphaeroides+2+4+1&wp_f=&wp_r=&wp_size=2000&wp_perfect=15&wp_good=15&wp_showPage=true&hgsid=159191 ''In silico'' PCR of ''R. sphaeroides'' 2.41 genome]<br />
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=='''Regulation'''==<br />
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[[Image:pucregulation-fullpanels.png|950px]]<br />
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'''The Natural System (Left)''' Under high oxygen tension, PpsR prevents transcription from the puc promoter of the pucBA genes, and thus prevents the expression of LH2(1st panel). Under low oxygen tension, this repression is removed by activated AppA, thus allowing expression of LH2 from pucBA(2nd panel).<br><br />
'''Synthetic Regulation System (Right)''' Our system takes advantage of constitutive protein expression from the puc promoter at low oxygen tension, and replaces pucBA with Cph8 at this site. Placing pucBA expression under the control of the ompC promoter allows for Cph8/OmpR control of LH2 expression under varying light conditions. Under high intensity of incident light, Cph8 will not be autophosphorylated, preventing expression of pucBA (3rd panel). When light intensity is lower, Cph8 will autophosphorylate, thus phosphorylating and activating OmpR and promoting expression of LH2 from the ompC promoter.<br><br />
This regulation system was designed such that light is able to penetrate deeper into and through photobioreactors where light is plentiful.<br />
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<br>[https://2009.igem.org/Team:Wash_U/Project Back To Top]<br />
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== '''Results''' ==<br />
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For methods, materials, and procedures regarding these results, please see [https://2009.igem.org/Team:Wash_U/Protocol#Tissue_Flask_Experiment Tissue Flask Experiment] or [https://2009.igem.org/Team:Wash_U/Biological_Parts#Modeling Modeling]<br />
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'''Figure 1'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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[[Image:Tissue Flask 2b.png| 450 px| left]][[Image:Tissue Flask 1b.png| 450px | right]]<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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[[Image:Tissue Flask 3b.png| 450px | left]][[Image:Tissue Flask 4b.png| 450px | right]]<br />
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'''e''' Tissue Flask Position 5 Optical Density of Wild Type and DBComega<br />
[[Image:Tissue Flask 5b.png| 450px | center]]<br><br />
Figure 1a,b,c,d,e are the optical density of a tissue flask, as assessed at 600 nm, over the course of the 6 days of the experiment.<br />
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'''Figure 2'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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[[Image:3D 241.png| 450 px | left]][[Image:3D DBCOmega2.png| 450 px | right]]<br />
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Figure 2a and 2b display the growth of the tissue flasks as measured by OD assessed as absorbance at 600 nm. The graph is organized such that those closest to the "days" axis were closest to the light source (i.e. flask 1 is closer than flask 2).<br />
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'''Figure 3'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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[[Image:Bar 241 b.png| 450px | left]][[Image:Bar DBCOmega b.png| 450px | right]]<br />
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Figure 3a and 3b display the cumulative growth of the tissue flasks as measured by the sum of the optical density of the respective cell type's tissue flasks at a given day. Furthermore, the contribution of a given tissue flask to cumulative growth is displayed.<br><br />
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'''Figure 4'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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[[image:sphaeroides tissue flask.jpg|450 px|left|]][[image:dbc tissue flask.jpg|450px|right]]<br />
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Figure 4A and 4B display the appearance of the tissue flasks for both strains on day 6. They are arranged from left to right with left being closed to the light source (flask 1) and right furthest (flask 5)<br />
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'''Figure 5'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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[[Image: WT irradiance 3d.png| 450 px | left]][[Image: DBComega irradiance 3d.png| 450 px | right]]<br />
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Contour of Absolute Irradiance by Flash distance from Source<br />
{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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[[Image: r sphaeroides day 2 a.jpg| 450px | left]][[Image: dbc day 4b.jpg| 450px | right]]<br />
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'''e''' Wild Type Absolute Irradiance Behind Flask 1: Day 6<br />
[[Image: irradiance WT flask 1 day6.png| 450px | center]]<br />
Figure 5 shows the results of the absolute irradiance data taken using the sprectroradiomter. A and B shows absolute irradiance on a given flask at each wavelength. Figure C and D represent the data for the same days as A and B in two dimensions. Figure E is the absolute irradiance behind only flask 1 on day 6. <br><br />
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Nonlinear least-squares estimation of WT LH2 saturation curve<br><br />
[[Image: saturation for WT as inferred.png| 450 px]]<br />
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Figure 6 was derived from the absolute irradiance data taken from the tissue flask experiment for the Wild Type.<br><br><br />
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'''Figure 7'''<br><br />
Simulated Optical Density of Mutant and Wild Type Bioreactors<br><br />
Layers One and Two . Layers Three, Four and Five<br />
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[[Image: OD after flask 1 and 2.jpg| 200px ]][[Image: OD after 3,4, and 5.jpg| 200px ]]<br><br />
Figure 7 shows the results of modelling the wild type vs. our mutant regulation system. <br />
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[https://2009.igem.org/Team:Wash_U/Project Back To Top]<br />
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== '''Analysis''' ==<br />
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Though we were not able to have our mutant functionally express our system, the tissue flask experiment was an interesting way to observe the effects of a full knockout of the Light Harvesting Antenna Complex 2 (LH2) vs the Wild Type on an array of consecutive photobioreactors. A couple of conclusions can be drawn from this data that can be used in modeling our synthetic regulation system for LH2 and in the future design of systems for the optimization of Light Harvesting Antenna size for photobioreactors . <br><br><br />
The most striking difference between the Wild Type Rhodobacter Sphaeroides 2.41 and DBCOmega (LH2 knockout) growth patterns is that the first flask in the WT (closest to the light source) grew less than the second flask, while the converse was true in DBCOmega (figure 2a and 2b). This can be attributed to the photosystem saturation curves for the respective cultures. The Wild Type has an LH2 complex, meaning that their antenna size is inherently larger and that their photosystem will be saturated at lower light intensities than the LH2 deficient mutant. We observed that the incident light intensity used in this experiment led to oversaturation for the first WT tissue flask and resulted in growth-inhibiting photodamage, as is evidenced by its lesser growth relative to the second culture. In contrast, photodamage was not observed in DBCOmega as is evidenced by the first tissue flask that grew at the fastest respective rate. Furthermore, it appears that this photodamage slowed the growth of the first WT tissue flask to the point that the OD of this tissue flask was nearly equal to that of DBCOmega after 5 days of growth(.987 vs. .967) (figure 1a). As such, the contribution of LH2 to the growth of the WT in flask 1 can be assessed to be little after day 3 when the growth curve slows- though, this light (at 800 and 850 nm) was still greatly absorbed by flask 1 (Figure 5e). It is likely that much of this light was then wasted through Non-Photochemical Quenching (NPQ), depleting the flasks behind flask 1 of photons at the LH2 absorbtion peaks and decreasing the cumulative productivity of flasks 1-5. This effect of wasting photons though NPQ under high light intensities is one that we sought to minimize in the design of our synthetic regulation for the pucB/A genes. <br><br><br />
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A further a comparison of the WT and DBCOmega experiments reveals that the 3rd tissue flask in each had almost identical growth rates (figure 1c), while the DBCOmega 4th and 5th tissue flask outperformed that of the WT (figure 1d and 1e). This can simply be attributed to the higher density of cells in the 2nd flask of the WT that led to an overall lesser quantity of photons passing through to the 4th and 5th flasks in the WT experiment and likely resulting in heterotrophic growth (though this will need to be confirmed with a "dark control" experiment that should be completed in time for the Jamboree). This decrease in available photons for the WT can be seen by viewing the absolute irradiance data (incident light intensity at a given wavelength) after the third culture for the WT (figure 5a and 5c). This decrease in irradiance is the most pronounced for the LH2 absorption bands at 800 and 850 nm, as was expected. Our synthetic regulation system for pucB/A was designed with the intention of allowing greater penetration of light through the first couple of flasks at the LH2 wavelengths, as expression of pucB/A is inversely correlated to incident light intensity.<br><br><br />
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Overall, the cumulative culture growth of the WT exceeded that of DBCOmega (figure 3) due to the performance of the second tissue flask of the WT relative to that of DBCOmega (figure 1b) despite the photodamage that occurred in tissue flask 1 for the WT. This cumulative culture growth can be considered photosynthetic productivty and is proportional to the amount of energy/desired product that can be obtained from these cultures. <br><br><br />
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It is an interesting exercise to model how our mutant system would perform under these same conditions. To do so, the LH2 saturation curve for the WT (figure 6) and the relative contribution of LH1/LH2 light absorbance to growth was derived from our empirical data and applied to a model that took into account incident light and gave the relative growth in terms of optical density as an output. In this model, our synthetic regulation system of pucB/A allowed our mutant to outperform the wild type species (figure 7). This was due to the performance of the 3rd, 4th, and 5th tissue flask relative to those of the wild type (see figure 7 layers 1,2 vs 1-5). The reason for this is that the wild type LH2 light saturation curve does not vary with changes in incident light intensity as the wild type expresses pucB/A at a uniform level under low oxygen conditions. As a result, the first two layers of Wild Type bioreactors absorb nearly all the available light and starve the bioreactors behind them for photons. <br><br><br />
<br />
Conversely, our mutant was modeled to have a dynamic expression of pucB/A based on the incident light intensity. This light response curve was modeled as an inverse exponential for the Cph8/OmpR ompC Promoter system and the saturation curve changes for each tissue flask in accordance with the incident light intensity- as light intensity decreases, the expression pucB/A increases. This results in more light passing through the first 2 flasks and facilitating greater growth in flasks 3, 4 and 5 as they express large antenna complexes. Nonetheless, we believe that growth across the bioreactors could be further maximized by increasing antenna size in the first 2 bioreactors and sacrificing growth in the 4th and 5th. This could be achieved with a feedback mechanism that sets a minimum level for LH2 expression. <br />
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='''Conclusion'''=<br />
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Though we were not able to conduct the tissue flask growth experiment on our mutant, the results of this experiment on our controls (WT and DBComega) match the assumptions that we had laid out at the beginning of the project, allowed us to draw further conclusions about the efficiency of light-based growth for a series of bioreactors, and to create a model for our mutant based on empirical data. <br> <br />
<br />
*Based on this model, we have concluded that it would be favorable to have a feedback regulation mechanism that sets a minimum expression level for pucB/A to further improve photosynthetic productivity. <br><br />
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*We intend to conduct the tissue flask w DBComega+prkcbc3 before jamboree and compare/interpret these results using our characterization data for the pucB/A and the puc promoter.<br />
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*We will conduct the tissue flask experiment with our functioning mutant in future and compare the results to our model<br />
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*Finally, we would like to characterize the puc promoter under various light conditions and additional oxygen tensions. <br><br />
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'''Overall- our project has demonstrated the potential for a synthetically regulated light harvesting antenna to improve photosynthetic productivity for a series of photobioreactors vs. the wild type.''' <br />
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[https://2009.igem.org/Team:Wash_U/Project Back To Top]<br />
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== '''References''' ==<br />
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# "2007-2008 Catalog & Technical Reference." <u>New England BioLabs Inc.</u> (2007).<br />
# Alon, Uri. <u>Introduction to systems biology and the design principles of biological networks</u>. Boca Raton, FL: Chapman & Hall, 2006.<br />
# Ausubel, Fred et al. <u>Short Protocols in Molecular Biology: Third Edition.</u> Canada: John Wiley and Sons, Inc., 1999.<br />
# "BioBrick Assembly Help." <u>Registry of Standard Biological Parts.</u> 2009. 6 July 2009. <http://partsregistry.org/Main_Page>.<br />
# Bower, James M. <u>Computational Modeling of Genetic and Biochemical Networks (Computational Molecular Biology).</u> New York: M.I.T. PRESS, 2001.<br />
# Braatsch, Stephan et al. "A single Flavoprotein AppA, Integrates Both Redox and Light Signals in ''Rhodobacter sphaeroides.''" <u>Molecular Microbiology.</u> Vol. 45.3 (2002): 827-836.<br />
# Brown, Stanley B. et al. "Bile Pigment Synthesis in Plants: Incorporation of Haem into Phycocyanobilin and Phycocyanobiliproteins in Cyanidium Caldarium." <u>Biochemistry.</u> Vol. 194 (1981): 137-147.<br />
# Dragnea, Vladimira et al. "Time-Resolved Spectroscopic Studies of the AppA Blue-Light Receptor BLUF Domain from ''Rhodobacter sphaeroides'' <u>Biochemistry.</u> Vol. 44 (2005): 15,978-15,985.<br />
# "Entrez Nucleotide Search." <u>National Center for Biotechnology Information.</u> 2009. 6 June 2009 <http://www.ncbi.nlm.nih.gov/nuccore/49175990?from=3533887&to=3534606&report=gbwithparts>.<br />
# Fowler, Gregory J. S. and C. Neil Hunter. "The Synthesis and Assembly of Functional High and Low Light LH2 Antenna Complexes from ''Rhodopseudomonas palustris'' in ''Rhodobacter sphaeroides.''" <u>The Journal of Biological Chemistry.</u> Vol. 271.23 (1996): 13,356-13,361.<br />
# Gabrielsen, Mads et al. "Peripheral Complexes of Purple Bacteria." <u>The Purple Phototropic Bacteria.</u> (2009): 135-153.<br />
# "GenElute HP Plasmid Miniprep Kit: User Guide." <u>Sigma-Aldrich</u>. (2008).<br />
# Harwood, Caroline S. "Nitrogenase-Catalyzed Hydrogen Production by Purple Nonsulfur Photosynthetic Bacteria." <u>Bioenergy.</u> (2008): 259-271.<br />
# Jager, Andreas et al. "The AppA and PpsR Proteins from ''Rhodobacter sphaeroides'' Can Establish a Redox-Dependent Signal Chain but Fail to Transmit Blue-Light Signals in Other Bacteria." <u>Journal of Bacteriology.</u> Vol. 189.6 (2007): 2,274-2,282.<br />
# Jones, M. R. et al. "Mutants of ''Rhodobacter sphaeroides'' Lacking One or More Pigment-Protein Complexes and Complementation with Reaction-Centre, LH1, and LH2 Genes." <u>Molecular Microbiology.</u> Vol. 6.9 (1992): 1,173-1,184.<br />
# Kondo, Toshihiko et al. "Enhancement of Hydrogen Production by a Photosynthetic Bacterium Mutant with Reduced Pigment." <u>Journal of Bioscience and Bioengineering.</u> Vol. 93.2 (2002): 145-150.<br />
# Lagarias, J. Clark. <u>PCB from Spirulina</u>. Personal Communication. June 2009.<br />
# Laible, Philip D. "Foreign Gene Expression in Photosynthetic Bacteria." <u>The Purple Phototropic Bacteria.</u> (2009): 839-860.<br />
# Lee, Jeong K. and Samuel Kaplan. "Transcriptional Regulation of ''puc'' Operon Expression in ''Rhodobacter sphaeroides.''" <u>Journal of Biological Chemistry.</u> Vol. 270.35 (1995): 20,453-20,458.<br />
# Levskaya, Anselmetal et al. "Engineering ''Escherichia coli'' to See Light." <u>Nature.</u> Vol. 438 (2005): 441-442.<br />
# "Life Sciences Catalog 2009-2010." <u>National Diagnostics.</u> (2009).<br />
# Melis, Anastasios. "Solar Energy Conversion Efficiencies in Photosynthesis: Minimizing the Chlorophyll Antennae to Maximize Efficiency." <u>Plant Science.</u> (2009).<br />
# Mizuno, Takeshi and Shoji Mizushima. "Characterization by Deletion and Localized Mutagenesis in Vitro of the Promoter Region of the ''Escherichia coli'' OmpC Gene and Importance of the Upstream DNA Domain in Positive Regulation by the OmpR Protein." <u>Journal of Bacteriology.</u> Vol. 168.1 (1986): 86-95.<br />
# Moskvin, Oleg V. et al. "Transcriptome Analysis of the ''Rhodobacter sphaeroides'' PspR Regulon: PspR as a Master Regulator of Photosystem Development." <u>Journal of Bacteriology.</u> Vol. 187.6 (2005): 2,148-2,156.<br />
# Mussgnug, Jan H. et al. "Engineering photosynthetic light capture: impacts on improved solar energy to biomass conversion." <u> Plant Biotechnology Journal</u> Vol. 5 Issue 6. (2007) pp. 802-814<br />
# "NEBCutter V2.0." <u>New England BioLabs.</u> 2009. 6 June 2009 <http://tools.neb.com/NEBcutter2/index.php>.<br />
# "OligoAnalyzer 3.1." <u>Integrated DNA Technologies.</u> 2009. 6 June 2009. <http://www.idtdna.com/analyzer/Applications/OligoAnalyzer/><br />
# Sambrook, Joseph and David W. Russell. <u>Molecular Cloning: A Laboratory Manual.</u> Vol. 1-3. New York: Cold Springs Harbor Laboratory Press, 2001.<br />
# Scheuring, Simon and Sturgis, James N. "Chromatic Adaptation of Photosynthetic Membranes" <u>Science</u> Vol. 309 (2005) pp.484-487<br />
# Sener, Melih K. et al. "Atomic-level structural and functional model of a bacterial photosynthetic membrane vesicle" <u>Proceedings of the National Academy of Sciences of the United States of America.</u> Vol. 104, Number 40,pp. 15723-15728<br />
# Shimizu-Sato, Sae et al. "A Light-Switchable Gene Promoter System." <u>Nature Publishing Group.</u> Vol. 20 (2002): 1,041-1044.<br />
# Smith, Harry and M. Geoffrey Holmes. <u>Techniquies in Photomorphogenesis.</u> London: Academic Press, Inc., 1984.<br />
# <u>System modeling in cellular biology from concepts to nuts and bolts.</u> Cambridge, MA: MIT P, 2006.<br />
# Terry, Matthew J. "Biosynthesis and Analysis of Bilins." <u>Heme, Chlorophyll, and Bilins: Methods and Protocols.</u> (2002) 273-291. <br />
# Wang, Wanneng et al. "Heterologous Synthesis and Assembly of Functional LHII Antenna Complexes from ''Rhodovulum sulfidophilum'' in ''Rhodobacter sphaeroides'' Mutant." <u>Molecular Biology Reports.</u> Vol. 12.3 (2008).<br />
# Walz, Thomas et al. "Projection Structures of Three Photosynthetic Complexes from Rhodobacter sphaeroides: LH2 at 6 A, LH1 and RC-LH1 at 25 A" <u>Journal of Molecular Biology</u> Vol. 282 pp. 833-845<br />
# Yakovlev, A. G. et al. "Light Control Over the Size of an Antenna Unit Building Block as an Efficient Strategy for Light Harvesting in Photosynthesis." <u>Federation of European Biochemical Societies.</u> (2002):129-132.<br />
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{{WashUbottom}}</div>Brendan1http://2009.igem.org/Team:Wash_U/ProjectTeam:Wash U/Project2009-11-10T00:20:48Z<p>Brendan1: /* Results */</p>
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__NOTOC__<br />
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== '''Introduction''' == <br />
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Our project goal is to maximize the photosynthetic productivity of a series of photobioreactors containing ''Rhodobacter sphaeroides'' under both high and low light intensities by synthetically regulating the size of the light-harvesting antenna complex LH2. We chose to undertake such a project using ''R. sphaeroides'' due to its well-characterized photosynthetic and genetic system. <br />
<br />
The antenna system functions to expand the spectrum of light available for photosynthesis by absorbing different wavelengths than that of the reaction center. Essentially, it is like the dish around the main receiver of any antenna. Marine bacteria, such as ''R. sphaeroides'', evolved very large antenna complexes to absorb light in a natural environment, where there is great competition for photons. As a result, the photosynthetic machinery is saturated at low light intensities in a synthetic non-competitive environment, such as a photobioreactor. This causes up to 95% of incidental photons to be dissipated as heat or fluorescence by bacteria through a process called Non-Photochemical Quenching (NPQ) (Mussgnug et al., 2007). In essence, these photons are being wasted as NPQ reduces light penetration into and across bioreactors and starves the shaded cells for for photons. One method that has been shown to improve photosynthetic productivity in photobioreactors is the reduction of light-harvesting antenna sizes (Polle et al., 2002, Mussgnug et al., 2007). Though, current approaches to this end are difficult to precisely control from the perspective of biological engineering and synthetic biology. <br />
<br />
Our intention is to create a dynamic system that varies antenna size, is dependent on incident light intensity, and can be readily optimized using bioengineering principles. This synthetic regulation of the LH2 complex should result in bacteria grown under high light intensities expressing fewer LH2 complexes than cells that are more shaded, leading to a reduction of wasted incident photons while maintaining a high overall absorbance across subsequent bioreactors. Consequently, we expect to see an increase in the total photosynthetic productivity of our mutant ''Rhodobacter sphaeroides'' when compared to the wild type grown under similar conditions. <br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:black;">'''Wild Type Energy Production in a series of Bioreactors'''<br />
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|<font size="2" style="color:black;">'''Synthetically Regulated Antenna Energy Production in Bioreactors'''<br />
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[[Image:Slide09b.jpg| 450px | left]][[Image:Slide10 a.jpg| 450px | right]]<br />
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[https://2009.igem.org/Team:Wash_U/Project Back To Top]<br />
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== '''Organism''' ==<br />
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''Rhodobacter sphaeroides'' is a purple Alphaproteobacteria. It is metabolically flexible: it can grow heterotrophically, via aerobic and anaerobic respiration, as well as phototrophically under anaerobic conditions with light. <br />
<br> <br><br />
''R. sphaeroides'' is one of the best understood photosynthetic organisms. The photosystem is located in intracytoplasmic membrane invaginations and the main components are the Light Haresting Complex 2 (LH2), Light Harvesting Complex 1 (LH1), and the Reaction Center (RC). These pigment-protein complexes non-covalently bind bacteriochlorophylls and carotenoids. LH1 and RC make up the core complex and are often found in the ratio of 1:1. LH2 is more peripheral and naturally ranges in the ratio to LH1/RC of 3.0:1 to 6.7:1 under varying light conditions (Scheuring et al., 2005). LH2 absorbs photons maximally at the wavelengths of 850 and 800 nm and funnels its energy to LH1 and the reaction center for photochemistry. (Scheuring et al., 2005). <br />
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The two subunits of LH2 are coded for by the pucB/A genes and are naturally promoted by the puc promoter. <br />
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<br><div style="text-align: center;">'''ICM of ''R. sphaeroides'' '''[[Image:R sphaeroides photosystem.png| 500 px | center]]<center>source:Sener et al.2007<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:black;">'''Plate of ''R. sphaeroides'' 2.4.1'''<br />
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[[Image:R sphaeroides aborbance.png| 450px | left]][[Image:R sphaeroides 0986.png| 450px | right]]<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br>source: Walz et al. 1998<br><br />
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====Online Resources for ''R. sphaeroides''====<br />
[http://www.kazusa.or.jp/codon/cgi-bin/showcodon.cgi?species=272943 Codon Usage Table for ''R. sphaeroides'' 2.41]<br />
<br> [http://www.ncbi.nlm.nih.gov/sites/entrez?db=genome&cmd=Retrieve&dopt=Overview&list_uids=18843 ''R. sphaeroides'' 2.41 NCBI Genome Tools]<br />
<br> [http://archaea.ucsc.edu/cgi-bin/hgPcr?wp_target=&db=0&org=Rhodobacter+sphaeroides+2+4+1&wp_f=&wp_r=&wp_size=2000&wp_perfect=15&wp_good=15&wp_showPage=true&hgsid=159191 ''In silico'' PCR of ''R. sphaeroides'' 2.41 genome]<br />
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[https://2009.igem.org/Team:Wash_U/Project Back To Top]<br />
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=='''Regulation'''==<br />
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[[Image:pucregulation-fullpanels.png|950px]]<br />
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'''The Natural System (Left)''' Under high oxygen tension, PpsR prevents transcription from the puc promoter of the pucBA genes, and thus prevents the expression of LH2(1st panel). Under low oxygen tension, this repression is removed by activated AppA, thus allowing expression of LH2 from pucBA(2nd panel).<br><br />
'''Synthetic Regulation System (Right)''' Our system takes advantage of constitutive protein expression from the puc promoter at low oxygen tension, and replaces pucBA with Cph8 at this site. Placing pucBA expression under the control of the ompC promoter allows for Cph8/OmpR control of LH2 expression under varying light conditions. Under high intensity of incident light, Cph8 will not be autophosphorylated, preventing expression of pucBA (3rd panel). When light intensity is lower, Cph8 will autophosphorylate, thus phosphorylating and activating OmpR and promoting expression of LH2 from the ompC promoter.<br><br />
This regulation system was designed such that light is able to penetrate deeper into and through photobioreactors where light is plentiful.<br />
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<br>[https://2009.igem.org/Team:Wash_U/Project Back To Top]<br />
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== '''Results''' ==<br />
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For methods, materials, and procedures regarding these results, please see [https://2009.igem.org/Team:Wash_U/Protocol#Tissue_Flask_Experiment Tissue Flask Experiment] or [https://2009.igem.org/Team:Wash_U/Biological_Parts#Modeling Modeling]<br />
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<div style="text-align: center;"> <br />
'''Figure 1'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:#black;">'''a''' Tissue Flask Position 1 Optical Density of Wild Type and DCBomega<br />
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|<font size="2" style="color:#black;">'''b''' Tissue Flask Position 2 Optical Density of Wild Type and DCBomega<br />
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[[Image:Tissue Flask 2b.png| 450 px| left]][[Image:Tissue Flask 1b.png| 450px | right]]<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:#black;">'''c''' Tissue Flask Position 3 Optical Density of Wild Type and DCBomega<br />
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|<font size="2" style="color:#black;">'''d''' Tissue Flask Position 4 Optical Density of Wild Type and DCBomega<br />
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[[Image:Tissue Flask 3b.png| 450px | left]][[Image:Tissue Flask 4b.png| 450px | right]]<br />
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'''e''' Tissue Flask Position 5 Optical Density of Wild Type and DBComega<br />
[[Image:Tissue Flask 5b.png| 450px | center]]<br><br />
Figure 1a,b,c,d,e are the optical density of a tissue flask, as assessed at 600 nm, over the course of the 6 days of the experiment.<br />
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'''Figure 2'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:#black;">'''a''' The Change in the Optical Density of the Tissue Flasks for the WT<br />
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[[Image:3D 241.png| 450 px | left]][[Image:3D DBCOmega2.png| 450 px | right]]<br />
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Figure 2a and 2b display the growth of the tissue flasks as measured by OD assessed as absorbance at 600 nm. The graph is organized such that those closest to the "days" axis were closest to the light source (i.e. flask 1 is closer than flask 2).<br />
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'''Figure 3'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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[[Image:Bar 241 b.png| 450px | left]][[Image:Bar DBCOmega b.png| 450px | right]]<br />
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Figure 3a and 3b display the cumulative growth of the tissue flasks as measured by the sum of the optical density of the respective cell type's tissue flasks at a given day. Furthermore, the contribution of a given tissue flask to cumulative growth is displayed.<br><br />
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'''Figure 4'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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[[image:sphaeroides tissue flask.jpg|450 px|left|]][[image:dbc tissue flask.jpg|450px|right]]<br />
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Figure 4A and 4B display the appearance of the tissue flasks for both strains on day 6. They are arranged from left to right with left being closed to the light source (flask 1) and right furthest (flask 5)<br />
<div style="text-align: center;"> <br />
'''Figure 5'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:#black;">'''a''' R. sphaeroides Spectrum by Flask Distance from Source <br />
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|<font size="2" style="color:#black;">'''b'''DBComega Spectrum by Flask Distance from Source<br />
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[[Image: WT irradiance 3d.png| 450 px | left]][[Image: DBComega irradiance 3d.png| 450 px | right]]<br />
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Contour of Absolute Irradiance by Flash distance from Source<br />
{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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[[Image: r sphaeroides day 2 a.jpg| 450px | left]][[Image: dbc day 4b.jpg| 450px | right]]<br />
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<div style="text-align: center;"><br />
'''e''' Wild Type Absolute Irradiance Behind Flask 1: Day 6<br />
[[Image: irradiance WT flask 1 day6.png| 450px | center]]<br />
Figure 5 shows the results of the absolute irradiance data taken using the sprectroradiomter. A and B shows absolute irradiance on a given flask at each wavelength. Figure C and D represent the data for the same days as A and B in two dimensions. Figure E is the absolute irradiance behind only flask 1 on day 6. <br><br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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'''Figure 6'''<br><br />
Nonlinear least-squares estimation of WT LH2 saturation curve<br><br />
[[Image: saturation for WT as inferred.png| 450 px]]<br />
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Figure 6 was derived from the absolute irradiance data taken from the tissue flask experiment for the Wild Type.<br><br><br />
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'''Figure 7'''<br><br />
Simulated Optical Density of Mutant and Wild Type Bioreactors<br><br />
Layers One and Two . Layers Three, Four and Five<br />
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[[Image: OD after flask 1 and 2.jpg| 200px ]][[Image: OD after 3,4, and 5.jpg| 200px ]]<br><br />
Figure 7 shows the results of modelling the wild type vs. our mutant regulation system. <br />
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[https://2009.igem.org/Team:Wash_U/Project Back To Top]<br />
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== '''Analysis''' ==<br />
<font size="2"><br />
<br />
Though we were not able to have our mutant functionally express our system, the tissue flask experiment was an interesting way to observe the effects of a full knockout of the Light Harvesting Antenna Complex 2 (LH2) vs the Wild Type on an array of consecutive photobioreactors. A couple of conclusions can be drawn from this data that can be used in modeling our synthetic regulation system for LH2 and in the future design of systems for the optimization of Light Harvesting Antenna size for photobioreactors . <br><br><br />
The most striking difference between the Wild Type Rhodobacter Sphaeroides 2.41 and DBCOmega (LH2 knockout) growth patterns is that the first flask in the WT (closest to the light source) grew less than the second flask, while the converse was true in DBCOmega (figure 2a and 2b). This can be attributed to the photosystem saturation curves for the respective cultures. The Wild Type has an LH2 complex, meaning that their antenna size is inherently larger and that their photosystem will be saturated at lower light intensities than the LH2 deficient mutant. We observed that the incident light intensity used in this experiment led to oversaturation for the first WT tissue flask and resulted in growth-inhibiting photodamage, as is evidenced by its lesser growth relative to the second culture. In contrast, photodamage was not observed in DBCOmega as is evidenced by the first tissue flask that grew at the fastest respective rate. Furthermore, it appears that this photodamage slowed the growth of the first WT tissue flask to the point that the OD of this tissue flask was nearly equal to that of DBCOmega after 5 days of growth(.987 vs. .967) (figure 1a). As such, the contribution of LH2 to the growth of the WT in flask 1 can be assessed to be little after day 3 when the growth curve slows- though, this light (at 800 and 850 nm) was still greatly absorbed by flask 1 (Figure 5e). It is likely that much of this light was then wasted through Non-Photochemical Quenching (NPQ), depleting the flasks behind flask 1 of photons at the LH2 absorbtion peaks and decreasing the cumulative productivity of flasks 1-5. This effect of wasting photons though NPQ under high light intensities is one that we sought to minimize in the design of our synthetic regulation for the pucB/A genes. <br><br><br />
<br />
A further a comparison of the WT and DBCOmega experiments reveals that the 3rd tissue flask in each had almost identical growth rates (figure 1c), while the DBCOmega 4th and 5th tissue flask outperformed that of the WT (figure 1d and 1e). This can simply be attributed to the higher density of cells in the 2nd flask of the WT that led to an overall lesser quantity of photons passing through to the 4th and 5th flasks in the WT experiment and likely resulting in heterotrophic growth (though this will need to be confirmed with a "dark control" experiment that should be completed in time for the Jamboree). This decrease in available photons for the WT can be seen by viewing the absolute irradiance data (incident light intensity at a given wavelength) after the third culture for the WT (figure 5a and 5c). This decrease in irradiance is the most pronounced for the LH2 absorption bands at 800 and 850 nm, as was expected. Our synthetic regulation system for pucB/A was designed with the intention of allowing greater penetration of light through the first couple of flasks at the LH2 wavelengths, as expression of pucB/A is inversely correlated to incident light intensity.<br><br><br />
<br />
Overall, the cumulative culture growth of the WT exceeded that of DBCOmega (figure 3) due to the performance of the second tissue flask of the WT relative to that of DBCOmega (figure 1b) despite the photodamage that occurred in tissue flask 1 for the WT. This cumulative culture growth can be considered photosynthetic productivty and is proportional to the amount of energy/desired product that can be obtained from these cultures. <br><br><br />
<br />
It is an interesting exercise to model how our mutant system would perform under these same conditions. To do so, the LH2 saturation curve for the WT (figure 6) and the relative contribution of LH1/LH2 light absorbance to growth was derived from our empirical data and applied to a model that took into account incident light and gave the relative growth in terms of optical density as an output. In this model, our synthetic regulation system of pucB/A allowed our mutant to outperform the wild type species (figure 7). This was due to the performance of the 3rd, 4th, and 5th tissue flask relative to those of the wild type (see figure 7 layers 1,2 vs 1-5). The reason for this is that the wild type LH2 light saturation curve does not vary with changes in incident light intensity as the wild type expresses pucB/A at a uniform level under low oxygen conditions. As a result, the first two layers of Wild Type bioreactors absorb nearly all the available light and starve the bioreactors behind them for photons. <br><br><br />
<br />
Conversely, our mutant was modeled to have a dynamic expression of pucB/A based on the incident light intensity. This light response curve was modeled as an inverse exponential for the Cph8/OmpR ompC Promoter system and the saturation curve changes for each tissue flask in accordance with the incident light intensity- as light intensity decreases, the expression pucB/A increases. This results in more light passing through the first 2 flasks and facilitating greater growth in flasks 3, 4 and 5 as they express large antenna complexes. Nonetheless, we believe that growth across the bioreactors could be further maximized by increasing antenna size in the first 2 bioreactors and sacrificing growth in the 4th and 5th. This could be achieved with a feedback mechanism that sets a minimum level for LH2 expression. <br />
<br />
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='''Conclusion'''=<br />
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Though we were not able to conduct the tissue flask growth experiment on our mutant, the results of this experiment on our controls (WT and DBComega) match the assumptions that we had laid out at the beginning of the project, allowed us to draw further conclusions about the efficiency of light-based growth for a series of bioreactors, and to create a model for our mutant based on empirical data. <br> <br />
<br />
*Based on this model, we have concluded that it would be favorable to have a feedback regulation mechanism that sets a minimum expression level for pucB/A to further improve photosynthetic productivity. <br><br />
<br />
*We intend to conduct the tissue flask w DBComega+prkcbc3 before jamboree and compare/interpret these results using our characterization data for the pucB/A and the puc promoter.<br />
<br />
*We will conduct the tissue flask experiment with our functioning mutant in future and compare the results to our model<br />
<br />
*Finally, we would like to characterize the puc promoter under various light conditions and additional oxygen tensions. <br><br />
<br />
'''Overall- our project has demonstrated the potential for a synthetically regulated light harvesting antenna to improve photosynthetic productivity for a series of photobioreactors vs. the wild type.''' <br />
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== '''References''' ==<br />
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# "2007-2008 Catalog & Technical Reference." <u>New England BioLabs Inc.</u> (2007).<br />
# Alon, Uri. <u>Introduction to systems biology and the design principles of biological networks</u>. Boca Raton, FL: Chapman & Hall, 2006.<br />
# Ausubel, Fred et al. <u>Short Protocols in Molecular Biology: Third Edition.</u> Canada: John Wiley and Sons, Inc., 1999.<br />
# "BioBrick Assembly Help." <u>Registry of Standard Biological Parts.</u> 2009. 6 July 2009. <http://partsregistry.org/Main_Page>.<br />
# Bower, James M. <u>Computational Modeling of Genetic and Biochemical Networks (Computational Molecular Biology).</u> New York: M.I.T. PRESS, 2001.<br />
# Braatsch, Stephan et al. "A single Flavoprotein AppA, Integrates Both Redox and Light Signals in ''Rhodobacter sphaeroides.''" <u>Molecular Microbiology.</u> Vol. 45.3 (2002): 827-836.<br />
# Brown, Stanley B. et al. "Bile Pigment Synthesis in Plants: Incorporation of Haem into Phycocyanobilin and Phycocyanobiliproteins in Cyanidium Caldarium." <u>Biochemistry.</u> Vol. 194 (1981): 137-147.<br />
# Dragnea, Vladimira et al. "Time-Resolved Spectroscopic Studies of the AppA Blue-Light Receptor BLUF Domain from ''Rhodobacter sphaeroides'' <u>Biochemistry.</u> Vol. 44 (2005): 15,978-15,985.<br />
# "Entrez Nucleotide Search." <u>National Center for Biotechnology Information.</u> 2009. 6 June 2009 <http://www.ncbi.nlm.nih.gov/nuccore/49175990?from=3533887&to=3534606&report=gbwithparts>.<br />
# Fowler, Gregory J. S. and C. Neil Hunter. "The Synthesis and Assembly of Functional High and Low Light LH2 Antenna Complexes from ''Rhodopseudomonas palustris'' in ''Rhodobacter sphaeroides.''" <u>The Journal of Biological Chemistry.</u> Vol. 271.23 (1996): 13,356-13,361.<br />
# Gabrielsen, Mads et al. "Peripheral Complexes of Purple Bacteria." <u>The Purple Phototropic Bacteria.</u> (2009): 135-153.<br />
# "GenElute HP Plasmid Miniprep Kit: User Guide." <u>Sigma-Aldrich</u>. (2008).<br />
# Harwood, Caroline S. "Nitrogenase-Catalyzed Hydrogen Production by Purple Nonsulfur Photosynthetic Bacteria." <u>Bioenergy.</u> (2008): 259-271.<br />
# Jager, Andreas et al. "The AppA and PpsR Proteins from ''Rhodobacter sphaeroides'' Can Establish a Redox-Dependent Signal Chain but Fail to Transmit Blue-Light Signals in Other Bacteria." <u>Journal of Bacteriology.</u> Vol. 189.6 (2007): 2,274-2,282.<br />
# Jones, M. R. et al. "Mutants of ''Rhodobacter sphaeroides'' Lacking One or More Pigment-Protein Complexes and Complementation with Reaction-Centre, LH1, and LH2 Genes." <u>Molecular Microbiology.</u> Vol. 6.9 (1992): 1,173-1,184.<br />
# Kondo, Toshihiko et al. "Enhancement of Hydrogen Production by a Photosynthetic Bacterium Mutant with Reduced Pigment." <u>Journal of Bioscience and Bioengineering.</u> Vol. 93.2 (2002): 145-150.<br />
# Lagarias, J. Clark. <u>PCB from Spirulina</u>. Personal Communication. June 2009.<br />
# Laible, Philip D. "Foreign Gene Expression in Photosynthetic Bacteria." <u>The Purple Phototropic Bacteria.</u> (2009): 839-860.<br />
# Lee, Jeong K. and Samuel Kaplan. "Transcriptional Regulation of ''puc'' Operon Expression in ''Rhodobacter sphaeroides.''" <u>Journal of Biological Chemistry.</u> Vol. 270.35 (1995): 20,453-20,458.<br />
# Levskaya, Anselmetal et al. "Engineering ''Escherichia coli'' to See Light." <u>Nature.</u> Vol. 438 (2005): 441-442.<br />
# "Life Sciences Catalog 2009-2010." <u>National Diagnostics.</u> (2009).<br />
# Melis, Anastasios. "Solar Energy Conversion Efficiencies in Photosynthesis: Minimizing the Chlorophyll Antennae to Maximize Efficiency." <u>Plant Science.</u> (2009).<br />
# Mizuno, Takeshi and Shoji Mizushima. "Characterization by Deletion and Localized Mutagenesis in Vitro of the Promoter Region of the ''Escherichia coli'' OmpC Gene and Importance of the Upstream DNA Domain in Positive Regulation by the OmpR Protein." <u>Journal of Bacteriology.</u> Vol. 168.1 (1986): 86-95.<br />
# Moskvin, Oleg V. et al. "Transcriptome Analysis of the ''Rhodobacter sphaeroides'' PspR Regulon: PspR as a Master Regulator of Photosystem Development." <u>Journal of Bacteriology.</u> Vol. 187.6 (2005): 2,148-2,156.<br />
# Mussgnug, Jan H. et al. "Engineering photosynthetic light capture: impacts on improved solar energy to biomass conversion." <u> Plant Biotechnology Journal</u> Vol. 5 Issue 6. (2007) pp. 802-814<br />
# "NEBCutter V2.0." <u>New England BioLabs.</u> 2009. 6 June 2009 <http://tools.neb.com/NEBcutter2/index.php>.<br />
# "OligoAnalyzer 3.1." <u>Integrated DNA Technologies.</u> 2009. 6 June 2009. <http://www.idtdna.com/analyzer/Applications/OligoAnalyzer/><br />
# Sambrook, Joseph and David W. Russell. <u>Molecular Cloning: A Laboratory Manual.</u> Vol. 1-3. New York: Cold Springs Harbor Laboratory Press, 2001.<br />
# Scheuring, Simon and Sturgis, James N. "Chromatic Adaptation of Photosynthetic Membranes" <u>Science</u> Vol. 309 (2005) pp.484-487<br />
# Sener, Melih K. et al. "Atomic-level structural and functional model of a bacterial photosynthetic membrane vesicle" <u>Proceedings of the National Academy of Sciences of the United States of America.</u> Vol. 104, Number 40,pp. 15723-15728<br />
# Shimizu-Sato, Sae et al. "A Light-Switchable Gene Promoter System." <u>Nature Publishing Group.</u> Vol. 20 (2002): 1,041-1044.<br />
# Smith, Harry and M. Geoffrey Holmes. <u>Techniquies in Photomorphogenesis.</u> London: Academic Press, Inc., 1984.<br />
# <u>System modeling in cellular biology from concepts to nuts and bolts.</u> Cambridge, MA: MIT P, 2006.<br />
# Terry, Matthew J. "Biosynthesis and Analysis of Bilins." <u>Heme, Chlorophyll, and Bilins: Methods and Protocols.</u> (2002) 273-291. <br />
# Wang, Wanneng et al. "Heterologous Synthesis and Assembly of Functional LHII Antenna Complexes from ''Rhodovulum sulfidophilum'' in ''Rhodobacter sphaeroides'' Mutant." <u>Molecular Biology Reports.</u> Vol. 12.3 (2008).<br />
# Walz, Thomas et al. "Projection Structures of Three Photosynthetic Complexes from Rhodobacter sphaeroides: LH2 at 6 A, LH1 and RC-LH1 at 25 A" <u>Journal of Molecular Biology</u> Vol. 282 pp. 833-845<br />
# Yakovlev, A. G. et al. "Light Control Over the Size of an Antenna Unit Building Block as an Efficient Strategy for Light Harvesting in Photosynthesis." <u>Federation of European Biochemical Societies.</u> (2002):129-132.<br />
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{{WashUbottom}}</div>Brendan1http://2009.igem.org/Team:Wash_UTeam:Wash U2009-11-10T00:15:37Z<p>Brendan1: </p>
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Welcome to the 2009 Wash U iGEM team wiki!<br><br />
[[Image:Examplegold medal.jpg|20px]] Gold Medal Team [[Image:Examplegold medal.jpg|20px]]<br><br />
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|<html><object width="425" height="344"><param name="movie" value="http://www.youtube.com/v/mbzObmQKJSo&hl=en&fs=1"></param><param name="allowFullScreen" value="true"></param><param name="allowscriptaccess" value="always"></param><embed src="http://www.youtube.com/v/mbzObmQKJSo&hl=en&fs=1" type="application/x-shockwave-flash" allowscriptaccess="always" allowfullscreen="true" width="425" height="344"></embed></object><br />
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== ''' Team''' == <br />
<font size="2"><br />
The first ever Washington University iGEM team is composed of nine undergraduate juniors and seniors majoring in Biology, Molecular Biology and Biochemistry, Biomedical Engineering and Chemical Engineering. Under the leadership of Dr. Blankenship (Biology and Chemistry departments), our team plans to synthetically regulate expression of a light harvesting antenna, which we believe is a first for both iGEM and synthetic biology. To learn more about our highly motivated and well-trained team, please click [https://2009.igem.org/Team:Wash_U/Team here].<br />
<font size="4"><br />
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== '''Project Abstract''' ==<br />
<font size="2"><br />
<br />
'''Improved Photosynthetic Productivity for ''Rhodobacter sphaeroides'' via Synthetic Regulation of the Light Harvesting Antenna LH2'''<br><br />
Photosynthetic light harvesting antennas function to collect light and transfer energy to a reaction center for photochemistry. Phototrophs evolved large antennas to compete for photons in natural environments where light is scarce. Consequently, cells at the surface of photobioreactors over-absorb light, leading to attenuated photobioreactor light penetration and starving interior cells of photons. This reduction of photosynthetic productivity has been identified as the primary impediment to improving photobioreactor efficiency. While reduction of antenna size improves photosynthetic productivity, current approaches to this uniformly truncate antennas and are difficult to manipulate from the perspective of bioengineering. We aim to create a modifiable system to optimize antenna size throughout the bioreactor by utilizing a synthetic regulatory mechanism that correlates expression of the pucB/A LH2 antenna genes with incident light intensity. This new application of synthetic biology serves to transform the science of antenna reduction into the engineering of antenna optimization. To learn more about our project, please click [https://2009.igem.org/Team:Wash_U/Project here].<br />
<br />
<font size="4"><br />
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=='''What Is iGEM?'''==<br />
<font size="2"><br />
The [https://2009.igem.org/Main_Page International Genetically Engineered Machine competition (iGEM)] is the premiere undergraduate Synthetic Biology competition. Student teams are given a kit of biological parts at the beginning of the summer from the [http://partsregistry.org/Main_Page Registry of Standard Biological Parts]. Working at their own schools over the summer, they use these parts and new parts of their own design to build biological systems and operate them in living cells.<br />
<br>The burgeoning field of [http://en.wikipedia.org/wiki/Synthetic_biology Synthetic Biology] is the culmination of the previous thirty years of research into recombinant DNA and biological engineering technology. It is fundamentally about the union of biology and engineering, thereby encouraging the collaboration of geneticists, molecular biologists, biochemists, and biomedical, chemical, and computer science engineers. Researchers in this field mainly seek to A) design and construct new biological parts, devices and systems or B) re-design existing, natural biological systems for useful purposes. <br />
<br />
<font size="4"><br />
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== '''Contact''' == <br />
<font size="2"><br />
Please feel free to contact us with any questions or concerns at <html><a href="mailto:washu.igem@gmail.com">washu.igem@gmail.com</a></html><br />
<br>Or feel free to leave a comment on our [https://2009.igem.org/Team:Wash_U/Comments wall].<br><br><br />
'''Interested in joining the 2010 team?''' iGEM is an excellent way for undergraduate students to get involved in hands on research and learn important laboratory skills. Build your resume next summer with a research job in Bioengineering and take part in planning, conducting, and presenting relevant results in synthetic biology. Have fun working with peers and take an active role in deciding the project instead of working individually on a project assigned to you by a professor. During the school year, meetings will occur approximately once a month for planning and learning purposes. Actual laboratory work is conducted over 10 weeks in the summer that may carry over into the next semester until results are presented at an international forum at the end of October. While iGEM does not directly provide stipends or funding for students, most students are paid for their work over the summer through the Summer Undergraduate Research Fund (SURF) program, or university credit may be earned.<br />
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<br><br></div>Brendan1http://2009.igem.org/Team:Wash_UTeam:Wash U2009-11-10T00:14:29Z<p>Brendan1: </p>
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<div style="text-align: center;"><font size="4"><br />
Welcome to the 2009 Wash U iGEM team wiki!<br><br />
[[Image:gold medal.jpg|20px]] Gold Medal Team [[Image:gold medal.jpg|20px]]<br><br />
<div style="text-align: center;"><br />
{|style="background-color:#ffefd5; width:100%" <font size="6"><br />
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|<font size="5" style="color:#8B0000;">'''Our Team'''<br />
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|<html><a href="https://2009.igem.org/Team:Wash_U/Team"><img height=344px width=505px src="https://static.igem.org/mediawiki/2009/2/2a/Teampicwashu.jpg"></a><br />
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<td></html><br />
|}<br />
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<font size="4"><br />
<div style="text-align: left;"><br />
<br />
== ''' Team''' == <br />
<font size="2"><br />
The first ever Washington University iGEM team is composed of nine undergraduate juniors and seniors majoring in Biology, Molecular Biology and Biochemistry, Biomedical Engineering and Chemical Engineering. Under the leadership of Dr. Blankenship (Biology and Chemistry departments), our team plans to synthetically regulate expression of a light harvesting antenna, which we believe is a first for both iGEM and synthetic biology. To learn more about our highly motivated and well-trained team, please click [https://2009.igem.org/Team:Wash_U/Team here].<br />
<font size="4"><br />
<br />
== '''Project Abstract''' ==<br />
<font size="2"><br />
<br />
'''Improved Photosynthetic Productivity for ''Rhodobacter sphaeroides'' via Synthetic Regulation of the Light Harvesting Antenna LH2'''<br><br />
Photosynthetic light harvesting antennas function to collect light and transfer energy to a reaction center for photochemistry. Phototrophs evolved large antennas to compete for photons in natural environments where light is scarce. Consequently, cells at the surface of photobioreactors over-absorb light, leading to attenuated photobioreactor light penetration and starving interior cells of photons. This reduction of photosynthetic productivity has been identified as the primary impediment to improving photobioreactor efficiency. While reduction of antenna size improves photosynthetic productivity, current approaches to this uniformly truncate antennas and are difficult to manipulate from the perspective of bioengineering. We aim to create a modifiable system to optimize antenna size throughout the bioreactor by utilizing a synthetic regulatory mechanism that correlates expression of the pucB/A LH2 antenna genes with incident light intensity. This new application of synthetic biology serves to transform the science of antenna reduction into the engineering of antenna optimization. To learn more about our project, please click [https://2009.igem.org/Team:Wash_U/Project here].<br />
<br />
<font size="4"><br />
<br />
=='''What Is iGEM?'''==<br />
<font size="2"><br />
The [https://2009.igem.org/Main_Page International Genetically Engineered Machine competition (iGEM)] is the premiere undergraduate Synthetic Biology competition. Student teams are given a kit of biological parts at the beginning of the summer from the [http://partsregistry.org/Main_Page Registry of Standard Biological Parts]. Working at their own schools over the summer, they use these parts and new parts of their own design to build biological systems and operate them in living cells.<br />
<br>The burgeoning field of [http://en.wikipedia.org/wiki/Synthetic_biology Synthetic Biology] is the culmination of the previous thirty years of research into recombinant DNA and biological engineering technology. It is fundamentally about the union of biology and engineering, thereby encouraging the collaboration of geneticists, molecular biologists, biochemists, and biomedical, chemical, and computer science engineers. Researchers in this field mainly seek to A) design and construct new biological parts, devices and systems or B) re-design existing, natural biological systems for useful purposes. <br />
<br />
<font size="4"><br />
<br />
== '''Contact''' == <br />
<font size="2"><br />
Please feel free to contact us with any questions or concerns at <html><a href="mailto:washu.igem@gmail.com">washu.igem@gmail.com</a></html><br />
<br>Or feel free to leave a comment on our [https://2009.igem.org/Team:Wash_U/Comments wall].<br><br><br />
'''Interested in joining the 2010 team?''' iGEM is an excellent way for undergraduate students to get involved in hands on research and learn important laboratory skills. Build your resume next summer with a research job in Bioengineering and take part in planning, conducting, and presenting relevant results in synthetic biology. Have fun working with peers and take an active role in deciding the project instead of working individually on a project assigned to you by a professor. During the school year, meetings will occur approximately once a month for planning and learning purposes. Actual laboratory work is conducted over 10 weeks in the summer that may carry over into the next semester until results are presented at an international forum at the end of October. While iGEM does not directly provide stipends or funding for students, most students are paid for their work over the summer through the Summer Undergraduate Research Fund (SURF) program, or university credit may be earned.<br />
<br />
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<br><br></div>Brendan1http://2009.igem.org/Team:Wash_U/ProjectTeam:Wash U/Project2009-11-08T23:27:39Z<p>Brendan1: /* Results */</p>
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__NOTOC__<br />
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== '''Introduction''' == <br />
<font size="2"><br />
Our project goal is to maximize the photosynthetic productivity of a series of photobioreactors containing ''Rhodobacter sphaeroides'' under both high and low light intensities by synthetically regulating the size of the light-harvesting antenna complex LH2. We chose to undertake such a project using ''R. sphaeroides'' due to its well-characterized photosynthetic and genetic system. <br />
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The antenna system functions to expand the spectrum of light available for photosynthesis by absorbing different wavelengths than that of the reaction center. Essentially, it is like the dish around the main receiver of any antenna. Marine bacteria, such as ''R. sphaeroides'', evolved very large antenna complexes to absorb light in a natural environment, where there is great competition for photons. As a result, the photosynthetic machinery is saturated at low light intensities in a synthetic non-competitive environment, such as a photobioreactor. This causes up to 95% of incidental photons to be dissipated as heat or fluorescence by bacteria through a process called Non-Photochemical Quenching (NPQ) (Mussgnug et al., 2007). In essence, these photons are being wasted as NPQ reduces light penetration into and across bioreactors and starves the shaded cells for for photons. One method that has been shown to improve photosynthetic productivity in photobioreactors is the reduction of light-harvesting antenna sizes (Polle et al., 2002, Mussgnug et al., 2007). Though, current approaches to this end are difficult to precisely control from the perspective of biological engineering and synthetic biology. <br />
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Our intention is to create a dynamic system that varies antenna size, is dependent on incident light intensity, and can be readily optimized using bioengineering principles. This synthetic regulation of the LH2 complex should result in bacteria grown under high light intensities expressing fewer LH2 complexes than cells that are more shaded, leading to a reduction of wasted incident photons while maintaining a high overall absorbance across subsequent bioreactors. Consequently, we expect to see an increase in the total photosynthetic productivity of our mutant ''Rhodobacter sphaeroides'' when compared to the wild type grown under similar conditions. <br />
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|<font size="2" style="color:black;">'''Wild Type Energy Production in a series of Bioreactors'''<br />
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|<font size="2" style="color:black;">'''Synthetically Regulated Antenna Energy Production in Bioreactors'''<br />
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[[Image:Slide09b.jpg| 450px | left]][[Image:Slide10 a.jpg| 450px | right]]<br />
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[https://2009.igem.org/Team:Wash_U/Project Back To Top]<br />
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== '''Organism''' ==<br />
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''Rhodobacter sphaeroides'' is a purple Alphaproteobacteria. It is metabolically flexible: it can grow heterotrophically, via aerobic and anaerobic respiration, as well as phototrophically under anaerobic conditions with light. <br />
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''R. sphaeroides'' is one of the best understood photosynthetic organisms. The photosystem is located in intracytoplasmic membrane invaginations and the main components are the Light Haresting Complex 2 (LH2), Light Harvesting Complex 1 (LH1), and the Reaction Center (RC). These pigment-protein complexes non-covalently bind bacteriochlorophylls and carotenoids. LH1 and RC make up the core complex and are often found in the ratio of 1:1. LH2 is more peripheral and naturally ranges in the ratio to LH1/RC of 3.0:1 to 6.7:1 under varying light conditions (Scheuring et al., 2005). LH2 absorbs photons maximally at the wavelengths of 850 and 800 nm and funnels its energy to LH1 and the reaction center for photochemistry. (Scheuring et al., 2005). <br />
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The two subunits of LH2 are coded for by the pucB/A genes and are naturally promoted by the puc promoter. <br />
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<br><div style="text-align: center;">'''ICM of ''R. sphaeroides'' '''[[Image:R sphaeroides photosystem.png| 500 px | center]]<center>source:Sener et al.2007<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:black;">'''Plate of ''R. sphaeroides'' 2.4.1'''<br />
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[[Image:R sphaeroides aborbance.png| 450px | left]][[Image:R sphaeroides 0986.png| 450px | right]]<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br>source: Walz et al. 1998<br><br />
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====Online Resources for ''R. sphaeroides''====<br />
[http://www.kazusa.or.jp/codon/cgi-bin/showcodon.cgi?species=272943 Codon Usage Table for ''R. sphaeroides'' 2.41]<br />
<br> [http://www.ncbi.nlm.nih.gov/sites/entrez?db=genome&cmd=Retrieve&dopt=Overview&list_uids=18843 ''R. sphaeroides'' 2.41 NCBI Genome Tools]<br />
<br> [http://archaea.ucsc.edu/cgi-bin/hgPcr?wp_target=&db=0&org=Rhodobacter+sphaeroides+2+4+1&wp_f=&wp_r=&wp_size=2000&wp_perfect=15&wp_good=15&wp_showPage=true&hgsid=159191 ''In silico'' PCR of ''R. sphaeroides'' 2.41 genome]<br />
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[https://2009.igem.org/Team:Wash_U/Project Back To Top]<br />
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=='''Regulation'''==<br />
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[[Image:pucregulation-fullpanels.png|950px]]<br />
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'''The Natural System (Left)''' Under high oxygen tension, PpsR prevents transcription from the puc promoter of the pucBA genes, and thus prevents the expression of LH2(1st panel). Under low oxygen tension, this repression is removed by activated AppA, thus allowing expression of LH2 from pucBA(2nd panel).<br><br />
'''Synthetic Regulation System (Right)''' Our system takes advantage of constitutive protein expression from the puc promoter at low oxygen tension, and replaces pucBA with Cph8 at this site. Placing pucBA expression under the control of the ompC promoter allows for Cph8/OmpR control of LH2 expression under varying light conditions. Under high intensity of incident light, Cph8 will not be autophosphorylated, preventing expression of pucBA (3rd panel). When light intensity is lower, Cph8 will autophosphorylate, thus phosphorylating and activating OmpR and promoting expression of LH2 from the ompC promoter.<br><br />
This regulation system was designed such that light is able to penetrate deeper into and through photobioreactors where light is plentiful.<br />
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<br>[https://2009.igem.org/Team:Wash_U/Project Back To Top]<br />
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== '''Results''' ==<br />
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For methods, materials, and procedures regarding these results, please see [https://2009.igem.org/Team:Wash_U/Protocol#Tissue_Flask_Experiment Tissue Flask Experiment] or [https://2009.igem.org/Team:Wash_U/Biological_Parts#Modeling Modeling]<br />
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'''Figure 1'''<br />
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|<font size="2" style="color:#black;">'''a''' Tissue Flask Position 1 Optical Density of Wild Type and DCBomega<br />
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|<font size="2" style="color:#black;">'''b''' Tissue Flask Position 2 Optical Density of Wild Type and DCBomega<br />
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[[Image:Tissue Flask 2b.png| 450 px| left]][[Image:Tissue Flask 1b.png| 450px | right]]<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:#black;">'''c''' Tissue Flask Position 3 Optical Density of Wild Type and DCBomega<br />
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|<font size="2" style="color:#black;">'''d''' Tissue Flask Position 4 Optical Density of Wild Type and DCBomega<br />
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[[Image:Tissue Flask 3b.png| 450px | left]][[Image:Tissue Flask 4b.png| 450px | right]]<br />
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'''e''' Tissue Flask Position 5 Optical Density of Wild Type and DBComega<br />
[[Image:Tissue Flask 5b.png| 450px | center]]<br><br />
Figure 1a,b,c,d,e are the optical density of a tissue flask, as assessed at 600 nm, over the course of the 6 days of the experiment.<br />
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'''Figure 2'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:#black;">'''b''' The Change in the Optical Density of the Tissue Flasks for DBComega<br />
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[[Image:3D 241.png| 450 px | left]][[Image:3D DBCOmega2.png| 450 px | right]]<br />
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Figure 2a and 2b display the growth of the tissue flasks as measured by OD assessed as absorbance at 600 nm. The graph is organized such that those closest to the "days" axis were closest to the light source (i.e. flask 1 is closer than flask 2).<br />
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'''Figure 3'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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[[Image:Bar 241 b.png| 450px | left]][[Image:Bar DBCOmega b.png| 450px | right]]<br />
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Figure 3a and 3b display the cumulative growth of the tissue flasks as measured by the sum of the optical density of the respective cell type's tissue flasks at a given day. Furthermore, the contribution of a given tissue flask to cumulative growth is displayed.<br><br />
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'''Figure 4'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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[[image:sphaeroides tissue flask.jpg|450 px|left|]][[image:dbc tissue flask.jpg|450px|right]]<br />
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Figure 4A and 4B display the appearance of the tissue flasks for both strains on day 6. They are arranged from left to right with left being closed to the light source (flask 1) and right furthest (flask 5)<br />
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'''Figure 5'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:#black;">'''a''' R. sphaeroides Spectrum by Flask Distance from Source <br />
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|<font size="2" style="color:#black;">'''b'''DBComega Spectrum by Flask Distance from Source<br />
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[[Image: WT irradiance 3d.png| 450 px | left]][[Image: DBComega irradiance 3d.png| 450 px | right]]<br />
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Contour of Absolute Irradiance by Flash distance from Source<br />
{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:#black;">'''c''' R. Sphaeroides 2.4.1<br />
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[[Image: r sphaeroides day 2 a.jpg| 450px | left]][[Image: dbc day 4b.jpg| 450px | right]]<br />
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'''e''' Wild Type Absolute Irradiance Behind Flask 1: Day 6<br />
[[Image: irradiance WT flask 1 day6.png| 450px | center]]<br />
Figure 5 shows the results of the absolute irradiance data taken using the sprectroradiomter. A and B shows absolute irradiance on a given flask at each wavelength. Figure C and D represent the data for the same days as A and B in two dimensions. Figure E is the absolute irradiance behind only flask 1 on day 6. <br><br />
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'''Figure 6'''<br><br />
Nonlinear least-squares estimation of WT LH2 saturation curve<br><br />
[[Image: saturation for WT as inferred.png| 450 px]]<br />
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Figure 6 was derived from the absolute irradiance data taken from the tissue flask experiment for the Wild Type.<br><br><br />
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'''Figure 7'''<br><br />
Simulated Optical Density of Mutant and Wild Type Bioreactors<br><br />
Layers One and Two . Layers Three, Four and Five<br />
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[[Image: OD after flask 1 and 2.jpg| 200px ]][[Image: OD after 3,4, and 5.jpg| 200px ]]<br><br />
Figure 7 shows the results of modelling the wild type vs. our mutant regulation system. <br />
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[https://2009.igem.org/Team:Wash_U/Project Back To Top]<br />
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== '''Analysis''' ==<br />
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Though we were not able to have our mutant functionally express our system, the tissue flask experiment was an interesting way to observe the effects of a full knockout of the Light Harvesting Antenna Complex 2 (LH2) vs the Wild Type on an array of consecutive photobioreactors. A couple of conclusions can be drawn from this data that can be used in modeling our synthetic regulation system for LH2 and in the future design of systems for the optimization of Light Harvesting Antenna size for photobioreactors . <br><br><br />
The most striking difference between the Wild Type Rhodobacter Sphaeroides 2.41 and DBCOmega (LH2 knockout) growth patterns is that the first flask in the WT (closest to the light source) grew less than the second flask, while the converse was true in DBCOmega (figure 2a and 2b). This can be attributed to the photosystem saturation curves for the respective cultures. The Wild Type has an LH2 complex, meaning that their antenna size is inherently larger and that their photosystem will be saturated at lower light intensities than the LH2 deficient mutant. We observed that the incident light intensity used in this experiment led to oversaturation for the first WT tissue flask and resulted in growth-inhibiting photodamage, as is evidenced by its lesser growth relative to the second culture. In contrast, photodamage was not observed in DBCOmega as is evidenced by the first tissue flask that grew at the fastest respective rate. Furthermore, it appears that this photodamage slowed the growth of the first WT tissue flask to the point that the OD of this tissue flask was nearly equal to that of DBCOmega after 5 days of growth(.987 vs. .967) (figure 1a). As such, the contribution of LH2 to the growth of the WT in flask 1 can be assessed to be little after day 3 when the growth curve slows- though, this light (at 800 and 850 nm) was still greatly absorbed by flask 1 (Figure 5e). It is likely that much of this light was then wasted through Non-Photochemical Quenching (NPQ), depleting the flasks behind flask 1 of photons at the LH2 absorbtion peaks and decreasing the cumulative productivity of flasks 1-5. This effect of wasting photons though NPQ under high light intensities is one that we sought to minimize in the design of our synthetic regulation for the pucB/A genes. <br><br><br />
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A further a comparison of the WT and DBCOmega experiments reveals that the 3rd tissue flask in each had almost identical growth rates (figure 1c), while the DBCOmega 4th and 5th tissue flask outperformed that of the WT (figure 1d and 1e). This can simply be attributed to the higher density of cells in the 2nd flask of the WT that led to an overall lesser quantity of photons passing through to the 4th and 5th flasks in the WT experiment and likely resulting in heterotrophic growth (though this will need to be confirmed with a "dark control" experiment that should be completed in time for the Jamboree). This decrease in available photons for the WT can be seen by viewing the absolute irradiance data (incident light intensity at a given wavelength) after the third culture for the WT (figure 5a and 5c). This decrease in irradiance is the most pronounced for the LH2 absorption bands at 800 and 850 nm, as was expected. Our synthetic regulation system for pucB/A was designed with the intention of allowing greater penetration of light through the first couple of flasks at the LH2 wavelengths, as expression of pucB/A is inversely correlated to incident light intensity.<br><br><br />
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Overall, the cumulative culture growth of the WT exceeded that of DBCOmega (figure 3) due to the performance of the second tissue flask of the WT relative to that of DBCOmega (figure 1b) despite the photodamage that occurred in tissue flask 1 for the WT. This cumulative culture growth can be considered photosynthetic productivty and is proportional to the amount of energy/desired product that can be obtained from these cultures. <br><br><br />
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It is an interesting exercise to model how our mutant system would perform under these same conditions. To do so, the LH2 saturation curve for the WT (figure 6) and the relative contribution of LH1/LH2 light absorbance to growth was derived from our empirical data and applied to a model that took into account incident light and gave the relative growth in terms of optical density as an output. In this model, our synthetic regulation system of pucB/A allowed our mutant to outperform the wild type species (figure 7). This was due to the performance of the 3rd, 4th, and 5th tissue flask relative to those of the wild type (see figure 7 layers 1,2 vs 1-5). The reason for this is that the wild type LH2 light saturation curve does not vary with changes in incident light intensity as the wild type expresses pucB/A at a uniform level under low oxygen conditions. As a result, the first two layers of Wild Type bioreactors absorb nearly all the available light and starve the bioreactors behind them for photons. <br><br><br />
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Conversely, our mutant was modeled to have a dynamic expression of pucB/A based on the incident light intensity. This light response curve was modeled as an inverse exponential for the Cph8/OmpR ompC Promoter system and the saturation curve changes for each tissue flask in accordance with the incident light intensity- as light intensity decreases, the expression pucB/A increases. This results in more light passing through the first 2 flasks and facilitating greater growth in flasks 3, 4 and 5 as they express large antenna complexes. Nonetheless, we believe that growth across the bioreactors could be further maximized by increasing antenna size in the first 2 bioreactors and sacrificing growth in the 4th and 5th. This could be achieved with a feedback mechanism that sets a minimum level for LH2 expression. <br />
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='''Conclusion'''=<br />
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Though we were not able to conduct the tissue flask growth experiment on our mutant, the results of this experiment on our controls (WT and DBComega) match the assumptions that we had laid out at the beginning of the project, allowed us to draw further conclusions about the efficiency of light-based growth for a series of bioreactors, and to create a model for our mutant based on empirical data. <br> <br />
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*Based on this model, we have concluded that it would be favorable to have a feedback regulation mechanism that sets a minimum expression level for pucB/A to further improve photosynthetic productivity. <br><br />
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*We intend to conduct the tissue flask w DBComega+prkcbc3 before jamboree and compare/interpret these results using our characterization data for the pucB/A and the puc promoter.<br />
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*We will conduct the tissue flask experiment with our functioning mutant in future and compare the results to our model<br />
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*Finally, we would like to characterize the puc promoter under various light conditions and additional oxygen tensions. <br><br />
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'''Overall- our project has demonstrated the potential for a synthetically regulated light harvesting antenna to improve photosynthetic productivity for a series of photobioreactors vs. the wild type.''' <br />
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[https://2009.igem.org/Team:Wash_U/Project Back To Top]<br />
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== '''References''' ==<br />
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# "2007-2008 Catalog & Technical Reference." <u>New England BioLabs Inc.</u> (2007).<br />
# Alon, Uri. <u>Introduction to systems biology and the design principles of biological networks</u>. Boca Raton, FL: Chapman & Hall, 2006.<br />
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# Bower, James M. <u>Computational Modeling of Genetic and Biochemical Networks (Computational Molecular Biology).</u> New York: M.I.T. PRESS, 2001.<br />
# Braatsch, Stephan et al. "A single Flavoprotein AppA, Integrates Both Redox and Light Signals in ''Rhodobacter sphaeroides.''" <u>Molecular Microbiology.</u> Vol. 45.3 (2002): 827-836.<br />
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# Kondo, Toshihiko et al. "Enhancement of Hydrogen Production by a Photosynthetic Bacterium Mutant with Reduced Pigment." <u>Journal of Bioscience and Bioengineering.</u> Vol. 93.2 (2002): 145-150.<br />
# Lagarias, J. Clark. <u>PCB from Spirulina</u>. Personal Communication. June 2009.<br />
# Laible, Philip D. "Foreign Gene Expression in Photosynthetic Bacteria." <u>The Purple Phototropic Bacteria.</u> (2009): 839-860.<br />
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# Levskaya, Anselmetal et al. "Engineering ''Escherichia coli'' to See Light." <u>Nature.</u> Vol. 438 (2005): 441-442.<br />
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# Melis, Anastasios. "Solar Energy Conversion Efficiencies in Photosynthesis: Minimizing the Chlorophyll Antennae to Maximize Efficiency." <u>Plant Science.</u> (2009).<br />
# Mizuno, Takeshi and Shoji Mizushima. "Characterization by Deletion and Localized Mutagenesis in Vitro of the Promoter Region of the ''Escherichia coli'' OmpC Gene and Importance of the Upstream DNA Domain in Positive Regulation by the OmpR Protein." <u>Journal of Bacteriology.</u> Vol. 168.1 (1986): 86-95.<br />
# Moskvin, Oleg V. et al. "Transcriptome Analysis of the ''Rhodobacter sphaeroides'' PspR Regulon: PspR as a Master Regulator of Photosystem Development." <u>Journal of Bacteriology.</u> Vol. 187.6 (2005): 2,148-2,156.<br />
# Mussgnug, Jan H. et al. "Engineering photosynthetic light capture: impacts on improved solar energy to biomass conversion." <u> Plant Biotechnology Journal</u> Vol. 5 Issue 6. (2007) pp. 802-814<br />
# "NEBCutter V2.0." <u>New England BioLabs.</u> 2009. 6 June 2009 <http://tools.neb.com/NEBcutter2/index.php>.<br />
# "OligoAnalyzer 3.1." <u>Integrated DNA Technologies.</u> 2009. 6 June 2009. <http://www.idtdna.com/analyzer/Applications/OligoAnalyzer/><br />
# Sambrook, Joseph and David W. Russell. <u>Molecular Cloning: A Laboratory Manual.</u> Vol. 1-3. New York: Cold Springs Harbor Laboratory Press, 2001.<br />
# Scheuring, Simon and Sturgis, James N. "Chromatic Adaptation of Photosynthetic Membranes" <u>Science</u> Vol. 309 (2005) pp.484-487<br />
# Sener, Melih K. et al. "Atomic-level structural and functional model of a bacterial photosynthetic membrane vesicle" <u>Proceedings of the National Academy of Sciences of the United States of America.</u> Vol. 104, Number 40,pp. 15723-15728<br />
# Shimizu-Sato, Sae et al. "A Light-Switchable Gene Promoter System." <u>Nature Publishing Group.</u> Vol. 20 (2002): 1,041-1044.<br />
# Smith, Harry and M. Geoffrey Holmes. <u>Techniquies in Photomorphogenesis.</u> London: Academic Press, Inc., 1984.<br />
# <u>System modeling in cellular biology from concepts to nuts and bolts.</u> Cambridge, MA: MIT P, 2006.<br />
# Terry, Matthew J. "Biosynthesis and Analysis of Bilins." <u>Heme, Chlorophyll, and Bilins: Methods and Protocols.</u> (2002) 273-291. <br />
# Wang, Wanneng et al. "Heterologous Synthesis and Assembly of Functional LHII Antenna Complexes from ''Rhodovulum sulfidophilum'' in ''Rhodobacter sphaeroides'' Mutant." <u>Molecular Biology Reports.</u> Vol. 12.3 (2008).<br />
# Walz, Thomas et al. "Projection Structures of Three Photosynthetic Complexes from Rhodobacter sphaeroides: LH2 at 6 A, LH1 and RC-LH1 at 25 A" <u>Journal of Molecular Biology</u> Vol. 282 pp. 833-845<br />
# Yakovlev, A. G. et al. "Light Control Over the Size of an Antenna Unit Building Block as an Efficient Strategy for Light Harvesting in Photosynthesis." <u>Federation of European Biochemical Societies.</u> (2002):129-132.<br />
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{{WashUbottom}}</div>Brendan1http://2009.igem.org/Team:Wash_UTeam:Wash U2009-11-08T23:14:40Z<p>Brendan1: /* Contact */</p>
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Welcome to the 2009 Wash U iGEM team wiki!<br><br />
[[Image:Examplegold medal.jpg|20px]] Gold medal team [[Image:Examplegold medal.jpg|20px]]<br><br />
<div style="text-align: center;"><br />
{|style="background-color:#ffefd5; width:100%" <font size="6"><br />
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|<font size="5" style="color:#8B0000;">'''Our Team'''<br />
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|<html><a href="https://2009.igem.org/Team:Wash_U/Team"><img height=344px width=505px src="https://static.igem.org/mediawiki/2009/2/2a/Teampicwashu.jpg"></a><br />
<td></html><br />
|<html><object width="425" height="344"><param name="movie" value="http://www.youtube.com/v/mbzObmQKJSo&hl=en&fs=1"></param><param name="allowFullScreen" value="true"></param><param name="allowscriptaccess" value="always"></param><embed src="http://www.youtube.com/v/mbzObmQKJSo&hl=en&fs=1" type="application/x-shockwave-flash" allowscriptaccess="always" allowfullscreen="true" width="425" height="344"></embed></object><br />
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|}<br />
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<font size="4"><br />
<div style="text-align: left;"><br />
<br />
== ''' Team''' == <br />
<font size="2"><br />
The first ever Washington University iGEM team is composed of nine undergraduate juniors and seniors majoring in Biology, Molecular Biology and Biochemistry, Biomedical Engineering and Chemical Engineering. Under the leadership of Dr. Blankenship (Biology and Chemistry departments), our team plans to synthetically regulate expression of a light harvesting antenna, which we believe is a first for both iGEM and synthetic biology. To learn more about our highly motivated and well-trained team, please click [https://2009.igem.org/Team:Wash_U/Team here].<br />
<font size="4"><br />
<br />
== '''Project Abstract''' ==<br />
<font size="2"><br />
<br />
'''Improved Photosynthetic Productivity for ''Rhodobacter sphaeroides'' via Synthetic Regulation of the Light Harvesting Antenna LH2'''<br><br />
Photosynthetic light harvesting antennas function to collect light and transfer energy to a reaction center for photochemistry. Phototrophs evolved large antennas to compete for photons in natural environments where light is scarce. Consequently, cells at the surface of photobioreactors over-absorb light, leading to attenuated photobioreactor light penetration and starving interior cells of photons. This reduction of photosynthetic productivity has been identified as the primary impediment to improving photobioreactor efficiency. While reduction of antenna size improves photosynthetic productivity, current approaches to this uniformly truncate antennas and are difficult to manipulate from the perspective of bioengineering. We aim to create a modifiable system to optimize antenna size throughout the bioreactor by utilizing a synthetic regulatory mechanism that correlates expression of the pucB/A LH2 antenna genes with incident light intensity. This new application of synthetic biology serves to transform the science of antenna reduction into the engineering of antenna optimization. To learn more about our project, please click [https://2009.igem.org/Team:Wash_U/Project here].<br />
<br />
<font size="4"><br />
<br />
=='''What Is iGEM?'''==<br />
<font size="2"><br />
The [https://2009.igem.org/Main_Page International Genetically Engineered Machine competition (iGEM)] is the premiere undergraduate Synthetic Biology competition. Student teams are given a kit of biological parts at the beginning of the summer from the [http://partsregistry.org/Main_Page Registry of Standard Biological Parts]. Working at their own schools over the summer, they use these parts and new parts of their own design to build biological systems and operate them in living cells.<br />
<br>The burgeoning field of [http://en.wikipedia.org/wiki/Synthetic_biology Synthetic Biology] is the culmination of the previous thirty years of research into recombinant DNA and biological engineering technology. It is fundamentally about the union of biology and engineering, thereby encouraging the collaboration of geneticists, molecular biologists, biochemists, and biomedical, chemical, and computer science engineers. Researchers in this field mainly seek to A) design and construct new biological parts, devices and systems or B) re-design existing, natural biological systems for useful purposes. <br />
<br />
<font size="4"><br />
<br />
== '''Contact''' == <br />
<font size="2"><br />
Please feel free to contact us with any questions or concerns at <html><a href="mailto:washu.igem@gmail.com">washu.igem@gmail.com</a></html><br />
<br>Or feel free to leave a comment on our [https://2009.igem.org/Team:Wash_U/Comments wall].<br><br><br />
'''Interested in joining the 2010 team?''' iGEM is an excellent way for undergraduate students to get involved in hands on research and learn important laboratory skills. Build your resume next summer with a research job in Bioengineering and take part in planning, conducting, and presenting relevant results in synthetic biology. Have fun working with peers and take an active role in deciding the project instead of working individually on a project assigned to you by a professor. During the school year, meetings will occur approximately once a month for planning and learning purposes. Actual laboratory work is conducted over 10 weeks in the summer that may carry over into the next semester until results are presented at an international forum at the end of October. While iGEM does not directly provide stipends or funding for students, most students are paid for their work over the summer through the Summer Undergraduate Research Fund (SURF) program, or university credit may be earned.<br />
<br />
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<br><br></div>Brendan1http://2009.igem.org/Team:Wash_UTeam:Wash U2009-11-08T23:12:23Z<p>Brendan1: /* Contact */</p>
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<div style="text-align: center;"><font size="4"><br />
Welcome to the 2009 Wash U iGEM team wiki!<br><br />
[[Image:Examplegold medal.jpg|20px]] Gold medal team [[Image:Examplegold medal.jpg|20px]]<br><br />
<div style="text-align: center;"><br />
{|style="background-color:#ffefd5; width:100%" <font size="6"><br />
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|<font size="5" style="color:#8B0000;">'''Our Team'''<br />
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{|style="background-color:#000000;"<br />
|<html><a href="https://2009.igem.org/Team:Wash_U/Team"><img height=344px width=505px src="https://static.igem.org/mediawiki/2009/2/2a/Teampicwashu.jpg"></a><br />
<td></html><br />
|<html><object width="425" height="344"><param name="movie" value="http://www.youtube.com/v/mbzObmQKJSo&hl=en&fs=1"></param><param name="allowFullScreen" value="true"></param><param name="allowscriptaccess" value="always"></param><embed src="http://www.youtube.com/v/mbzObmQKJSo&hl=en&fs=1" type="application/x-shockwave-flash" allowscriptaccess="always" allowfullscreen="true" width="425" height="344"></embed></object><br />
<td></html><br />
|}<br />
<br />
<font size="4"><br />
<div style="text-align: left;"><br />
<br />
== ''' Team''' == <br />
<font size="2"><br />
The first ever Washington University iGEM team is composed of nine undergraduate juniors and seniors majoring in Biology, Molecular Biology and Biochemistry, Biomedical Engineering and Chemical Engineering. Under the leadership of Dr. Blankenship (Biology and Chemistry departments), our team plans to synthetically regulate expression of a light harvesting antenna, which we believe is a first for both iGEM and synthetic biology. To learn more about our highly motivated and well-trained team, please click [https://2009.igem.org/Team:Wash_U/Team here].<br />
<font size="4"><br />
<br />
== '''Project Abstract''' ==<br />
<font size="2"><br />
<br />
'''Improved Photosynthetic Productivity for ''Rhodobacter sphaeroides'' via Synthetic Regulation of the Light Harvesting Antenna LH2'''<br><br />
Photosynthetic light harvesting antennas function to collect light and transfer energy to a reaction center for photochemistry. Phototrophs evolved large antennas to compete for photons in natural environments where light is scarce. Consequently, cells at the surface of photobioreactors over-absorb light, leading to attenuated photobioreactor light penetration and starving interior cells of photons. This reduction of photosynthetic productivity has been identified as the primary impediment to improving photobioreactor efficiency. While reduction of antenna size improves photosynthetic productivity, current approaches to this uniformly truncate antennas and are difficult to manipulate from the perspective of bioengineering. We aim to create a modifiable system to optimize antenna size throughout the bioreactor by utilizing a synthetic regulatory mechanism that correlates expression of the pucB/A LH2 antenna genes with incident light intensity. This new application of synthetic biology serves to transform the science of antenna reduction into the engineering of antenna optimization. To learn more about our project, please click [https://2009.igem.org/Team:Wash_U/Project here].<br />
<br />
<font size="4"><br />
<br />
=='''What Is iGEM?'''==<br />
<font size="2"><br />
The [https://2009.igem.org/Main_Page International Genetically Engineered Machine competition (iGEM)] is the premiere undergraduate Synthetic Biology competition. Student teams are given a kit of biological parts at the beginning of the summer from the [http://partsregistry.org/Main_Page Registry of Standard Biological Parts]. Working at their own schools over the summer, they use these parts and new parts of their own design to build biological systems and operate them in living cells.<br />
<br>The burgeoning field of [http://en.wikipedia.org/wiki/Synthetic_biology Synthetic Biology] is the culmination of the previous thirty years of research into recombinant DNA and biological engineering technology. It is fundamentally about the union of biology and engineering, thereby encouraging the collaboration of geneticists, molecular biologists, biochemists, and biomedical, chemical, and computer science engineers. Researchers in this field mainly seek to A) design and construct new biological parts, devices and systems or B) re-design existing, natural biological systems for useful purposes. <br />
<br />
<font size="4"><br />
<br />
== '''Contact''' == <br />
<font size="2"><br />
Please feel free to contact us with any questions or concerns at <html><a href="mailto:washu.igem@gmail.com">washu.igem@gmail.com</a></html><br />
<br>Or feel free to leave a comment on our [https://2009.igem.org/Team:Wash_U/Comments wall].<br><br><br />
Interested in joining the 2010 team? iGEM is an excellent way for undergraduate students to get involved in hands on research and learn important laboratory skills. Build your resume next summer with a research job in Bioengineering and take part in planning, conducting, and presenting relevant results in synthetic biology. Have fun working with peers and take an active role in deciding the project instead of working individually on a project assigned to you by a professor. During the school year, meetings will occur approximately once a month for planning and learning purposes. Actual laboratory work is conducted over 10 weeks in the summer that may carry over into the next semester until results are presented at an international forum at the end of October. While iGEM does not directly provide stipends or funding for students, most students are paid for their work over the summer through the Summer Undergraduate Research Fund (SURF) program, or university credit may be earned.<br />
<br />
<br />
{{WashUbottom}}<br />
<br />
<br />
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<html><center><br />
<a href="http://www3.clustrmaps.com/counter/maps.php?url=https://2009.igem.org/Team:Wash_U" id="clustrMapsLink"><img src="http://www3.clustrmaps.com/counter/index2.php?url=https://2009.igem.org/Team:Wash_U" style="border:0px;" alt="Locations of visitors to this page" title="Locations of visitors to this page" id="clustrMapsImg" onerror="this.onerror=null; this.src='http://www2.clustrmaps.com/images/clustrmaps-back-soon.jpg'; document.getElementById('clustrMapsLink').href='http://www2.clustrmaps.com';" /><br />
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<br><br></div>Brendan1http://2009.igem.org/Team:Wash_UTeam:Wash U2009-11-08T23:06:31Z<p>Brendan1: </p>
<hr />
<div>{{WashUback}}<br />
<br />
<div style="text-align: center;"><font size="4"><br />
Welcome to the 2009 Wash U iGEM team wiki!<br><br />
[[Image:Examplegold medal.jpg|20px]] Gold medal team [[Image:Examplegold medal.jpg|20px]]<br><br />
<div style="text-align: center;"><br />
{|style="background-color:#ffefd5; width:100%" <font size="6"><br />
|<br />
|<br />
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|<font size="5" style="color:#8B0000;">'''Our Team'''<br />
|<br />
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| <br />
|<font size="5" style="color:#8B0000;">'''Our Project'''<br />
|<br />
|<br />
|<br />
|}<br />
{|style="background-color:#000000;"<br />
|<html><a href="https://2009.igem.org/Team:Wash_U/Team"><img height=344px width=505px src="https://static.igem.org/mediawiki/2009/2/2a/Teampicwashu.jpg"></a><br />
<td></html><br />
|<html><object width="425" height="344"><param name="movie" value="http://www.youtube.com/v/mbzObmQKJSo&hl=en&fs=1"></param><param name="allowFullScreen" value="true"></param><param name="allowscriptaccess" value="always"></param><embed src="http://www.youtube.com/v/mbzObmQKJSo&hl=en&fs=1" type="application/x-shockwave-flash" allowscriptaccess="always" allowfullscreen="true" width="425" height="344"></embed></object><br />
<td></html><br />
|}<br />
<br />
<font size="4"><br />
<div style="text-align: left;"><br />
<br />
== ''' Team''' == <br />
<font size="2"><br />
The first ever Washington University iGEM team is composed of nine undergraduate juniors and seniors majoring in Biology, Molecular Biology and Biochemistry, Biomedical Engineering and Chemical Engineering. Under the leadership of Dr. Blankenship (Biology and Chemistry departments), our team plans to synthetically regulate expression of a light harvesting antenna, which we believe is a first for both iGEM and synthetic biology. To learn more about our highly motivated and well-trained team, please click [https://2009.igem.org/Team:Wash_U/Team here].<br />
<font size="4"><br />
<br />
== '''Project Abstract''' ==<br />
<font size="2"><br />
<br />
'''Improved Photosynthetic Productivity for ''Rhodobacter sphaeroides'' via Synthetic Regulation of the Light Harvesting Antenna LH2'''<br><br />
Photosynthetic light harvesting antennas function to collect light and transfer energy to a reaction center for photochemistry. Phototrophs evolved large antennas to compete for photons in natural environments where light is scarce. Consequently, cells at the surface of photobioreactors over-absorb light, leading to attenuated photobioreactor light penetration and starving interior cells of photons. This reduction of photosynthetic productivity has been identified as the primary impediment to improving photobioreactor efficiency. While reduction of antenna size improves photosynthetic productivity, current approaches to this uniformly truncate antennas and are difficult to manipulate from the perspective of bioengineering. We aim to create a modifiable system to optimize antenna size throughout the bioreactor by utilizing a synthetic regulatory mechanism that correlates expression of the pucB/A LH2 antenna genes with incident light intensity. This new application of synthetic biology serves to transform the science of antenna reduction into the engineering of antenna optimization. To learn more about our project, please click [https://2009.igem.org/Team:Wash_U/Project here].<br />
<br />
<font size="4"><br />
<br />
=='''What Is iGEM?'''==<br />
<font size="2"><br />
The [https://2009.igem.org/Main_Page International Genetically Engineered Machine competition (iGEM)] is the premiere undergraduate Synthetic Biology competition. Student teams are given a kit of biological parts at the beginning of the summer from the [http://partsregistry.org/Main_Page Registry of Standard Biological Parts]. Working at their own schools over the summer, they use these parts and new parts of their own design to build biological systems and operate them in living cells.<br />
<br>The burgeoning field of [http://en.wikipedia.org/wiki/Synthetic_biology Synthetic Biology] is the culmination of the previous thirty years of research into recombinant DNA and biological engineering technology. It is fundamentally about the union of biology and engineering, thereby encouraging the collaboration of geneticists, molecular biologists, biochemists, and biomedical, chemical, and computer science engineers. Researchers in this field mainly seek to A) design and construct new biological parts, devices and systems or B) re-design existing, natural biological systems for useful purposes. <br />
<br />
<font size="4"><br />
<br />
== '''Contact''' == <br />
<font size="2"><br />
Please feel free to contact us with any questions or concerns at <html><a href="mailto:washu.igem@gmail.com">washu.igem@gmail.com</a></html><br />
<br>Or feel free to leave a comment on our [https://2009.igem.org/Team:Wash_U/Comments wall].<br />
<br />
<br />
{{WashUbottom}}<br />
<br />
<br />
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<html><center><br />
<a href="http://www3.clustrmaps.com/counter/maps.php?url=https://2009.igem.org/Team:Wash_U" id="clustrMapsLink"><img src="http://www3.clustrmaps.com/counter/index2.php?url=https://2009.igem.org/Team:Wash_U" style="border:0px;" alt="Locations of visitors to this page" title="Locations of visitors to this page" id="clustrMapsImg" onerror="this.onerror=null; this.src='http://www2.clustrmaps.com/images/clustrmaps-back-soon.jpg'; document.getElementById('clustrMapsLink').href='http://www2.clustrmaps.com';" /><br />
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<br><br></div>Brendan1http://2009.igem.org/Team:Wash_UTeam:Wash U2009-11-08T23:05:52Z<p>Brendan1: </p>
<hr />
<div>{{WashUback}}<br />
<br />
<div style="text-align: center;"><font size="4"><br />
Welcome to the 2009 Wash U team wiki!<br><br />
[[Image:Examplegold medal.jpg|20px]] Gold medal team [[Image:Examplegold medal.jpg|20px]]<br><br />
<div style="text-align: center;"><br />
{|style="background-color:#ffefd5; width:100%" <font size="6"><br />
|<br />
|<br />
|<br />
|<font size="5" style="color:#8B0000;">'''Our Team'''<br />
|<br />
|<br />
| <br />
|<font size="5" style="color:#8B0000;">'''Our Project'''<br />
|<br />
|<br />
|<br />
|}<br />
{|style="background-color:#000000;"<br />
|<html><a href="https://2009.igem.org/Team:Wash_U/Team"><img height=344px width=505px src="https://static.igem.org/mediawiki/2009/2/2a/Teampicwashu.jpg"></a><br />
<td></html><br />
|<html><object width="425" height="344"><param name="movie" value="http://www.youtube.com/v/mbzObmQKJSo&hl=en&fs=1"></param><param name="allowFullScreen" value="true"></param><param name="allowscriptaccess" value="always"></param><embed src="http://www.youtube.com/v/mbzObmQKJSo&hl=en&fs=1" type="application/x-shockwave-flash" allowscriptaccess="always" allowfullscreen="true" width="425" height="344"></embed></object><br />
<td></html><br />
|}<br />
<br />
<font size="4"><br />
<div style="text-align: left;"><br />
<br />
== ''' Team''' == <br />
<font size="2"><br />
The first ever Washington University iGEM team is composed of nine undergraduate juniors and seniors majoring in Biology, Molecular Biology and Biochemistry, Biomedical Engineering and Chemical Engineering. Under the leadership of Dr. Blankenship (Biology and Chemistry departments), our team plans to synthetically regulate expression of a light harvesting antenna, which we believe is a first for both iGEM and synthetic biology. To learn more about our highly motivated and well-trained team, please click [https://2009.igem.org/Team:Wash_U/Team here].<br />
<font size="4"><br />
<br />
== '''Project Abstract''' ==<br />
<font size="2"><br />
<br />
'''Improved Photosynthetic Productivity for ''Rhodobacter sphaeroides'' via Synthetic Regulation of the Light Harvesting Antenna LH2'''<br><br />
Photosynthetic light harvesting antennas function to collect light and transfer energy to a reaction center for photochemistry. Phototrophs evolved large antennas to compete for photons in natural environments where light is scarce. Consequently, cells at the surface of photobioreactors over-absorb light, leading to attenuated photobioreactor light penetration and starving interior cells of photons. This reduction of photosynthetic productivity has been identified as the primary impediment to improving photobioreactor efficiency. While reduction of antenna size improves photosynthetic productivity, current approaches to this uniformly truncate antennas and are difficult to manipulate from the perspective of bioengineering. We aim to create a modifiable system to optimize antenna size throughout the bioreactor by utilizing a synthetic regulatory mechanism that correlates expression of the pucB/A LH2 antenna genes with incident light intensity. This new application of synthetic biology serves to transform the science of antenna reduction into the engineering of antenna optimization. To learn more about our project, please click [https://2009.igem.org/Team:Wash_U/Project here].<br />
<br />
<font size="4"><br />
<br />
=='''What Is iGEM?'''==<br />
<font size="2"><br />
The [https://2009.igem.org/Main_Page International Genetically Engineered Machine competition (iGEM)] is the premiere undergraduate Synthetic Biology competition. Student teams are given a kit of biological parts at the beginning of the summer from the [http://partsregistry.org/Main_Page Registry of Standard Biological Parts]. Working at their own schools over the summer, they use these parts and new parts of their own design to build biological systems and operate them in living cells.<br />
<br>The burgeoning field of [http://en.wikipedia.org/wiki/Synthetic_biology Synthetic Biology] is the culmination of the previous thirty years of research into recombinant DNA and biological engineering technology. It is fundamentally about the union of biology and engineering, thereby encouraging the collaboration of geneticists, molecular biologists, biochemists, and biomedical, chemical, and computer science engineers. Researchers in this field mainly seek to A) design and construct new biological parts, devices and systems or B) re-design existing, natural biological systems for useful purposes. <br />
<br />
<font size="4"><br />
<br />
== '''Contact''' == <br />
<font size="2"><br />
Please feel free to contact us with any questions or concerns at <html><a href="mailto:washu.igem@gmail.com">washu.igem@gmail.com</a></html><br />
<br>Or feel free to leave a comment on our [https://2009.igem.org/Team:Wash_U/Comments wall].<br />
<br />
<br />
{{WashUbottom}}<br />
<br />
<br />
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<html><center><br />
<a href="http://www3.clustrmaps.com/counter/maps.php?url=https://2009.igem.org/Team:Wash_U" id="clustrMapsLink"><img src="http://www3.clustrmaps.com/counter/index2.php?url=https://2009.igem.org/Team:Wash_U" style="border:0px;" alt="Locations of visitors to this page" title="Locations of visitors to this page" id="clustrMapsImg" onerror="this.onerror=null; this.src='http://www2.clustrmaps.com/images/clustrmaps-back-soon.jpg'; document.getElementById('clustrMapsLink').href='http://www2.clustrmaps.com';" /><br />
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<br><br></div>Brendan1http://2009.igem.org/Team:Wash_UTeam:Wash U2009-11-08T23:05:25Z<p>Brendan1: </p>
<hr />
<div>{{WashUback}}<br />
<br />
<div style="text-align: center;"><font size="4"><br />
Welcome to the 2009 Wash U team wiki!<br><br />
[[Image:Examplegold medal.jpg|30px]] Gold medal team [[Image:Examplegold medal.jpg|30px]]<br><br />
<div style="text-align: center;"><br />
{|style="background-color:#ffefd5; width:100%" <font size="6"><br />
|<br />
|<br />
|<br />
|<font size="5" style="color:#8B0000;">'''Our Team'''<br />
|<br />
|<br />
| <br />
|<font size="5" style="color:#8B0000;">'''Our Project'''<br />
|<br />
|<br />
|<br />
|}<br />
{|style="background-color:#000000;"<br />
|<html><a href="https://2009.igem.org/Team:Wash_U/Team"><img height=344px width=505px src="https://static.igem.org/mediawiki/2009/2/2a/Teampicwashu.jpg"></a><br />
<td></html><br />
|<html><object width="425" height="344"><param name="movie" value="http://www.youtube.com/v/mbzObmQKJSo&hl=en&fs=1"></param><param name="allowFullScreen" value="true"></param><param name="allowscriptaccess" value="always"></param><embed src="http://www.youtube.com/v/mbzObmQKJSo&hl=en&fs=1" type="application/x-shockwave-flash" allowscriptaccess="always" allowfullscreen="true" width="425" height="344"></embed></object><br />
<td></html><br />
|}<br />
<br />
<font size="4"><br />
<div style="text-align: left;"><br />
<br />
== ''' Team''' == <br />
<font size="2"><br />
The first ever Washington University iGEM team is composed of nine undergraduate juniors and seniors majoring in Biology, Molecular Biology and Biochemistry, Biomedical Engineering and Chemical Engineering. Under the leadership of Dr. Blankenship (Biology and Chemistry departments), our team plans to synthetically regulate expression of a light harvesting antenna, which we believe is a first for both iGEM and synthetic biology. To learn more about our highly motivated and well-trained team, please click [https://2009.igem.org/Team:Wash_U/Team here].<br />
<font size="4"><br />
<br />
== '''Project Abstract''' ==<br />
<font size="2"><br />
<br />
'''Improved Photosynthetic Productivity for ''Rhodobacter sphaeroides'' via Synthetic Regulation of the Light Harvesting Antenna LH2'''<br><br />
Photosynthetic light harvesting antennas function to collect light and transfer energy to a reaction center for photochemistry. Phototrophs evolved large antennas to compete for photons in natural environments where light is scarce. Consequently, cells at the surface of photobioreactors over-absorb light, leading to attenuated photobioreactor light penetration and starving interior cells of photons. This reduction of photosynthetic productivity has been identified as the primary impediment to improving photobioreactor efficiency. While reduction of antenna size improves photosynthetic productivity, current approaches to this uniformly truncate antennas and are difficult to manipulate from the perspective of bioengineering. We aim to create a modifiable system to optimize antenna size throughout the bioreactor by utilizing a synthetic regulatory mechanism that correlates expression of the pucB/A LH2 antenna genes with incident light intensity. This new application of synthetic biology serves to transform the science of antenna reduction into the engineering of antenna optimization. To learn more about our project, please click [https://2009.igem.org/Team:Wash_U/Project here].<br />
<br />
<font size="4"><br />
<br />
=='''What Is iGEM?'''==<br />
<font size="2"><br />
The [https://2009.igem.org/Main_Page International Genetically Engineered Machine competition (iGEM)] is the premiere undergraduate Synthetic Biology competition. Student teams are given a kit of biological parts at the beginning of the summer from the [http://partsregistry.org/Main_Page Registry of Standard Biological Parts]. Working at their own schools over the summer, they use these parts and new parts of their own design to build biological systems and operate them in living cells.<br />
<br>The burgeoning field of [http://en.wikipedia.org/wiki/Synthetic_biology Synthetic Biology] is the culmination of the previous thirty years of research into recombinant DNA and biological engineering technology. It is fundamentally about the union of biology and engineering, thereby encouraging the collaboration of geneticists, molecular biologists, biochemists, and biomedical, chemical, and computer science engineers. Researchers in this field mainly seek to A) design and construct new biological parts, devices and systems or B) re-design existing, natural biological systems for useful purposes. <br />
<br />
<font size="4"><br />
<br />
== '''Contact''' == <br />
<font size="2"><br />
Please feel free to contact us with any questions or concerns at <html><a href="mailto:washu.igem@gmail.com">washu.igem@gmail.com</a></html><br />
<br>Or feel free to leave a comment on our [https://2009.igem.org/Team:Wash_U/Comments wall].<br />
<br />
<br />
{{WashUbottom}}<br />
<br />
<br />
<br />
<html><center><br />
<a href="http://www3.clustrmaps.com/counter/maps.php?url=https://2009.igem.org/Team:Wash_U" id="clustrMapsLink"><img src="http://www3.clustrmaps.com/counter/index2.php?url=https://2009.igem.org/Team:Wash_U" style="border:0px;" alt="Locations of visitors to this page" title="Locations of visitors to this page" id="clustrMapsImg" onerror="this.onerror=null; this.src='http://www2.clustrmaps.com/images/clustrmaps-back-soon.jpg'; document.getElementById('clustrMapsLink').href='http://www2.clustrmaps.com';" /><br />
</a><br />
</html><br />
<br><br></div>Brendan1http://2009.igem.org/Team:Wash_UTeam:Wash U2009-11-08T23:04:49Z<p>Brendan1: </p>
<hr />
<div>{{WashUback}}<br />
<br />
<div style="text-align: center;"><font size="4"><br />
Welcome to the 2009 Wash U team wiki!<br><br />
[[Image:Examplegold medal.jpg|50px]]Gold medal team[[Image:Examplegold medal.jpg|50px]]<br><br />
<div style="text-align: center;"><br />
{|style="background-color:#ffefd5; width:100%" <font size="6"><br />
|<br />
|<br />
|<br />
|<font size="5" style="color:#8B0000;">'''Our Team'''<br />
|<br />
|<br />
| <br />
|<font size="5" style="color:#8B0000;">'''Our Project'''<br />
|<br />
|<br />
|<br />
|}<br />
{|style="background-color:#000000;"<br />
|<html><a href="https://2009.igem.org/Team:Wash_U/Team"><img height=344px width=505px src="https://static.igem.org/mediawiki/2009/2/2a/Teampicwashu.jpg"></a><br />
<td></html><br />
|<html><object width="425" height="344"><param name="movie" value="http://www.youtube.com/v/mbzObmQKJSo&hl=en&fs=1"></param><param name="allowFullScreen" value="true"></param><param name="allowscriptaccess" value="always"></param><embed src="http://www.youtube.com/v/mbzObmQKJSo&hl=en&fs=1" type="application/x-shockwave-flash" allowscriptaccess="always" allowfullscreen="true" width="425" height="344"></embed></object><br />
<td></html><br />
|}<br />
<br />
<font size="4"><br />
<div style="text-align: left;"><br />
<br />
== ''' Team''' == <br />
<font size="2"><br />
The first ever Washington University iGEM team is composed of nine undergraduate juniors and seniors majoring in Biology, Molecular Biology and Biochemistry, Biomedical Engineering and Chemical Engineering. Under the leadership of Dr. Blankenship (Biology and Chemistry departments), our team plans to synthetically regulate expression of a light harvesting antenna, which we believe is a first for both iGEM and synthetic biology. To learn more about our highly motivated and well-trained team, please click [https://2009.igem.org/Team:Wash_U/Team here].<br />
<font size="4"><br />
<br />
== '''Project Abstract''' ==<br />
<font size="2"><br />
<br />
'''Improved Photosynthetic Productivity for ''Rhodobacter sphaeroides'' via Synthetic Regulation of the Light Harvesting Antenna LH2'''<br><br />
Photosynthetic light harvesting antennas function to collect light and transfer energy to a reaction center for photochemistry. Phototrophs evolved large antennas to compete for photons in natural environments where light is scarce. Consequently, cells at the surface of photobioreactors over-absorb light, leading to attenuated photobioreactor light penetration and starving interior cells of photons. This reduction of photosynthetic productivity has been identified as the primary impediment to improving photobioreactor efficiency. While reduction of antenna size improves photosynthetic productivity, current approaches to this uniformly truncate antennas and are difficult to manipulate from the perspective of bioengineering. We aim to create a modifiable system to optimize antenna size throughout the bioreactor by utilizing a synthetic regulatory mechanism that correlates expression of the pucB/A LH2 antenna genes with incident light intensity. This new application of synthetic biology serves to transform the science of antenna reduction into the engineering of antenna optimization. To learn more about our project, please click [https://2009.igem.org/Team:Wash_U/Project here].<br />
<br />
<font size="4"><br />
<br />
=='''What Is iGEM?'''==<br />
<font size="2"><br />
The [https://2009.igem.org/Main_Page International Genetically Engineered Machine competition (iGEM)] is the premiere undergraduate Synthetic Biology competition. Student teams are given a kit of biological parts at the beginning of the summer from the [http://partsregistry.org/Main_Page Registry of Standard Biological Parts]. Working at their own schools over the summer, they use these parts and new parts of their own design to build biological systems and operate them in living cells.<br />
<br>The burgeoning field of [http://en.wikipedia.org/wiki/Synthetic_biology Synthetic Biology] is the culmination of the previous thirty years of research into recombinant DNA and biological engineering technology. It is fundamentally about the union of biology and engineering, thereby encouraging the collaboration of geneticists, molecular biologists, biochemists, and biomedical, chemical, and computer science engineers. Researchers in this field mainly seek to A) design and construct new biological parts, devices and systems or B) re-design existing, natural biological systems for useful purposes. <br />
<br />
<font size="4"><br />
<br />
== '''Contact''' == <br />
<font size="2"><br />
Please feel free to contact us with any questions or concerns at <html><a href="mailto:washu.igem@gmail.com">washu.igem@gmail.com</a></html><br />
<br>Or feel free to leave a comment on our [https://2009.igem.org/Team:Wash_U/Comments wall].<br />
<br />
<br />
{{WashUbottom}}<br />
<br />
<br />
<br />
<html><center><br />
<a href="http://www3.clustrmaps.com/counter/maps.php?url=https://2009.igem.org/Team:Wash_U" id="clustrMapsLink"><img src="http://www3.clustrmaps.com/counter/index2.php?url=https://2009.igem.org/Team:Wash_U" style="border:0px;" alt="Locations of visitors to this page" title="Locations of visitors to this page" id="clustrMapsImg" onerror="this.onerror=null; this.src='http://www2.clustrmaps.com/images/clustrmaps-back-soon.jpg'; document.getElementById('clustrMapsLink').href='http://www2.clustrmaps.com';" /><br />
</a><br />
</html><br />
<br><br></div>Brendan1http://2009.igem.org/File:Examplegold_medal.jpgFile:Examplegold medal.jpg2009-11-08T23:03:59Z<p>Brendan1: </p>
<hr />
<div></div>Brendan1http://2009.igem.org/Team:Wash_UTeam:Wash U2009-11-08T23:03:45Z<p>Brendan1: </p>
<hr />
<div>{{WashUback}}<br />
<br />
<div style="text-align: center;"><font size="4"><br />
Welcome to the 2009 Wash U team wiki!<br><br />
[[Image:Examplegold medal.jpg]]Gold medal team[[Image:Examplegold medal.jpg]]<br />
<div style="text-align: center;"><br />
{|style="background-color:#ffefd5; width:100%" <font size="6"><br />
|<br />
|<br />
|<br />
|<font size="5" style="color:#8B0000;">'''Our Team'''<br />
|<br />
|<br />
| <br />
|<font size="5" style="color:#8B0000;">'''Our Project'''<br />
|<br />
|<br />
|<br />
|}<br />
{|style="background-color:#000000;"<br />
|<html><a href="https://2009.igem.org/Team:Wash_U/Team"><img height=344px width=505px src="https://static.igem.org/mediawiki/2009/2/2a/Teampicwashu.jpg"></a><br />
<td></html><br />
|<html><object width="425" height="344"><param name="movie" value="http://www.youtube.com/v/mbzObmQKJSo&hl=en&fs=1"></param><param name="allowFullScreen" value="true"></param><param name="allowscriptaccess" value="always"></param><embed src="http://www.youtube.com/v/mbzObmQKJSo&hl=en&fs=1" type="application/x-shockwave-flash" allowscriptaccess="always" allowfullscreen="true" width="425" height="344"></embed></object><br />
<td></html><br />
|}<br />
<br />
<font size="4"><br />
<div style="text-align: left;"><br />
<br />
== ''' Team''' == <br />
<font size="2"><br />
The first ever Washington University iGEM team is composed of nine undergraduate juniors and seniors majoring in Biology, Molecular Biology and Biochemistry, Biomedical Engineering and Chemical Engineering. Under the leadership of Dr. Blankenship (Biology and Chemistry departments), our team plans to synthetically regulate expression of a light harvesting antenna, which we believe is a first for both iGEM and synthetic biology. To learn more about our highly motivated and well-trained team, please click [https://2009.igem.org/Team:Wash_U/Team here].<br />
<font size="4"><br />
<br />
== '''Project Abstract''' ==<br />
<font size="2"><br />
<br />
'''Improved Photosynthetic Productivity for ''Rhodobacter sphaeroides'' via Synthetic Regulation of the Light Harvesting Antenna LH2'''<br><br />
Photosynthetic light harvesting antennas function to collect light and transfer energy to a reaction center for photochemistry. Phototrophs evolved large antennas to compete for photons in natural environments where light is scarce. Consequently, cells at the surface of photobioreactors over-absorb light, leading to attenuated photobioreactor light penetration and starving interior cells of photons. This reduction of photosynthetic productivity has been identified as the primary impediment to improving photobioreactor efficiency. While reduction of antenna size improves photosynthetic productivity, current approaches to this uniformly truncate antennas and are difficult to manipulate from the perspective of bioengineering. We aim to create a modifiable system to optimize antenna size throughout the bioreactor by utilizing a synthetic regulatory mechanism that correlates expression of the pucB/A LH2 antenna genes with incident light intensity. This new application of synthetic biology serves to transform the science of antenna reduction into the engineering of antenna optimization. To learn more about our project, please click [https://2009.igem.org/Team:Wash_U/Project here].<br />
<br />
<font size="4"><br />
<br />
=='''What Is iGEM?'''==<br />
<font size="2"><br />
The [https://2009.igem.org/Main_Page International Genetically Engineered Machine competition (iGEM)] is the premiere undergraduate Synthetic Biology competition. Student teams are given a kit of biological parts at the beginning of the summer from the [http://partsregistry.org/Main_Page Registry of Standard Biological Parts]. Working at their own schools over the summer, they use these parts and new parts of their own design to build biological systems and operate them in living cells.<br />
<br>The burgeoning field of [http://en.wikipedia.org/wiki/Synthetic_biology Synthetic Biology] is the culmination of the previous thirty years of research into recombinant DNA and biological engineering technology. It is fundamentally about the union of biology and engineering, thereby encouraging the collaboration of geneticists, molecular biologists, biochemists, and biomedical, chemical, and computer science engineers. Researchers in this field mainly seek to A) design and construct new biological parts, devices and systems or B) re-design existing, natural biological systems for useful purposes. <br />
<br />
<font size="4"><br />
<br />
== '''Contact''' == <br />
<font size="2"><br />
Please feel free to contact us with any questions or concerns at <html><a href="mailto:washu.igem@gmail.com">washu.igem@gmail.com</a></html><br />
<br>Or feel free to leave a comment on our [https://2009.igem.org/Team:Wash_U/Comments wall].<br />
<br />
<br />
{{WashUbottom}}<br />
<br />
<br />
<br />
<html><center><br />
<a href="http://www3.clustrmaps.com/counter/maps.php?url=https://2009.igem.org/Team:Wash_U" id="clustrMapsLink"><img src="http://www3.clustrmaps.com/counter/index2.php?url=https://2009.igem.org/Team:Wash_U" style="border:0px;" alt="Locations of visitors to this page" title="Locations of visitors to this page" id="clustrMapsImg" onerror="this.onerror=null; this.src='http://www2.clustrmaps.com/images/clustrmaps-back-soon.jpg'; document.getElementById('clustrMapsLink').href='http://www2.clustrmaps.com';" /><br />
</a><br />
</html><br />
<br><br></div>Brendan1http://2009.igem.org/Team:Wash_UTeam:Wash U2009-11-08T23:03:20Z<p>Brendan1: </p>
<hr />
<div>{{WashUback}}<br />
<br />
<div style="text-align: center;"><font size="4"><br />
Welcome to the 2009 Wash U team wiki!<br />
[[Image:Examplegold medal.jpg]]Gold medal team[[Image:Examplegold medal.jpg]]<br />
<div style="text-align: center;"><br />
{|style="background-color:#ffefd5; width:100%" <font size="6"><br />
|<br />
|<br />
|<br />
|<font size="5" style="color:#8B0000;">'''Our Team'''<br />
|<br />
|<br />
| <br />
|<font size="5" style="color:#8B0000;">'''Our Project'''<br />
|<br />
|<br />
|<br />
|}<br />
{|style="background-color:#000000;"<br />
|<html><a href="https://2009.igem.org/Team:Wash_U/Team"><img height=344px width=505px src="https://static.igem.org/mediawiki/2009/2/2a/Teampicwashu.jpg"></a><br />
<td></html><br />
|<html><object width="425" height="344"><param name="movie" value="http://www.youtube.com/v/mbzObmQKJSo&hl=en&fs=1"></param><param name="allowFullScreen" value="true"></param><param name="allowscriptaccess" value="always"></param><embed src="http://www.youtube.com/v/mbzObmQKJSo&hl=en&fs=1" type="application/x-shockwave-flash" allowscriptaccess="always" allowfullscreen="true" width="425" height="344"></embed></object><br />
<td></html><br />
|}<br />
<br />
<font size="4"><br />
<div style="text-align: left;"><br />
<br />
== ''' Team''' == <br />
<font size="2"><br />
The first ever Washington University iGEM team is composed of nine undergraduate juniors and seniors majoring in Biology, Molecular Biology and Biochemistry, Biomedical Engineering and Chemical Engineering. Under the leadership of Dr. Blankenship (Biology and Chemistry departments), our team plans to synthetically regulate expression of a light harvesting antenna, which we believe is a first for both iGEM and synthetic biology. To learn more about our highly motivated and well-trained team, please click [https://2009.igem.org/Team:Wash_U/Team here].<br />
<font size="4"><br />
<br />
== '''Project Abstract''' ==<br />
<font size="2"><br />
<br />
'''Improved Photosynthetic Productivity for ''Rhodobacter sphaeroides'' via Synthetic Regulation of the Light Harvesting Antenna LH2'''<br><br />
Photosynthetic light harvesting antennas function to collect light and transfer energy to a reaction center for photochemistry. Phototrophs evolved large antennas to compete for photons in natural environments where light is scarce. Consequently, cells at the surface of photobioreactors over-absorb light, leading to attenuated photobioreactor light penetration and starving interior cells of photons. This reduction of photosynthetic productivity has been identified as the primary impediment to improving photobioreactor efficiency. While reduction of antenna size improves photosynthetic productivity, current approaches to this uniformly truncate antennas and are difficult to manipulate from the perspective of bioengineering. We aim to create a modifiable system to optimize antenna size throughout the bioreactor by utilizing a synthetic regulatory mechanism that correlates expression of the pucB/A LH2 antenna genes with incident light intensity. This new application of synthetic biology serves to transform the science of antenna reduction into the engineering of antenna optimization. To learn more about our project, please click [https://2009.igem.org/Team:Wash_U/Project here].<br />
<br />
<font size="4"><br />
<br />
=='''What Is iGEM?'''==<br />
<font size="2"><br />
The [https://2009.igem.org/Main_Page International Genetically Engineered Machine competition (iGEM)] is the premiere undergraduate Synthetic Biology competition. Student teams are given a kit of biological parts at the beginning of the summer from the [http://partsregistry.org/Main_Page Registry of Standard Biological Parts]. Working at their own schools over the summer, they use these parts and new parts of their own design to build biological systems and operate them in living cells.<br />
<br>The burgeoning field of [http://en.wikipedia.org/wiki/Synthetic_biology Synthetic Biology] is the culmination of the previous thirty years of research into recombinant DNA and biological engineering technology. It is fundamentally about the union of biology and engineering, thereby encouraging the collaboration of geneticists, molecular biologists, biochemists, and biomedical, chemical, and computer science engineers. Researchers in this field mainly seek to A) design and construct new biological parts, devices and systems or B) re-design existing, natural biological systems for useful purposes. <br />
<br />
<font size="4"><br />
<br />
== '''Contact''' == <br />
<font size="2"><br />
Please feel free to contact us with any questions or concerns at <html><a href="mailto:washu.igem@gmail.com">washu.igem@gmail.com</a></html><br />
<br>Or feel free to leave a comment on our [https://2009.igem.org/Team:Wash_U/Comments wall].<br />
<br />
<br />
{{WashUbottom}}<br />
<br />
<br />
<br />
<html><center><br />
<a href="http://www3.clustrmaps.com/counter/maps.php?url=https://2009.igem.org/Team:Wash_U" id="clustrMapsLink"><img src="http://www3.clustrmaps.com/counter/index2.php?url=https://2009.igem.org/Team:Wash_U" style="border:0px;" alt="Locations of visitors to this page" title="Locations of visitors to this page" id="clustrMapsImg" onerror="this.onerror=null; this.src='http://www2.clustrmaps.com/images/clustrmaps-back-soon.jpg'; document.getElementById('clustrMapsLink').href='http://www2.clustrmaps.com';" /><br />
</a><br />
</html><br />
<br><br></div>Brendan1http://2009.igem.org/Team:Wash_UTeam:Wash U2009-11-08T22:35:13Z<p>Brendan1: </p>
<hr />
<div>{{WashUback}}<br />
<br />
<div style="text-align: center;"><font size="4"><br />
Welcome to the 2009 Wash U team wiki<br />
<div style="text-align: center;"><br />
{|style="background-color:#ffefd5; width:100%" <font size="6"><br />
|<br />
|<br />
|<br />
|<font size="5" style="color:#8B0000;">'''Our Team'''<br />
|<br />
|<br />
| <br />
|<font size="5" style="color:#8B0000;">'''Our Project'''<br />
|<br />
|<br />
|<br />
|}<br />
{|style="background-color:#000000;"<br />
|<html><a href="https://2009.igem.org/Team:Wash_U/Team"><img height=344px width=505px src="https://static.igem.org/mediawiki/2009/2/2a/Teampicwashu.jpg"></a><br />
<td></html><br />
|<html><object width="425" height="344"><param name="movie" value="http://www.youtube.com/v/mbzObmQKJSo&hl=en&fs=1"></param><param name="allowFullScreen" value="true"></param><param name="allowscriptaccess" value="always"></param><embed src="http://www.youtube.com/v/mbzObmQKJSo&hl=en&fs=1" type="application/x-shockwave-flash" allowscriptaccess="always" allowfullscreen="true" width="425" height="344"></embed></object><br />
<td></html><br />
|}<br />
<br />
<font size="4"><br />
<div style="text-align: left;"><br />
<br />
== ''' Team''' == <br />
<font size="2"><br />
The first ever Washington University iGEM team is composed of nine undergraduate juniors and seniors majoring in Biology, Molecular Biology and Biochemistry, Biomedical Engineering and Chemical Engineering. Under the leadership of Dr. Blankenship (Biology and Chemistry departments), our team plans to synthetically regulate expression of a light harvesting antenna, which we believe is a first for both iGEM and synthetic biology. To learn more about our highly motivated and well-trained team, please click [https://2009.igem.org/Team:Wash_U/Team here].<br />
<font size="4"><br />
<br />
== '''Project Abstract''' ==<br />
<font size="2"><br />
<br />
'''Improved Photosynthetic Productivity for ''Rhodobacter sphaeroides'' via Synthetic Regulation of the Light Harvesting Antenna LH2'''<br><br />
Photosynthetic light harvesting antennas function to collect light and transfer energy to a reaction center for photochemistry. Phototrophs evolved large antennas to compete for photons in natural environments where light is scarce. Consequently, cells at the surface of photobioreactors over-absorb light, leading to attenuated photobioreactor light penetration and starving interior cells of photons. This reduction of photosynthetic productivity has been identified as the primary impediment to improving photobioreactor efficiency. While reduction of antenna size improves photosynthetic productivity, current approaches to this uniformly truncate antennas and are difficult to manipulate from the perspective of bioengineering. We aim to create a modifiable system to optimize antenna size throughout the bioreactor by utilizing a synthetic regulatory mechanism that correlates expression of the pucB/A LH2 antenna genes with incident light intensity. This new application of synthetic biology serves to transform the science of antenna reduction into the engineering of antenna optimization. To learn more about our project, please click [https://2009.igem.org/Team:Wash_U/Project here].<br />
<br />
<font size="4"><br />
<br />
=='''What Is iGEM?'''==<br />
<font size="2"><br />
The [https://2009.igem.org/Main_Page International Genetically Engineered Machine competition (iGEM)] is the premiere undergraduate Synthetic Biology competition. Student teams are given a kit of biological parts at the beginning of the summer from the [http://partsregistry.org/Main_Page Registry of Standard Biological Parts]. Working at their own schools over the summer, they use these parts and new parts of their own design to build biological systems and operate them in living cells.<br />
<br>The burgeoning field of [http://en.wikipedia.org/wiki/Synthetic_biology Synthetic Biology] is the culmination of the previous thirty years of research into recombinant DNA and biological engineering technology. It is fundamentally about the union of biology and engineering, thereby encouraging the collaboration of geneticists, molecular biologists, biochemists, and biomedical, chemical, and computer science engineers. Researchers in this field mainly seek to A) design and construct new biological parts, devices and systems or B) re-design existing, natural biological systems for useful purposes. <br />
<br />
<font size="4"><br />
<br />
== '''Contact''' == <br />
<font size="2"><br />
Please feel free to contact us with any questions or concerns at <html><a href="mailto:washu.igem@gmail.com">washu.igem@gmail.com</a></html><br />
<br>Or feel free to leave a comment on our [https://2009.igem.org/Team:Wash_U/Comments wall].<br />
<br />
<br />
{{WashUbottom}}<br />
<br />
<br />
<br />
<html><center><br />
<a href="http://www3.clustrmaps.com/counter/maps.php?url=https://2009.igem.org/Team:Wash_U" id="clustrMapsLink"><img src="http://www3.clustrmaps.com/counter/index2.php?url=https://2009.igem.org/Team:Wash_U" style="border:0px;" alt="Locations of visitors to this page" title="Locations of visitors to this page" id="clustrMapsImg" onerror="this.onerror=null; this.src='http://www2.clustrmaps.com/images/clustrmaps-back-soon.jpg'; document.getElementById('clustrMapsLink').href='http://www2.clustrmaps.com';" /><br />
</a><br />
</html><br />
<br><br></div>Brendan1http://2009.igem.org/Team:Wash_UTeam:Wash U2009-11-08T22:33:52Z<p>Brendan1: </p>
<hr />
<div>{{WashUback}}<br />
<br />
<div style="text-align: center;"><br />
Welcome to the Wash U 2009 Gold Medal iGEM team wiki<br />
<div style="text-align: center;"><br />
{|style="background-color:#ffefd5; width:100%" <font size="6"><br />
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|<font size="5" style="color:#8B0000;">'''Our Team'''<br />
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|<font size="5" style="color:#8B0000;">'''Our Project'''<br />
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== ''' Team''' == <br />
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The first ever Washington University iGEM team is composed of nine undergraduate juniors and seniors majoring in Biology, Molecular Biology and Biochemistry, Biomedical Engineering and Chemical Engineering. Under the leadership of Dr. Blankenship (Biology and Chemistry departments), our team plans to synthetically regulate expression of a light harvesting antenna, which we believe is a first for both iGEM and synthetic biology. To learn more about our highly motivated and well-trained team, please click [https://2009.igem.org/Team:Wash_U/Team here].<br />
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== '''Project Abstract''' ==<br />
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'''Improved Photosynthetic Productivity for ''Rhodobacter sphaeroides'' via Synthetic Regulation of the Light Harvesting Antenna LH2'''<br><br />
Photosynthetic light harvesting antennas function to collect light and transfer energy to a reaction center for photochemistry. Phototrophs evolved large antennas to compete for photons in natural environments where light is scarce. Consequently, cells at the surface of photobioreactors over-absorb light, leading to attenuated photobioreactor light penetration and starving interior cells of photons. This reduction of photosynthetic productivity has been identified as the primary impediment to improving photobioreactor efficiency. While reduction of antenna size improves photosynthetic productivity, current approaches to this uniformly truncate antennas and are difficult to manipulate from the perspective of bioengineering. We aim to create a modifiable system to optimize antenna size throughout the bioreactor by utilizing a synthetic regulatory mechanism that correlates expression of the pucB/A LH2 antenna genes with incident light intensity. This new application of synthetic biology serves to transform the science of antenna reduction into the engineering of antenna optimization. To learn more about our project, please click [https://2009.igem.org/Team:Wash_U/Project here].<br />
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=='''What Is iGEM?'''==<br />
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The [https://2009.igem.org/Main_Page International Genetically Engineered Machine competition (iGEM)] is the premiere undergraduate Synthetic Biology competition. Student teams are given a kit of biological parts at the beginning of the summer from the [http://partsregistry.org/Main_Page Registry of Standard Biological Parts]. Working at their own schools over the summer, they use these parts and new parts of their own design to build biological systems and operate them in living cells.<br />
<br>The burgeoning field of [http://en.wikipedia.org/wiki/Synthetic_biology Synthetic Biology] is the culmination of the previous thirty years of research into recombinant DNA and biological engineering technology. It is fundamentally about the union of biology and engineering, thereby encouraging the collaboration of geneticists, molecular biologists, biochemists, and biomedical, chemical, and computer science engineers. Researchers in this field mainly seek to A) design and construct new biological parts, devices and systems or B) re-design existing, natural biological systems for useful purposes. <br />
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== '''Contact''' == <br />
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Please feel free to contact us with any questions or concerns at <html><a href="mailto:washu.igem@gmail.com">washu.igem@gmail.com</a></html><br />
<br>Or feel free to leave a comment on our [https://2009.igem.org/Team:Wash_U/Comments wall].<br />
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<br><br></div>Brendan1http://2009.igem.org/Team:Wash_U/ProjectTeam:Wash U/Project2009-10-22T03:32:56Z<p>Brendan1: /* Conclusion */</p>
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== '''Introduction''' == <br />
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Our project goal is to maximize the photosynthetic productivity for a series of photobioreactors containing ''Rhodobacter sphaeroides'' under both high and low light intensities by synthetically regulating the size of the light-harvesting antenna complex LH2. We chose to undertake such a project in ''R. sphaeroides'' due to its well-characterized photosynthetic and genetic system. <br />
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The antenna system functions to expand the spectrum of light available for photosynthesis by absorbing different wavelengths than that of the reaction center. Essentially, it is like the dish around the main receiver of any antenna. Marine bacteria, such as ''R. sphaeroides'', evolved very large antenna complexes to absorb light in a natural environment where there is great competition for photons. As a result, the photosynthetic machinery is saturated at low light intensities in a synthetic non-competitive environment, such as a photobioreactor. This causes up to 95% of incidental photons to be dissipated as heat or fluorescence by bacteria through a process called Non-Photochemical Quenching (NPQ) (Mussgnug et al., 2007). In essence, these photons are being wasted as NPQ reduces light penetration into and across bioreactors and starves the shaded cells for for photons. One method that has been shown to improve photosynthetic productivity in photobioreactors is the reduction of light-harvesting antenna sizes (Polle et al., 2002, Mussgnug et al., 2007). Though, current approaches to this end are difficult to precisely control from the perspective of biological engineering and synthetic biology. <br />
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Our intention is to create a dynamic system to vary antenna size that is dependent on incident light intensity and that can be readily optimized using bioengineering principles. This synthetic regulation of the LH2 complex should result in the bacteria grown under high light intensities expressing fewer LH2 complexes than the cells that are more shaded, leading to a reduction of wasted incident photons while maintaining a high overall absorbance across subsequent bioreactors. Consequently, we expect to see an increase in the total photosynthetic productivity for our mutant ''Rhodobacter sphaeroides'' when compared to the wild type grown under similar conditions. <br />
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[[Image:Slide09b.jpg| 450px | left]][[Image:Slide10 a.jpg| 450px | right]]<br />
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[https://2009.igem.org/Team:Wash_U/Project Back To Top]<br />
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== '''Organism''' ==<br />
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''Rhodobacter sphaeroides'' is a purple Alphaproteobacteria. It is flexible metabolically and can grow heterotrophically via aerobic and anaerobic respiration, as well as phototrophically under anaerobic conditions with light. <br />
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''R. sphaeroides'' is one of the best understood photosynthetic organisms. The photosystem is located in intracytoplasmic membrane invaginations and the main components are the Light Haresting Complex 2 (LH2), Light Harvesting Complex 1 (LH1), and the Reaction Center (RC). These pigment-protein complexes non-covalently bind bacteriochlorophylls and carotenoids. LH1 and RC make up the core complex and are often found in the ratio 1:1. LH2 is more peripheral and naturally ranges in the ratio to LH1/RC of 3.0:1 to 6.7:1 under varying light conditions (Scheuring et al., 2005). LH2 absorbs photons maximally at the wavelengths of 850 and 800 nm and funnels its energy to LH1 and the reaction center for photochemistry. (Scheuring et al., 2005). <br />
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The two subunits of LH2 are coded for by the pucB/A genes and are naturally promoted by the puc promoter. <br />
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<br><div style="text-align: center;">'''ICM of ''R. sphaeroides'' '''[[Image:R sphaeroides photosystem.png| 500 px | center]]<center>source:Sener et al.2007<br />
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|<font size="2" style="color:black;">'''Plate of ''R. sphaeroides'' 2.4.1'''<br />
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[[Image:R sphaeroides aborbance.png| 450px | left]][[Image:R sphaeroides 0986.png| 450px | right]]<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br>source: Walz et al. 1998<br><br />
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====Online Resources for ''R. sphaeroides''====<br />
[http://www.kazusa.or.jp/codon/cgi-bin/showcodon.cgi?species=272943 Codon Usage Table for ''R. sphaeroides'' 2.41]<br />
<br> [http://www.ncbi.nlm.nih.gov/sites/entrez?db=genome&cmd=Retrieve&dopt=Overview&list_uids=18843 ''R. sphaeroides'' 2.41 NCBI Genome Tools]<br />
<br> [http://archaea.ucsc.edu/cgi-bin/hgPcr?wp_target=&db=0&org=Rhodobacter+sphaeroides+2+4+1&wp_f=&wp_r=&wp_size=2000&wp_perfect=15&wp_good=15&wp_showPage=true&hgsid=159191 ''In silico'' PCR of ''R. sphaeroides'' 2.41 genome]<br />
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[https://2009.igem.org/Team:Wash_U/Project Back To Top]<br />
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=='''Regulation'''==<br />
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[[Image:pucregulation-fullpanels.png|950px]]<br />
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'''The Natural System (Left)''' Under high oxygen tension, PpsR prevents transcription from the puc promoter of the pucBA genes, and thus prevents the expression of LH2(1st panel). Under low oxygen tension, this repression is removed by activated AppA, thus allowing expression of LH2 from pucBA(2nd panel).<br><br />
'''Synthetic Regulation System (Right)''' Our system takes advantage of constitutive protein expression from the puc promoter at low oxygen tension, and replaces pucBA with Cph8 at this site. Placing pucBA expression under the control of the ompC promoter allows for Cph8/OmpR control of LH2 expression under varying light conditions. Under high intensity of incident light, Cph8 will not be autophosphorylated, preventing expression of pucBA (3rd panel). When light intensity is lower, Cph8 will autophosphorylate, thus phosphorylating and activating OmpR and promoting expression of LH2 from the ompC promoter.<br><br />
This regulation system was designed such that light is able to penetrate deeper into and through photobioreactors where light is plentiful.<br />
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<br>[https://2009.igem.org/Team:Wash_U/Project Back To Top]<br />
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== '''Results''' ==<br />
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For methods, materials, and procedures regarding these results, please see [https://2009.igem.org/Team:Wash_U/Protocol#Tissue_Flask_Experiment Tissue Flask Experiment] or [https://2009.igem.org/Team:Wash_U/Biological_Parts#Modeling Modeling]<br />
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'''Figure 1'''<br />
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|<font size="2" style="color:#black;">'''a''' Tissue Flask Position 1 Optical Density of Wild Type and DCBomega<br />
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|<font size="2" style="color:#black;">'''b''' Tissue Flask Position 2 Optical Density of Wild Type and DCBomega<br />
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[[Image:Tissue Flask 1b.png| 450 px| left]][[Image:Tissue Flask 2b.png| 450px | right]]<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:#black;">'''c''' Tissue Flask Position 3 Optical Density of Wild Type and DCBomega<br />
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|<font size="2" style="color:#black;">'''d''' Tissue Flask Position 4 Optical Density of Wild Type and DCBomega<br />
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[[Image:Tissue Flask 3b.png| 450px | left]][[Image:Tissue Flask 4b.png| 450px | right]]<br />
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'''e''' Tissue Flask Position 5 Optical Density of Wild Type and DBComega<br />
[[Image:Tissue Flask 5b.png| 450px | center]]<br><br />
Figure 1a,b,c,d,e are the optical density of a tissue flask, as assessed at 600 nm, over the course of the 6 days of the experiment.<br />
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'''Figure 2'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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[[Image:3D 241.png| 450 px | left]][[Image:3D DBCOmega2.png| 450 px | right]]<br />
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Figure 2a and 2b display the growth of the tissue flasks as measured by OD assessed as absorbance at 600 nm. The graph is organized such that those closest to the "days" axis were closest to the light source (i.e. flask 1 is closer than flask 2).<br />
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'''Figure 3'''<br />
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[[Image:Bar 241 b.png| 450px | left]][[Image:Bar DBCOmega b.png| 450px | right]]<br />
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Figure 3a and 3b display the cumulative growth of the tissue flasks as measured by the sum of the optical density of the respective cell type's tissue flasks at a given day. Furthermore, the contribution of a given tissue flask to cumulative growth is displayed.<br><br />
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'''Figure 4'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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[[image:sphaeroides tissue flask.jpg|450 px|left|]][[image:dbc tissue flask.jpg|450px|right]]<br />
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Figure 4A and 4B display the appearance of the tissue flasks for both strains on day 6. They are arranged from left to right with left being closed to the light source (flask 1) and right furthest (flask 5)<br />
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'''Figure 5'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:#black;">'''a''' DBComega Spectrum by Flask Distance from Source<br />
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|<font size="2" style="color:#black;">'''b''' R. sphaeroides Spectrum by Flask Distance from Source<br />
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[[Image: WT irradiance 3d.png| 450 px | left]][[Image: DBComega irradiance 3d.png| 450 px | right]]<br />
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Contour of Absolute Irradiance by Flash distance from Source<br />
{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:#black;">'''c''' R. Sphaeroides 2.4.1<br />
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[[Image: r sphaeroides day 2 a.jpg| 450px | left]][[Image: dbc day 4b.jpg| 450px | right]]<br />
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'''e''' Wild Type Absolute Irradiance Behind Flask 1: Day 6<br />
[[Image: irradiance WT flask 1 day6.png| 450px | center]]<br />
Caption for 5e<br><br />
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'''Figure 6'''<br><br />
Nonlinear least-squares estimation of WT LH2 saturation curve<br><br />
[[Image: saturation for WT as inferred.png| 450 px]]<br />
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Caption for figure 6<br><br><br />
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'''Figure 7'''<br><br />
Simulated Optical Density of Mutant and Wild Type Bioreactors<br><br />
Layers One and Two . Layers Three, Four and Five<br />
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[[Image: OD after flask 1 and 2.jpg| 200px ]][[Image: OD after 3,4, and 5.jpg| 200px ]]<br><br />
Caption for Figure 7<br />
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[https://2009.igem.org/Team:Wash_U/Project Back To Top]<br />
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== '''Analysis''' ==<br />
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<br />
Though we were not able to have our mutant functionally express our system, the tissue flask experiment was an interesting way to observe the effects of a full knockout of the Light Harvesting Antenna Complex 2 (LH2) vs the Wild Type on an array of consecutive photobioreactors. A couple of conclusions can be drawn from this data that can be used in modeling our synthetic regulation system for LH2 and in the future design of systems for the optimization of Light Harvesting Antenna size for photobioreactors . <br><br><br />
The most striking difference between the Wild Type Rhodobacter Sphaeroides 2.41 and DBCOmega (LH2 knockout) growth patterns is that the first flask in the WT (closest to the light source) grew less than the second flask, while the converse was true in DBCOmega (figure 2a and 2b). This can be attributed to the photosystem saturation curves for the respective cultures. The Wild Type has an LH2 complex, meaning that their antenna size is inherently larger and that their photosystem will be saturated at lower light intensities than the LH2 deficient mutant. We observed that the incident light intensity used in this experiment led to oversaturation for the first WT tissue flask and resulted in growth-inhibiting photodamage, as is evidenced by its lesser growth relative to the second culture. In contrast, photodamage was not observed in DBCOmega as is evidenced by the first tissue flask that grew at the fastest respective rate. Furthermore, it appears that this photodamage slowed the growth of the first WT tissue flask to the point that the OD of this tissue flask was nearly equal to that of DBCOmega after 5 days of growth(.987 vs. .967) (figure 1a). As such, the contribution of LH2 to the growth of the WT in flask 1 can be assessed to be little after day 3 when the growth curve slows- though, this light (at 800 and 850 nm) was still greatly absorbed by flask 1 (Figure 5e). It is likely that much of this light was then wasted through Non-Photochemical Quenching (NPQ), depleting the flasks behind flask 1 of photons at the LH2 absorbtion peaks and decreasing the cumulative productivity of flasks 1-5. This effect of wasting photons though NPQ under high light intensities is one that we sought to minimize in the design of our synthetic regulation for the pucB/A genes. <br><br><br />
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A further a comparison of the WT and DBCOmega experiments reveals that the 3rd tissue flask in each had almost identical growth rates (figure 1c), while the DBCOmega 4th and 5th tissue flask outperformed that of the WT (figure 1d and 1e). This can simply be attributed to the higher density of cells in the 2nd flask of the WT that led to an overall lesser quantity of photons passing through to the 4th and 5th flasks in the WT experiment and likely resulting in heterotrophic growth (though this will need to be confirmed with a "dark control" experiment that should be completed in time for the Jamboree). This decrease in available photons for the WT can be seen by viewing the absolute irradiance data (incident light intensity at a given wavelength) after the third culture for the WT (figure 5a and 5c). This decrease in irradiance is the most pronounced for the LH2 absorption bands at 800 and 850 nm, as was expected. Our synthetic regulation system for pucB/A was designed with the intention of allowing greater penetration of light through the first couple of flasks at the LH2 wavelengths, as expression of pucB/A is inversely correlated to incident light intensity.<br><br><br />
'''Thomas, add in some further comments about the sprectroradiometer stuff. Specifically about how it changes in the WT vs. that of DBCOmega using some specific examples on the graphs'''<br><br><br />
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Overall, the cumulative culture growth of the WT exceeded that of DBCOmega (figure 3) due to the performance of the second tissue flask of the WT relative to that of DBCOmega (figure 1b) despite the photodamage that occurred in tissue flask 1 for the WT. This cumulative culture growth can be considered photosynthetic productivty and is proportional to the amount of energy/desired product that can be obtained from these cultures. <br><br><br />
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It is an interesting exercise to model how our mutant system would perform under these same conditions. To do so, the LH2 saturation curve for the WT (figure 6) and the relative contribution of LH1/LH2 light absorbance to growth was derived from our empirical data and applied to a model that took into account incident light and gave the relative growth in terms of optical density as an output. In this model, our synthetic regulation system of pucB/A allowed our mutant to outperform the wild type species (figure 7). This was due to the performance of the 3rd, 4th, and 5th tissue flask relative to those of the wild type (see figure 7 layers 1,2 vs 1-5). The reason for this is that the wild type LH2 light saturation curve does not vary with changes in incident light intensity as the wild type expresses pucB/A at a uniform level under low oxygen conditions. As a result, the first two layers of Wild Type bioreactors absorb nearly all the available light and starve the bioreactors behind them for photons. <br><br><br />
Conversely, our mutant was modeled to have a dynamic expression of pucB/A based on the incident light intensity. This light response curve was modeled as an inverse exponential for the Cph8/OmpR ompC Promoter system and the saturation curve changes for each tissue flask in accordance with the incident light intensity- as light intensity decreases, the expression pucB/A increases. This results in more light passing through the first 2 flasks and facilitating greater growth in flasks 3, 4 and 5 as they express large antenna complexes. <br />
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='''Conclusion'''=<br />
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Based on model, should have had feedback mechanism- should be something that sets a minimum expression level for LH2, that is...<br><br />
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-tissue flask w prkcbc3 before jamboree and compare/interpret using characterization data<br><br />
-tissue flask w. full mutant in future- if this doesn't reveal cph8 to responding in an exponentially decaying fashion to light intensity, try w. a system that would<br />
-characterize puc promoter under light conditions and additional oxygen tensions<br />
-complete other characterization work<br />
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[https://2009.igem.org/Team:Wash_U/Project Back To Top]<br />
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== '''References''' ==<br />
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# "2007-2008 Catalog & Technical Reference." <u>New England BioLabs Inc.</u> (2007).<br />
# Alon, Uri. <u>Introduction to systems biology and the design principles of biological networks</u>. Boca Raton, FL: Chapman & Hall, 2006.<br />
# Ausubel, Fred et al. <u>Short Protocols in Molecular Biology: Third Edition.</u> Canada: John Wiley and Sons, Inc., 1999.<br />
# "BioBrick Assembly Help." <u>Registry of Standard Biological Parts.</u> 2009. 6 July 2009. <http://partsregistry.org/Main_Page>.<br />
# Bower, James M. <u>Computational Modeling of Genetic and Biochemical Networks (Computational Molecular Biology).</u> New York: M.I.T. PRESS, 2001.<br />
# Braatsch, Stephan et al. "A single Flavoprotein AppA, Integrates Both Redox and Light Signals in ''Rhodobacter sphaeroides.''" <u>Molecular Microbiology.</u> Vol. 45.3 (2002): 827-836.<br />
# Brown, Stanley B. et al. "Bile Pigment Synthesis in Plants: Incorporation of Haem into Phycocyanobilin and Phycocyanobiliproteins in Cyanidium Caldarium." <u>Biochemistry.</u> Vol. 194 (1981): 137-147.<br />
# Dragnea, Vladimira et al. "Time-Resolved Spectroscopic Studies of the AppA Blue-Light Receptor BLUF Domain from ''Rhodobacter sphaeroides'' <u>Biochemistry.</u> Vol. 44 (2005): 15,978-15,985.<br />
# "Entrez Nucleotide Search." <u>National Center for Biotechnology Information.</u> 2009. 6 June 2009 <http://www.ncbi.nlm.nih.gov/nuccore/49175990?from=3533887&to=3534606&report=gbwithparts>.<br />
# Fowler, Gregory J. S. and C. Neil Hunter. "The Synthesis and Assembly of Functional High and Low Light LH2 Antenna Complexes from ''Rhodopseudomonas palustris'' in ''Rhodobacter sphaeroides.''" <u>The Journal of Biological Chemistry.</u> Vol. 271.23 (1996): 13,356-13,361.<br />
# Gabrielsen, Mads et al. "Peripheral Complexes of Purple Bacteria." <u>The Purple Phototropic Bacteria.</u> (2009): 135-153.<br />
# "GenElute HP Plasmid Miniprep Kit: User Guide." <u>Sigma-Aldrich</u>. (2008).<br />
# Harwood, Caroline S. "Nitrogenase-Catalyzed Hydrogen Production by Purple Nonsulfur Photosynthetic Bacteria." <u>Bioenergy.</u> (2008): 259-271.<br />
# Jager, Andreas et al. "The AppA and PpsR Proteins from ''Rhodobacter sphaeroides'' Can Establish a Redox-Dependent Signal Chain but Fail to Transmit Blue-Light Signals in Other Bacteria." <u>Journal of Bacteriology.</u> Vol. 189.6 (2007): 2,274-2,282.<br />
# Jones, M. R. et al. "Mutants of ''Rhodobacter sphaeroides'' Lacking One or More Pigment-Protein Complexes and Complementation with Reaction-Centre, LH1, and LH2 Genes." <u>Molecular Microbiology.</u> Vol. 6.9 (1992): 1,173-1,184.<br />
# Kondo, Toshihiko et al. "Enhancement of Hydrogen Production by a Photosynthetic Bacterium Mutant with Reduced Pigment." <u>Journal of Bioscience and Bioengineering.</u> Vol. 93.2 (2002): 145-150.<br />
# Lagarias, J. Clark. <u>PCB from Spirulina</u>. Personal Communication. June 2009.<br />
# Laible, Philip D. "Foreign Gene Expression in Photosynthetic Bacteria." <u>The Purple Phototropic Bacteria.</u> (2009): 839-860.<br />
# Lee, Jeong K. and Samuel Kaplan. "Transcriptional Regulation of ''puc'' Operon Expression in ''Rhodobacter sphaeroides.''" <u>Journal of Biological Chemistry.</u> Vol. 270.35 (1995): 20,453-20,458.<br />
# Levskaya, Anselmetal et al. "Engineering ''Escherichia coli'' to See Light." <u>Nature.</u> Vol. 438 (2005): 441-442.<br />
# "Life Sciences Catalog 2009-2010." <u>National Diagnostics.</u> (2009).<br />
# Melis, Anastasios. "Solar Energy Conversion Efficiencies in Photosynthesis: Minimizing the Chlorophyll Antennae to Maximize Efficiency." <u>Plant Science.</u> (2009).<br />
# Mizuno, Takeshi and Shoji Mizushima. "Characterization by Deletion and Localized Mutagenesis in Vitro of the Promoter Region of the ''Escherichia coli'' OmpC Gene and Importance of the Upstream DNA Domain in Positive Regulation by the OmpR Protein." <u>Journal of Bacteriology.</u> Vol. 168.1 (1986): 86-95.<br />
# Moskvin, Oleg V. et al. "Transcriptome Analysis of the ''Rhodobacter sphaeroides'' PspR Regulon: PspR as a Master Regulator of Photosystem Development." <u>Journal of Bacteriology.</u> Vol. 187.6 (2005): 2,148-2,156.<br />
# Mussgnug, Jan H. et al. "Engineering photosynthetic light capture: impacts on improved solar energy to biomass conversion." <u> Plant Biotechnology Journal</u> Vol. 5 Issue 6. (2007) pp. 802-814<br />
# "NEBCutter V2.0." <u>New England BioLabs.</u> 2009. 6 June 2009 <http://tools.neb.com/NEBcutter2/index.php>.<br />
# "OligoAnalyzer 3.1." <u>Integrated DNA Technologies.</u> 2009. 6 June 2009. <http://www.idtdna.com/analyzer/Applications/OligoAnalyzer/><br />
# Sambrook, Joseph and David W. Russell. <u>Molecular Cloning: A Laboratory Manual.</u> Vol. 1-3. New York: Cold Springs Harbor Laboratory Press, 2001.<br />
# Scheuring, Simon and Sturgis, James N. "Chromatic Adaptation of Photosynthetic Membranes" <u>Science</u> Vol. 309 (2005) pp.484-487<br />
# Sener, Melih K. et al. "Atomic-level structural and functional model of a bacterial photosynthetic membrane vesicle" <u>Proceedings of the National Academy of Sciences of the United States of America.</u> Vol. 104, Number 40,pp. 15723-15728<br />
# Shimizu-Sato, Sae et al. "A Light-Switchable Gene Promoter System." <u>Nature Publishing Group.</u> Vol. 20 (2002): 1,041-1044.<br />
# Smith, Harry and M. Geoffrey Holmes. <u>Techniquies in Photomorphogenesis.</u> London: Academic Press, Inc., 1984.<br />
# <u>System modeling in cellular biology from concepts to nuts and bolts.</u> Cambridge, MA: MIT P, 2006.<br />
# Terry, Matthew J. "Biosynthesis and Analysis of Bilins." <u>Heme, Chlorophyll, and Bilins: Methods and Protocols.</u> (2002) 273-291. <br />
# Wang, Wanneng et al. "Heterologous Synthesis and Assembly of Functional LHII Antenna Complexes from ''Rhodovulum sulfidophilum'' in ''Rhodobacter sphaeroides'' Mutant." <u>Molecular Biology Reports.</u> Vol. 12.3 (2008).<br />
# Walz, Thomas et al. "Projection Structures of Three Photosynthetic Complexes from Rhodobacter sphaeroides: LH2 at 6 A, LH1 and RC-LH1 at 25 A" <u>Journal of Molecular Biology</u> Vol. 282 pp. 833-845<br />
# Yakovlev, A. G. et al. "Light Control Over the Size of an Antenna Unit Building Block as an Efficient Strategy for Light Harvesting in Photosynthesis." <u>Federation of European Biochemical Societies.</u> (2002):129-132.<br />
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{{WashUbottom}}</div>Brendan1http://2009.igem.org/Team:Wash_U/ProjectTeam:Wash U/Project2009-10-22T03:32:18Z<p>Brendan1: /* Conclusion */</p>
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__NOTOC__<br />
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== '''Introduction''' == <br />
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Our project goal is to maximize the photosynthetic productivity for a series of photobioreactors containing ''Rhodobacter sphaeroides'' under both high and low light intensities by synthetically regulating the size of the light-harvesting antenna complex LH2. We chose to undertake such a project in ''R. sphaeroides'' due to its well-characterized photosynthetic and genetic system. <br />
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The antenna system functions to expand the spectrum of light available for photosynthesis by absorbing different wavelengths than that of the reaction center. Essentially, it is like the dish around the main receiver of any antenna. Marine bacteria, such as ''R. sphaeroides'', evolved very large antenna complexes to absorb light in a natural environment where there is great competition for photons. As a result, the photosynthetic machinery is saturated at low light intensities in a synthetic non-competitive environment, such as a photobioreactor. This causes up to 95% of incidental photons to be dissipated as heat or fluorescence by bacteria through a process called Non-Photochemical Quenching (NPQ) (Mussgnug et al., 2007). In essence, these photons are being wasted as NPQ reduces light penetration into and across bioreactors and starves the shaded cells for for photons. One method that has been shown to improve photosynthetic productivity in photobioreactors is the reduction of light-harvesting antenna sizes (Polle et al., 2002, Mussgnug et al., 2007). Though, current approaches to this end are difficult to precisely control from the perspective of biological engineering and synthetic biology. <br />
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Our intention is to create a dynamic system to vary antenna size that is dependent on incident light intensity and that can be readily optimized using bioengineering principles. This synthetic regulation of the LH2 complex should result in the bacteria grown under high light intensities expressing fewer LH2 complexes than the cells that are more shaded, leading to a reduction of wasted incident photons while maintaining a high overall absorbance across subsequent bioreactors. Consequently, we expect to see an increase in the total photosynthetic productivity for our mutant ''Rhodobacter sphaeroides'' when compared to the wild type grown under similar conditions. <br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:black;">'''Wild Type Energy Production in a series of Bioreactors'''<br />
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== '''Organism''' ==<br />
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''Rhodobacter sphaeroides'' is a purple Alphaproteobacteria. It is flexible metabolically and can grow heterotrophically via aerobic and anaerobic respiration, as well as phototrophically under anaerobic conditions with light. <br />
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''R. sphaeroides'' is one of the best understood photosynthetic organisms. The photosystem is located in intracytoplasmic membrane invaginations and the main components are the Light Haresting Complex 2 (LH2), Light Harvesting Complex 1 (LH1), and the Reaction Center (RC). These pigment-protein complexes non-covalently bind bacteriochlorophylls and carotenoids. LH1 and RC make up the core complex and are often found in the ratio 1:1. LH2 is more peripheral and naturally ranges in the ratio to LH1/RC of 3.0:1 to 6.7:1 under varying light conditions (Scheuring et al., 2005). LH2 absorbs photons maximally at the wavelengths of 850 and 800 nm and funnels its energy to LH1 and the reaction center for photochemistry. (Scheuring et al., 2005). <br />
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The two subunits of LH2 are coded for by the pucB/A genes and are naturally promoted by the puc promoter. <br />
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<br><div style="text-align: center;">'''ICM of ''R. sphaeroides'' '''[[Image:R sphaeroides photosystem.png| 500 px | center]]<center>source:Sener et al.2007<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:black;">'''Plate of ''R. sphaeroides'' 2.4.1'''<br />
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[[Image:R sphaeroides aborbance.png| 450px | left]][[Image:R sphaeroides 0986.png| 450px | right]]<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br>source: Walz et al. 1998<br><br />
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====Online Resources for ''R. sphaeroides''====<br />
[http://www.kazusa.or.jp/codon/cgi-bin/showcodon.cgi?species=272943 Codon Usage Table for ''R. sphaeroides'' 2.41]<br />
<br> [http://www.ncbi.nlm.nih.gov/sites/entrez?db=genome&cmd=Retrieve&dopt=Overview&list_uids=18843 ''R. sphaeroides'' 2.41 NCBI Genome Tools]<br />
<br> [http://archaea.ucsc.edu/cgi-bin/hgPcr?wp_target=&db=0&org=Rhodobacter+sphaeroides+2+4+1&wp_f=&wp_r=&wp_size=2000&wp_perfect=15&wp_good=15&wp_showPage=true&hgsid=159191 ''In silico'' PCR of ''R. sphaeroides'' 2.41 genome]<br />
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=='''Regulation'''==<br />
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[[Image:pucregulation-fullpanels.png|950px]]<br />
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'''The Natural System (Left)''' Under high oxygen tension, PpsR prevents transcription from the puc promoter of the pucBA genes, and thus prevents the expression of LH2(1st panel). Under low oxygen tension, this repression is removed by activated AppA, thus allowing expression of LH2 from pucBA(2nd panel).<br><br />
'''Synthetic Regulation System (Right)''' Our system takes advantage of constitutive protein expression from the puc promoter at low oxygen tension, and replaces pucBA with Cph8 at this site. Placing pucBA expression under the control of the ompC promoter allows for Cph8/OmpR control of LH2 expression under varying light conditions. Under high intensity of incident light, Cph8 will not be autophosphorylated, preventing expression of pucBA (3rd panel). When light intensity is lower, Cph8 will autophosphorylate, thus phosphorylating and activating OmpR and promoting expression of LH2 from the ompC promoter.<br><br />
This regulation system was designed such that light is able to penetrate deeper into and through photobioreactors where light is plentiful.<br />
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<br>[https://2009.igem.org/Team:Wash_U/Project Back To Top]<br />
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== '''Results''' ==<br />
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For methods, materials, and procedures regarding these results, please see [https://2009.igem.org/Team:Wash_U/Protocol#Tissue_Flask_Experiment Tissue Flask Experiment] or [https://2009.igem.org/Team:Wash_U/Biological_Parts#Modeling Modeling]<br />
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<div style="text-align: center;"> <br />
'''Figure 1'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:#black;">'''a''' Tissue Flask Position 1 Optical Density of Wild Type and DCBomega<br />
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|<font size="2" style="color:#black;">'''b''' Tissue Flask Position 2 Optical Density of Wild Type and DCBomega<br />
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[[Image:Tissue Flask 1b.png| 450 px| left]][[Image:Tissue Flask 2b.png| 450px | right]]<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:#black;">'''d''' Tissue Flask Position 4 Optical Density of Wild Type and DCBomega<br />
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[[Image:Tissue Flask 3b.png| 450px | left]][[Image:Tissue Flask 4b.png| 450px | right]]<br />
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'''e''' Tissue Flask Position 5 Optical Density of Wild Type and DBComega<br />
[[Image:Tissue Flask 5b.png| 450px | center]]<br><br />
Figure 1a,b,c,d,e are the optical density of a tissue flask, as assessed at 600 nm, over the course of the 6 days of the experiment.<br />
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'''Figure 2'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:#black;">'''b''' The Change in the Optical Density of the Tissue Flasks for the WT<br />
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[[Image:3D 241.png| 450 px | left]][[Image:3D DBCOmega2.png| 450 px | right]]<br />
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Figure 2a and 2b display the growth of the tissue flasks as measured by OD assessed as absorbance at 600 nm. The graph is organized such that those closest to the "days" axis were closest to the light source (i.e. flask 1 is closer than flask 2).<br />
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'''Figure 3'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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[[Image:Bar 241 b.png| 450px | left]][[Image:Bar DBCOmega b.png| 450px | right]]<br />
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Figure 3a and 3b display the cumulative growth of the tissue flasks as measured by the sum of the optical density of the respective cell type's tissue flasks at a given day. Furthermore, the contribution of a given tissue flask to cumulative growth is displayed.<br><br />
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'''Figure 4'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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[[image:sphaeroides tissue flask.jpg|450 px|left|]][[image:dbc tissue flask.jpg|450px|right]]<br />
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Figure 4A and 4B display the appearance of the tissue flasks for both strains on day 6. They are arranged from left to right with left being closed to the light source (flask 1) and right furthest (flask 5)<br />
<div style="text-align: center;"> <br />
'''Figure 5'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:#black;">'''a''' DBComega Spectrum by Flask Distance from Source<br />
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|<font size="2" style="color:#black;">'''b''' R. sphaeroides Spectrum by Flask Distance from Source<br />
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[[Image: WT irradiance 3d.png| 450 px | left]][[Image: DBComega irradiance 3d.png| 450 px | right]]<br />
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Contour of Absolute Irradiance by Flash distance from Source<br />
{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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[[Image: r sphaeroides day 2 a.jpg| 450px | left]][[Image: dbc day 4b.jpg| 450px | right]]<br />
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'''e''' Wild Type Absolute Irradiance Behind Flask 1: Day 6<br />
[[Image: irradiance WT flask 1 day6.png| 450px | center]]<br />
Caption for 5e<br><br />
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'''Figure 6'''<br><br />
Nonlinear least-squares estimation of WT LH2 saturation curve<br><br />
[[Image: saturation for WT as inferred.png| 450 px]]<br />
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Caption for figure 6<br><br><br />
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'''Figure 7'''<br><br />
Simulated Optical Density of Mutant and Wild Type Bioreactors<br><br />
Layers One and Two . Layers Three, Four and Five<br />
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[[Image: OD after flask 1 and 2.jpg| 200px ]][[Image: OD after 3,4, and 5.jpg| 200px ]]<br><br />
Caption for Figure 7<br />
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[https://2009.igem.org/Team:Wash_U/Project Back To Top]<br />
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== '''Analysis''' ==<br />
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<br />
Though we were not able to have our mutant functionally express our system, the tissue flask experiment was an interesting way to observe the effects of a full knockout of the Light Harvesting Antenna Complex 2 (LH2) vs the Wild Type on an array of consecutive photobioreactors. A couple of conclusions can be drawn from this data that can be used in modeling our synthetic regulation system for LH2 and in the future design of systems for the optimization of Light Harvesting Antenna size for photobioreactors . <br><br><br />
The most striking difference between the Wild Type Rhodobacter Sphaeroides 2.41 and DBCOmega (LH2 knockout) growth patterns is that the first flask in the WT (closest to the light source) grew less than the second flask, while the converse was true in DBCOmega (figure 2a and 2b). This can be attributed to the photosystem saturation curves for the respective cultures. The Wild Type has an LH2 complex, meaning that their antenna size is inherently larger and that their photosystem will be saturated at lower light intensities than the LH2 deficient mutant. We observed that the incident light intensity used in this experiment led to oversaturation for the first WT tissue flask and resulted in growth-inhibiting photodamage, as is evidenced by its lesser growth relative to the second culture. In contrast, photodamage was not observed in DBCOmega as is evidenced by the first tissue flask that grew at the fastest respective rate. Furthermore, it appears that this photodamage slowed the growth of the first WT tissue flask to the point that the OD of this tissue flask was nearly equal to that of DBCOmega after 5 days of growth(.987 vs. .967) (figure 1a). As such, the contribution of LH2 to the growth of the WT in flask 1 can be assessed to be little after day 3 when the growth curve slows- though, this light (at 800 and 850 nm) was still greatly absorbed by flask 1 (Figure 5e). It is likely that much of this light was then wasted through Non-Photochemical Quenching (NPQ), depleting the flasks behind flask 1 of photons at the LH2 absorbtion peaks and decreasing the cumulative productivity of flasks 1-5. This effect of wasting photons though NPQ under high light intensities is one that we sought to minimize in the design of our synthetic regulation for the pucB/A genes. <br><br><br />
<br />
A further a comparison of the WT and DBCOmega experiments reveals that the 3rd tissue flask in each had almost identical growth rates (figure 1c), while the DBCOmega 4th and 5th tissue flask outperformed that of the WT (figure 1d and 1e). This can simply be attributed to the higher density of cells in the 2nd flask of the WT that led to an overall lesser quantity of photons passing through to the 4th and 5th flasks in the WT experiment and likely resulting in heterotrophic growth (though this will need to be confirmed with a "dark control" experiment that should be completed in time for the Jamboree). This decrease in available photons for the WT can be seen by viewing the absolute irradiance data (incident light intensity at a given wavelength) after the third culture for the WT (figure 5a and 5c). This decrease in irradiance is the most pronounced for the LH2 absorption bands at 800 and 850 nm, as was expected. Our synthetic regulation system for pucB/A was designed with the intention of allowing greater penetration of light through the first couple of flasks at the LH2 wavelengths, as expression of pucB/A is inversely correlated to incident light intensity.<br><br><br />
'''Thomas, add in some further comments about the sprectroradiometer stuff. Specifically about how it changes in the WT vs. that of DBCOmega using some specific examples on the graphs'''<br><br><br />
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Overall, the cumulative culture growth of the WT exceeded that of DBCOmega (figure 3) due to the performance of the second tissue flask of the WT relative to that of DBCOmega (figure 1b) despite the photodamage that occurred in tissue flask 1 for the WT. This cumulative culture growth can be considered photosynthetic productivty and is proportional to the amount of energy/desired product that can be obtained from these cultures. <br><br><br />
<br />
It is an interesting exercise to model how our mutant system would perform under these same conditions. To do so, the LH2 saturation curve for the WT (figure 6) and the relative contribution of LH1/LH2 light absorbance to growth was derived from our empirical data and applied to a model that took into account incident light and gave the relative growth in terms of optical density as an output. In this model, our synthetic regulation system of pucB/A allowed our mutant to outperform the wild type species (figure 7). This was due to the performance of the 3rd, 4th, and 5th tissue flask relative to those of the wild type (see figure 7 layers 1,2 vs 1-5). The reason for this is that the wild type LH2 light saturation curve does not vary with changes in incident light intensity as the wild type expresses pucB/A at a uniform level under low oxygen conditions. As a result, the first two layers of Wild Type bioreactors absorb nearly all the available light and starve the bioreactors behind them for photons. <br><br><br />
Conversely, our mutant was modeled to have a dynamic expression of pucB/A based on the incident light intensity. This light response curve was modeled as an inverse exponential for the Cph8/OmpR ompC Promoter system and the saturation curve changes for each tissue flask in accordance with the incident light intensity- as light intensity decreases, the expression pucB/A increases. This results in more light passing through the first 2 flasks and facilitating greater growth in flasks 3, 4 and 5 as they express large antenna complexes. <br />
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='''Conclusion'''=<br />
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Based on model, should have had feedback mechanism- should be something that sets a minimum expression level for LH2, that is...<br><br />
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-tissue flask w prkcbc3 before jamboree and compare/interpret using characterization data<br><br />
-tissue flask w. full mutant in future- if this doesn't reveal cph8 to responding in an exponentially decaying fashion to light intensity, try w. a system that would<br />
-characterize puc promoter under light conditions and additional oxygen tensions<br />
-complete other characterization work<br />
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[https://2009.igem.org/Team:Wash_U/Project Back To Top]<br />
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== '''References''' ==<br />
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# "2007-2008 Catalog & Technical Reference." <u>New England BioLabs Inc.</u> (2007).<br />
# Alon, Uri. <u>Introduction to systems biology and the design principles of biological networks</u>. Boca Raton, FL: Chapman & Hall, 2006.<br />
# Ausubel, Fred et al. <u>Short Protocols in Molecular Biology: Third Edition.</u> Canada: John Wiley and Sons, Inc., 1999.<br />
# "BioBrick Assembly Help." <u>Registry of Standard Biological Parts.</u> 2009. 6 July 2009. <http://partsregistry.org/Main_Page>.<br />
# Bower, James M. <u>Computational Modeling of Genetic and Biochemical Networks (Computational Molecular Biology).</u> New York: M.I.T. PRESS, 2001.<br />
# Braatsch, Stephan et al. "A single Flavoprotein AppA, Integrates Both Redox and Light Signals in ''Rhodobacter sphaeroides.''" <u>Molecular Microbiology.</u> Vol. 45.3 (2002): 827-836.<br />
# Brown, Stanley B. et al. "Bile Pigment Synthesis in Plants: Incorporation of Haem into Phycocyanobilin and Phycocyanobiliproteins in Cyanidium Caldarium." <u>Biochemistry.</u> Vol. 194 (1981): 137-147.<br />
# Dragnea, Vladimira et al. "Time-Resolved Spectroscopic Studies of the AppA Blue-Light Receptor BLUF Domain from ''Rhodobacter sphaeroides'' <u>Biochemistry.</u> Vol. 44 (2005): 15,978-15,985.<br />
# "Entrez Nucleotide Search." <u>National Center for Biotechnology Information.</u> 2009. 6 June 2009 <http://www.ncbi.nlm.nih.gov/nuccore/49175990?from=3533887&to=3534606&report=gbwithparts>.<br />
# Fowler, Gregory J. S. and C. Neil Hunter. "The Synthesis and Assembly of Functional High and Low Light LH2 Antenna Complexes from ''Rhodopseudomonas palustris'' in ''Rhodobacter sphaeroides.''" <u>The Journal of Biological Chemistry.</u> Vol. 271.23 (1996): 13,356-13,361.<br />
# Gabrielsen, Mads et al. "Peripheral Complexes of Purple Bacteria." <u>The Purple Phototropic Bacteria.</u> (2009): 135-153.<br />
# "GenElute HP Plasmid Miniprep Kit: User Guide." <u>Sigma-Aldrich</u>. (2008).<br />
# Harwood, Caroline S. "Nitrogenase-Catalyzed Hydrogen Production by Purple Nonsulfur Photosynthetic Bacteria." <u>Bioenergy.</u> (2008): 259-271.<br />
# Jager, Andreas et al. "The AppA and PpsR Proteins from ''Rhodobacter sphaeroides'' Can Establish a Redox-Dependent Signal Chain but Fail to Transmit Blue-Light Signals in Other Bacteria." <u>Journal of Bacteriology.</u> Vol. 189.6 (2007): 2,274-2,282.<br />
# Jones, M. R. et al. "Mutants of ''Rhodobacter sphaeroides'' Lacking One or More Pigment-Protein Complexes and Complementation with Reaction-Centre, LH1, and LH2 Genes." <u>Molecular Microbiology.</u> Vol. 6.9 (1992): 1,173-1,184.<br />
# Kondo, Toshihiko et al. "Enhancement of Hydrogen Production by a Photosynthetic Bacterium Mutant with Reduced Pigment." <u>Journal of Bioscience and Bioengineering.</u> Vol. 93.2 (2002): 145-150.<br />
# Lagarias, J. Clark. <u>PCB from Spirulina</u>. Personal Communication. June 2009.<br />
# Laible, Philip D. "Foreign Gene Expression in Photosynthetic Bacteria." <u>The Purple Phototropic Bacteria.</u> (2009): 839-860.<br />
# Lee, Jeong K. and Samuel Kaplan. "Transcriptional Regulation of ''puc'' Operon Expression in ''Rhodobacter sphaeroides.''" <u>Journal of Biological Chemistry.</u> Vol. 270.35 (1995): 20,453-20,458.<br />
# Levskaya, Anselmetal et al. "Engineering ''Escherichia coli'' to See Light." <u>Nature.</u> Vol. 438 (2005): 441-442.<br />
# "Life Sciences Catalog 2009-2010." <u>National Diagnostics.</u> (2009).<br />
# Melis, Anastasios. "Solar Energy Conversion Efficiencies in Photosynthesis: Minimizing the Chlorophyll Antennae to Maximize Efficiency." <u>Plant Science.</u> (2009).<br />
# Mizuno, Takeshi and Shoji Mizushima. "Characterization by Deletion and Localized Mutagenesis in Vitro of the Promoter Region of the ''Escherichia coli'' OmpC Gene and Importance of the Upstream DNA Domain in Positive Regulation by the OmpR Protein." <u>Journal of Bacteriology.</u> Vol. 168.1 (1986): 86-95.<br />
# Moskvin, Oleg V. et al. "Transcriptome Analysis of the ''Rhodobacter sphaeroides'' PspR Regulon: PspR as a Master Regulator of Photosystem Development." <u>Journal of Bacteriology.</u> Vol. 187.6 (2005): 2,148-2,156.<br />
# Mussgnug, Jan H. et al. "Engineering photosynthetic light capture: impacts on improved solar energy to biomass conversion." <u> Plant Biotechnology Journal</u> Vol. 5 Issue 6. (2007) pp. 802-814<br />
# "NEBCutter V2.0." <u>New England BioLabs.</u> 2009. 6 June 2009 <http://tools.neb.com/NEBcutter2/index.php>.<br />
# "OligoAnalyzer 3.1." <u>Integrated DNA Technologies.</u> 2009. 6 June 2009. <http://www.idtdna.com/analyzer/Applications/OligoAnalyzer/><br />
# Sambrook, Joseph and David W. Russell. <u>Molecular Cloning: A Laboratory Manual.</u> Vol. 1-3. New York: Cold Springs Harbor Laboratory Press, 2001.<br />
# Scheuring, Simon and Sturgis, James N. "Chromatic Adaptation of Photosynthetic Membranes" <u>Science</u> Vol. 309 (2005) pp.484-487<br />
# Sener, Melih K. et al. "Atomic-level structural and functional model of a bacterial photosynthetic membrane vesicle" <u>Proceedings of the National Academy of Sciences of the United States of America.</u> Vol. 104, Number 40,pp. 15723-15728<br />
# Shimizu-Sato, Sae et al. "A Light-Switchable Gene Promoter System." <u>Nature Publishing Group.</u> Vol. 20 (2002): 1,041-1044.<br />
# Smith, Harry and M. Geoffrey Holmes. <u>Techniquies in Photomorphogenesis.</u> London: Academic Press, Inc., 1984.<br />
# <u>System modeling in cellular biology from concepts to nuts and bolts.</u> Cambridge, MA: MIT P, 2006.<br />
# Terry, Matthew J. "Biosynthesis and Analysis of Bilins." <u>Heme, Chlorophyll, and Bilins: Methods and Protocols.</u> (2002) 273-291. <br />
# Wang, Wanneng et al. "Heterologous Synthesis and Assembly of Functional LHII Antenna Complexes from ''Rhodovulum sulfidophilum'' in ''Rhodobacter sphaeroides'' Mutant." <u>Molecular Biology Reports.</u> Vol. 12.3 (2008).<br />
# Walz, Thomas et al. "Projection Structures of Three Photosynthetic Complexes from Rhodobacter sphaeroides: LH2 at 6 A, LH1 and RC-LH1 at 25 A" <u>Journal of Molecular Biology</u> Vol. 282 pp. 833-845<br />
# Yakovlev, A. G. et al. "Light Control Over the Size of an Antenna Unit Building Block as an Efficient Strategy for Light Harvesting in Photosynthesis." <u>Federation of European Biochemical Societies.</u> (2002):129-132.<br />
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{{WashUbottom}}</div>Brendan1http://2009.igem.org/Team:Wash_U/ProjectTeam:Wash U/Project2009-10-22T03:31:56Z<p>Brendan1: /* Conclusion and Future Work */</p>
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__NOTOC__<br />
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<div style="text-align: left;"> <font size="4" style="color:black"><br />
== '''Introduction''' == <br />
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Our project goal is to maximize the photosynthetic productivity for a series of photobioreactors containing ''Rhodobacter sphaeroides'' under both high and low light intensities by synthetically regulating the size of the light-harvesting antenna complex LH2. We chose to undertake such a project in ''R. sphaeroides'' due to its well-characterized photosynthetic and genetic system. <br />
<br />
The antenna system functions to expand the spectrum of light available for photosynthesis by absorbing different wavelengths than that of the reaction center. Essentially, it is like the dish around the main receiver of any antenna. Marine bacteria, such as ''R. sphaeroides'', evolved very large antenna complexes to absorb light in a natural environment where there is great competition for photons. As a result, the photosynthetic machinery is saturated at low light intensities in a synthetic non-competitive environment, such as a photobioreactor. This causes up to 95% of incidental photons to be dissipated as heat or fluorescence by bacteria through a process called Non-Photochemical Quenching (NPQ) (Mussgnug et al., 2007). In essence, these photons are being wasted as NPQ reduces light penetration into and across bioreactors and starves the shaded cells for for photons. One method that has been shown to improve photosynthetic productivity in photobioreactors is the reduction of light-harvesting antenna sizes (Polle et al., 2002, Mussgnug et al., 2007). Though, current approaches to this end are difficult to precisely control from the perspective of biological engineering and synthetic biology. <br />
<br />
Our intention is to create a dynamic system to vary antenna size that is dependent on incident light intensity and that can be readily optimized using bioengineering principles. This synthetic regulation of the LH2 complex should result in the bacteria grown under high light intensities expressing fewer LH2 complexes than the cells that are more shaded, leading to a reduction of wasted incident photons while maintaining a high overall absorbance across subsequent bioreactors. Consequently, we expect to see an increase in the total photosynthetic productivity for our mutant ''Rhodobacter sphaeroides'' when compared to the wild type grown under similar conditions. <br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:black;">'''Wild Type Energy Production in a series of Bioreactors'''<br />
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|<font size="2" style="color:black;">'''Synthetically Regulated Antenna Energy Production in Bioreactors'''<br />
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[[Image:Slide09b.jpg| 450px | left]][[Image:Slide10 a.jpg| 450px | right]]<br />
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[https://2009.igem.org/Team:Wash_U/Project Back To Top]<br />
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== '''Organism''' ==<br />
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''Rhodobacter sphaeroides'' is a purple Alphaproteobacteria. It is flexible metabolically and can grow heterotrophically via aerobic and anaerobic respiration, as well as phototrophically under anaerobic conditions with light. <br />
<br> <br><br />
''R. sphaeroides'' is one of the best understood photosynthetic organisms. The photosystem is located in intracytoplasmic membrane invaginations and the main components are the Light Haresting Complex 2 (LH2), Light Harvesting Complex 1 (LH1), and the Reaction Center (RC). These pigment-protein complexes non-covalently bind bacteriochlorophylls and carotenoids. LH1 and RC make up the core complex and are often found in the ratio 1:1. LH2 is more peripheral and naturally ranges in the ratio to LH1/RC of 3.0:1 to 6.7:1 under varying light conditions (Scheuring et al., 2005). LH2 absorbs photons maximally at the wavelengths of 850 and 800 nm and funnels its energy to LH1 and the reaction center for photochemistry. (Scheuring et al., 2005). <br />
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The two subunits of LH2 are coded for by the pucB/A genes and are naturally promoted by the puc promoter. <br />
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<br><div style="text-align: center;">'''ICM of ''R. sphaeroides'' '''[[Image:R sphaeroides photosystem.png| 500 px | center]]<center>source:Sener et al.2007<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:black;">'''LH2 Absorption Spectra'''<br />
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|<font size="2" style="color:black;">'''Plate of ''R. sphaeroides'' 2.4.1'''<br />
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[[Image:R sphaeroides aborbance.png| 450px | left]][[Image:R sphaeroides 0986.png| 450px | right]]<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br>source: Walz et al. 1998<br><br />
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====Online Resources for ''R. sphaeroides''====<br />
[http://www.kazusa.or.jp/codon/cgi-bin/showcodon.cgi?species=272943 Codon Usage Table for ''R. sphaeroides'' 2.41]<br />
<br> [http://www.ncbi.nlm.nih.gov/sites/entrez?db=genome&cmd=Retrieve&dopt=Overview&list_uids=18843 ''R. sphaeroides'' 2.41 NCBI Genome Tools]<br />
<br> [http://archaea.ucsc.edu/cgi-bin/hgPcr?wp_target=&db=0&org=Rhodobacter+sphaeroides+2+4+1&wp_f=&wp_r=&wp_size=2000&wp_perfect=15&wp_good=15&wp_showPage=true&hgsid=159191 ''In silico'' PCR of ''R. sphaeroides'' 2.41 genome]<br />
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[https://2009.igem.org/Team:Wash_U/Project Back To Top]<br />
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=='''Regulation'''==<br />
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[[Image:pucregulation-fullpanels.png|950px]]<br />
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'''The Natural System (Left)''' Under high oxygen tension, PpsR prevents transcription from the puc promoter of the pucBA genes, and thus prevents the expression of LH2(1st panel). Under low oxygen tension, this repression is removed by activated AppA, thus allowing expression of LH2 from pucBA(2nd panel).<br><br />
'''Synthetic Regulation System (Right)''' Our system takes advantage of constitutive protein expression from the puc promoter at low oxygen tension, and replaces pucBA with Cph8 at this site. Placing pucBA expression under the control of the ompC promoter allows for Cph8/OmpR control of LH2 expression under varying light conditions. Under high intensity of incident light, Cph8 will not be autophosphorylated, preventing expression of pucBA (3rd panel). When light intensity is lower, Cph8 will autophosphorylate, thus phosphorylating and activating OmpR and promoting expression of LH2 from the ompC promoter.<br><br />
This regulation system was designed such that light is able to penetrate deeper into and through photobioreactors where light is plentiful.<br />
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<br>[https://2009.igem.org/Team:Wash_U/Project Back To Top]<br />
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== '''Results''' ==<br />
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For methods, materials, and procedures regarding these results, please see [https://2009.igem.org/Team:Wash_U/Protocol#Tissue_Flask_Experiment Tissue Flask Experiment] or [https://2009.igem.org/Team:Wash_U/Biological_Parts#Modeling Modeling]<br />
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<div style="text-align: center;"> <br />
'''Figure 1'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:#black;">'''a''' Tissue Flask Position 1 Optical Density of Wild Type and DCBomega<br />
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|<font size="2" style="color:#black;">'''b''' Tissue Flask Position 2 Optical Density of Wild Type and DCBomega<br />
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<div style="text-align: left;"><br />
[[Image:Tissue Flask 1b.png| 450 px| left]][[Image:Tissue Flask 2b.png| 450px | right]]<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<div style="text-align: center;"><br />
<br />
{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:#black;">'''c''' Tissue Flask Position 3 Optical Density of Wild Type and DCBomega<br />
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|<font size="2" style="color:#black;">'''d''' Tissue Flask Position 4 Optical Density of Wild Type and DCBomega<br />
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[[Image:Tissue Flask 3b.png| 450px | left]][[Image:Tissue Flask 4b.png| 450px | right]]<br />
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<br><br><br><br><br><br><br><br><br><br><br><br><br><br><div style="text-align: center;"><br />
'''e''' Tissue Flask Position 5 Optical Density of Wild Type and DBComega<br />
[[Image:Tissue Flask 5b.png| 450px | center]]<br><br />
Figure 1a,b,c,d,e are the optical density of a tissue flask, as assessed at 600 nm, over the course of the 6 days of the experiment.<br />
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<div style="text-align: center;"><br />
'''Figure 2'''<br />
<div style="text-align: left;"><br />
<br />
{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:#black;">'''a''' The Change in the Optical Density of the Tissue Flasks for the WT<br />
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|<font size="2" style="color:#black;">'''b''' The Change in the Optical Density of the Tissue Flasks for the WT<br />
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[[Image:3D 241.png| 450 px | left]][[Image:3D DBCOmega2.png| 450 px | right]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
Figure 2a and 2b display the growth of the tissue flasks as measured by OD assessed as absorbance at 600 nm. The graph is organized such that those closest to the "days" axis were closest to the light source (i.e. flask 1 is closer than flask 2).<br />
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<div style="text-align: center;"><br />
'''Figure 3'''<br />
<br />
{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:#black;">'''a''' Cumulative Growth of Wild Type Tissue Flasks<br />
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|<font size="2" style="color:#black;">'''b''' Cumulative Growth of the DBComega Tissue Flasks<br />
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<div style="text-align: left;"><br />
[[Image:Bar 241 b.png| 450px | left]][[Image:Bar DBCOmega b.png| 450px | right]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
Figure 3a and 3b display the cumulative growth of the tissue flasks as measured by the sum of the optical density of the respective cell type's tissue flasks at a given day. Furthermore, the contribution of a given tissue flask to cumulative growth is displayed.<br><br />
<br />
<br />
<div style="text-align: center;"><br />
'''Figure 4'''<br />
<br />
{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:#black;">'''a''' Wild Type Tissue Flasks<br />
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|<font size="2" style="color:#black;">'''b''' The DBComega Tissue Flasks<br />
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<div style="text-align: left;"><br />
[[image:sphaeroides tissue flask.jpg|450 px|left|]][[image:dbc tissue flask.jpg|450px|right]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
Figure 4A and 4B display the appearance of the tissue flasks for both strains on day 6. They are arranged from left to right with left being closed to the light source (flask 1) and right furthest (flask 5)<br />
<div style="text-align: center;"> <br />
'''Figure 5'''<br />
<br />
{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:#black;">'''a''' DBComega Spectrum by Flask Distance from Source<br />
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|<font size="2" style="color:#black;">'''b''' R. sphaeroides Spectrum by Flask Distance from Source<br />
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<br />
[[Image: WT irradiance 3d.png| 450 px | left]][[Image: DBComega irradiance 3d.png| 450 px | right]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
Contour of Absolute Irradiance by Flash distance from Source<br />
{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:#black;">'''c''' R. Sphaeroides 2.4.1<br />
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|<font size="2" style="color:#black;">'''d''' DBComega<br />
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<div style="text-align: left;"><br />
[[Image: r sphaeroides day 2 a.jpg| 450px | left]][[Image: dbc day 4b.jpg| 450px | right]]<br />
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<div style="text-align: center;"><br />
'''e''' Wild Type Absolute Irradiance Behind Flask 1: Day 6<br />
[[Image: irradiance WT flask 1 day6.png| 450px | center]]<br />
Caption for 5e<br><br />
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<div style="text-align: center;"> <br />
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<br />
<div style="text-align: center;"> <br />
'''Figure 6'''<br><br />
Nonlinear least-squares estimation of WT LH2 saturation curve<br><br />
[[Image: saturation for WT as inferred.png| 450 px]]<br />
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Caption for figure 6<br><br><br />
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<div style="text-align: center;"> <br />
'''Figure 7'''<br><br />
Simulated Optical Density of Mutant and Wild Type Bioreactors<br><br />
Layers One and Two . Layers Three, Four and Five<br />
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<div style="text-align: center;"><br />
[[Image: OD after flask 1 and 2.jpg| 200px ]][[Image: OD after 3,4, and 5.jpg| 200px ]]<br><br />
Caption for Figure 7<br />
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[https://2009.igem.org/Team:Wash_U/Project Back To Top]<br />
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== '''Analysis''' ==<br />
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<br />
Though we were not able to have our mutant functionally express our system, the tissue flask experiment was an interesting way to observe the effects of a full knockout of the Light Harvesting Antenna Complex 2 (LH2) vs the Wild Type on an array of consecutive photobioreactors. A couple of conclusions can be drawn from this data that can be used in modeling our synthetic regulation system for LH2 and in the future design of systems for the optimization of Light Harvesting Antenna size for photobioreactors . <br><br><br />
The most striking difference between the Wild Type Rhodobacter Sphaeroides 2.41 and DBCOmega (LH2 knockout) growth patterns is that the first flask in the WT (closest to the light source) grew less than the second flask, while the converse was true in DBCOmega (figure 2a and 2b). This can be attributed to the photosystem saturation curves for the respective cultures. The Wild Type has an LH2 complex, meaning that their antenna size is inherently larger and that their photosystem will be saturated at lower light intensities than the LH2 deficient mutant. We observed that the incident light intensity used in this experiment led to oversaturation for the first WT tissue flask and resulted in growth-inhibiting photodamage, as is evidenced by its lesser growth relative to the second culture. In contrast, photodamage was not observed in DBCOmega as is evidenced by the first tissue flask that grew at the fastest respective rate. Furthermore, it appears that this photodamage slowed the growth of the first WT tissue flask to the point that the OD of this tissue flask was nearly equal to that of DBCOmega after 5 days of growth(.987 vs. .967) (figure 1a). As such, the contribution of LH2 to the growth of the WT in flask 1 can be assessed to be little after day 3 when the growth curve slows- though, this light (at 800 and 850 nm) was still greatly absorbed by flask 1 (Figure 5e). It is likely that much of this light was then wasted through Non-Photochemical Quenching (NPQ), depleting the flasks behind flask 1 of photons at the LH2 absorbtion peaks and decreasing the cumulative productivity of flasks 1-5. This effect of wasting photons though NPQ under high light intensities is one that we sought to minimize in the design of our synthetic regulation for the pucB/A genes. <br><br><br />
<br />
A further a comparison of the WT and DBCOmega experiments reveals that the 3rd tissue flask in each had almost identical growth rates (figure 1c), while the DBCOmega 4th and 5th tissue flask outperformed that of the WT (figure 1d and 1e). This can simply be attributed to the higher density of cells in the 2nd flask of the WT that led to an overall lesser quantity of photons passing through to the 4th and 5th flasks in the WT experiment and likely resulting in heterotrophic growth (though this will need to be confirmed with a "dark control" experiment that should be completed in time for the Jamboree). This decrease in available photons for the WT can be seen by viewing the absolute irradiance data (incident light intensity at a given wavelength) after the third culture for the WT (figure 5a and 5c). This decrease in irradiance is the most pronounced for the LH2 absorption bands at 800 and 850 nm, as was expected. Our synthetic regulation system for pucB/A was designed with the intention of allowing greater penetration of light through the first couple of flasks at the LH2 wavelengths, as expression of pucB/A is inversely correlated to incident light intensity.<br><br><br />
'''Thomas, add in some further comments about the sprectroradiometer stuff. Specifically about how it changes in the WT vs. that of DBCOmega using some specific examples on the graphs'''<br><br><br />
<br />
Overall, the cumulative culture growth of the WT exceeded that of DBCOmega (figure 3) due to the performance of the second tissue flask of the WT relative to that of DBCOmega (figure 1b) despite the photodamage that occurred in tissue flask 1 for the WT. This cumulative culture growth can be considered photosynthetic productivty and is proportional to the amount of energy/desired product that can be obtained from these cultures. <br><br><br />
<br />
It is an interesting exercise to model how our mutant system would perform under these same conditions. To do so, the LH2 saturation curve for the WT (figure 6) and the relative contribution of LH1/LH2 light absorbance to growth was derived from our empirical data and applied to a model that took into account incident light and gave the relative growth in terms of optical density as an output. In this model, our synthetic regulation system of pucB/A allowed our mutant to outperform the wild type species (figure 7). This was due to the performance of the 3rd, 4th, and 5th tissue flask relative to those of the wild type (see figure 7 layers 1,2 vs 1-5). The reason for this is that the wild type LH2 light saturation curve does not vary with changes in incident light intensity as the wild type expresses pucB/A at a uniform level under low oxygen conditions. As a result, the first two layers of Wild Type bioreactors absorb nearly all the available light and starve the bioreactors behind them for photons. <br><br><br />
Conversely, our mutant was modeled to have a dynamic expression of pucB/A based on the incident light intensity. This light response curve was modeled as an inverse exponential for the Cph8/OmpR ompC Promoter system and the saturation curve changes for each tissue flask in accordance with the incident light intensity- as light intensity decreases, the expression pucB/A increases. This results in more light passing through the first 2 flasks and facilitating greater growth in flasks 3, 4 and 5 as they express large antenna complexes. <br />
<br />
<br><br><br />
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==='''Conclusion'''===<br />
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Based on model, should have had feedback mechanism- should be something that sets a minimum expression level for LH2, that is...<br><br />
<br />
-tissue flask w prkcbc3 before jamboree and compare/interpret using characterization data<br><br />
-tissue flask w. full mutant in future- if this doesn't reveal cph8 to responding in an exponentially decaying fashion to light intensity, try w. a system that would<br />
-characterize puc promoter under light conditions and additional oxygen tensions<br />
-complete other characterization work<br />
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== '''References''' ==<br />
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# "2007-2008 Catalog & Technical Reference." <u>New England BioLabs Inc.</u> (2007).<br />
# Alon, Uri. <u>Introduction to systems biology and the design principles of biological networks</u>. Boca Raton, FL: Chapman & Hall, 2006.<br />
# Ausubel, Fred et al. <u>Short Protocols in Molecular Biology: Third Edition.</u> Canada: John Wiley and Sons, Inc., 1999.<br />
# "BioBrick Assembly Help." <u>Registry of Standard Biological Parts.</u> 2009. 6 July 2009. <http://partsregistry.org/Main_Page>.<br />
# Bower, James M. <u>Computational Modeling of Genetic and Biochemical Networks (Computational Molecular Biology).</u> New York: M.I.T. PRESS, 2001.<br />
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# Fowler, Gregory J. S. and C. Neil Hunter. "The Synthesis and Assembly of Functional High and Low Light LH2 Antenna Complexes from ''Rhodopseudomonas palustris'' in ''Rhodobacter sphaeroides.''" <u>The Journal of Biological Chemistry.</u> Vol. 271.23 (1996): 13,356-13,361.<br />
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# Harwood, Caroline S. "Nitrogenase-Catalyzed Hydrogen Production by Purple Nonsulfur Photosynthetic Bacteria." <u>Bioenergy.</u> (2008): 259-271.<br />
# Jager, Andreas et al. "The AppA and PpsR Proteins from ''Rhodobacter sphaeroides'' Can Establish a Redox-Dependent Signal Chain but Fail to Transmit Blue-Light Signals in Other Bacteria." <u>Journal of Bacteriology.</u> Vol. 189.6 (2007): 2,274-2,282.<br />
# Jones, M. R. et al. "Mutants of ''Rhodobacter sphaeroides'' Lacking One or More Pigment-Protein Complexes and Complementation with Reaction-Centre, LH1, and LH2 Genes." <u>Molecular Microbiology.</u> Vol. 6.9 (1992): 1,173-1,184.<br />
# Kondo, Toshihiko et al. "Enhancement of Hydrogen Production by a Photosynthetic Bacterium Mutant with Reduced Pigment." <u>Journal of Bioscience and Bioengineering.</u> Vol. 93.2 (2002): 145-150.<br />
# Lagarias, J. Clark. <u>PCB from Spirulina</u>. Personal Communication. June 2009.<br />
# Laible, Philip D. "Foreign Gene Expression in Photosynthetic Bacteria." <u>The Purple Phototropic Bacteria.</u> (2009): 839-860.<br />
# Lee, Jeong K. and Samuel Kaplan. "Transcriptional Regulation of ''puc'' Operon Expression in ''Rhodobacter sphaeroides.''" <u>Journal of Biological Chemistry.</u> Vol. 270.35 (1995): 20,453-20,458.<br />
# Levskaya, Anselmetal et al. "Engineering ''Escherichia coli'' to See Light." <u>Nature.</u> Vol. 438 (2005): 441-442.<br />
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# Melis, Anastasios. "Solar Energy Conversion Efficiencies in Photosynthesis: Minimizing the Chlorophyll Antennae to Maximize Efficiency." <u>Plant Science.</u> (2009).<br />
# Mizuno, Takeshi and Shoji Mizushima. "Characterization by Deletion and Localized Mutagenesis in Vitro of the Promoter Region of the ''Escherichia coli'' OmpC Gene and Importance of the Upstream DNA Domain in Positive Regulation by the OmpR Protein." <u>Journal of Bacteriology.</u> Vol. 168.1 (1986): 86-95.<br />
# Moskvin, Oleg V. et al. "Transcriptome Analysis of the ''Rhodobacter sphaeroides'' PspR Regulon: PspR as a Master Regulator of Photosystem Development." <u>Journal of Bacteriology.</u> Vol. 187.6 (2005): 2,148-2,156.<br />
# Mussgnug, Jan H. et al. "Engineering photosynthetic light capture: impacts on improved solar energy to biomass conversion." <u> Plant Biotechnology Journal</u> Vol. 5 Issue 6. (2007) pp. 802-814<br />
# "NEBCutter V2.0." <u>New England BioLabs.</u> 2009. 6 June 2009 <http://tools.neb.com/NEBcutter2/index.php>.<br />
# "OligoAnalyzer 3.1." <u>Integrated DNA Technologies.</u> 2009. 6 June 2009. <http://www.idtdna.com/analyzer/Applications/OligoAnalyzer/><br />
# Sambrook, Joseph and David W. Russell. <u>Molecular Cloning: A Laboratory Manual.</u> Vol. 1-3. New York: Cold Springs Harbor Laboratory Press, 2001.<br />
# Scheuring, Simon and Sturgis, James N. "Chromatic Adaptation of Photosynthetic Membranes" <u>Science</u> Vol. 309 (2005) pp.484-487<br />
# Sener, Melih K. et al. "Atomic-level structural and functional model of a bacterial photosynthetic membrane vesicle" <u>Proceedings of the National Academy of Sciences of the United States of America.</u> Vol. 104, Number 40,pp. 15723-15728<br />
# Shimizu-Sato, Sae et al. "A Light-Switchable Gene Promoter System." <u>Nature Publishing Group.</u> Vol. 20 (2002): 1,041-1044.<br />
# Smith, Harry and M. Geoffrey Holmes. <u>Techniquies in Photomorphogenesis.</u> London: Academic Press, Inc., 1984.<br />
# <u>System modeling in cellular biology from concepts to nuts and bolts.</u> Cambridge, MA: MIT P, 2006.<br />
# Terry, Matthew J. "Biosynthesis and Analysis of Bilins." <u>Heme, Chlorophyll, and Bilins: Methods and Protocols.</u> (2002) 273-291. <br />
# Wang, Wanneng et al. "Heterologous Synthesis and Assembly of Functional LHII Antenna Complexes from ''Rhodovulum sulfidophilum'' in ''Rhodobacter sphaeroides'' Mutant." <u>Molecular Biology Reports.</u> Vol. 12.3 (2008).<br />
# Walz, Thomas et al. "Projection Structures of Three Photosynthetic Complexes from Rhodobacter sphaeroides: LH2 at 6 A, LH1 and RC-LH1 at 25 A" <u>Journal of Molecular Biology</u> Vol. 282 pp. 833-845<br />
# Yakovlev, A. G. et al. "Light Control Over the Size of an Antenna Unit Building Block as an Efficient Strategy for Light Harvesting in Photosynthesis." <u>Federation of European Biochemical Societies.</u> (2002):129-132.<br />
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</html></div>Brendan1http://2009.igem.org/Team:Wash_U/Biological_PartsTeam:Wash U/Biological Parts2009-10-22T03:10:23Z<p>Brendan1: /* Simulating a Bioreactor */</p>
<hr />
<div>{{WashUback}}<br />
<br />
<br />
The Registry of Standard Biological Parts is a library of DNA sequences combined with online characterization resources. The Registry has created a standard protocol for making segments of DNA compatible with all other segments of DNA, regardless of order or size. BioBrick is the term given to such segments of DNA, a term alluding to the fact that any number of bricks may be combined in any order to produce complex, unique systems. This is accomplished by standardizing the restriction enzymes used to surround BioBricks, as well as the plasmids used to transform them. For a graphical representation of of the process, please click [http://ginkgobioworks.com/support/BioBrick_Assembly_Manual.jpg here]. A powerful online database provides information and characterization of all of the BioBricks in the Registry and uses the wiki format (the same one used in wikipedia) which encourages others to edit content directly on the page. Below are list of parts that were used/created for this project. <br />
<div style="text-align: left;"><font size="4" style="color:black"><br />
=='''Parts'''==<br />
<center><br />
<font size="2"><br />
Parts used to characterize and build our final project<br />
{|cellpadding="9" cellspacing="0" style="background-color:#FFEFD5;" border=1<br />
|'''Component'''<br />
|'''Part/Accession #'''<br />
|'''Base Pairs'''<br />
|'''Plasmid'''<br />
|'''Resistance'''<br />
|'''Well'''<br />
|-<br />
|RBS-34<br />
|[http://partsregistry.org/Part:BBa_B0034 BBa_B0034]<br />
|12<br />
|pSB1A2<br />
|Ampicillin<br />
|plate 1, 2M<br />
|-<br />
|Cph8<br />
|[http://partsregistry.org/Part:BBa_I15010 BBa_I15010]<br />
|2,238<br />
|pSB2K3<br />
|Kanamycin<br />
|N/A<br />
|-<br />
|RFP<br />
|[http://partsregistry.org/Part:BBa_J04051 BBa_J04051]<br />
|720<br />
|N/A<br />
|N/A<br />
|N/A<br />
|-<br />
|OmpR (E. coli)<br />
|[http://partsregistry.org/wiki/index.php?title=Part:BBa_K098011 BBa_K098011]<br />
|720<br />
|pSB1T3<br />
|Tetracycline<br />
|N/A<br />
|-<br />
|OmpR (R. sphaeroides)<br />
|[http://partsregistry.org/Part:BBa_K227010 BBa_K227010]<br />
|720<br />
|New<br />
|New<br />
|New<br />
|-<br />
|Terminator<br />
|[http://partsregistry.org/Part:BBa_B0015 BBa_B0015]<br />
|129<br />
|pSB1AK3<br />
|Ampicillin <br>and Kanamycin<br />
|plate 1, 23L<br />
|-<br />
|RBS +OmpR(sph) + Terminator<br />
includes prefix and suffix<br />
|[[media:OmpR_+_terminator.txt|sequence]]<br />
[http://partsregistry.org/Part:BBa_K227011 BBa_K227011]<br />
|875/916<br />
|pSB1k3<br />
|Kanamycin<br />
|synthesized<br />
|-<br />
|OmpC promoter<br />
|[http://partsregistry.org/Part:BBa_R0082 BBa_R0082]<br />
|108<br />
|pSB1A2<br />
|Ampicillin<br />
|plate 1, 16K<br />
|-<br />
|puc promoter<br />
|[http://partsregistry.org/Part:BBa_K227007 BBa_K227007]<br />
|651<br />
|pSB1k3<br />
|Kanamycin<br />
|New<br />
|-<br />
|puc BA<br />
|[http://partsregistry.org/Part:BBa_K227006 BBa_K227006]<br />
|336<br />
|pSB1k3<br />
|Kanamycin<br />
|New<br />
|-<br />
|puc B<br />
|[http://partsregistry.org/Part:BBa_K227005 BBa_K227005]<br />
|156<br />
|pSB1k3<br />
|Kanamycin<br />
|New<br />
|-<br />
|puc A<br />
|[http://partsregistry.org/Part:BBa_K227004 BBa_K227004]<br />
|165<br />
|pSB1k3<br />
|Kanamycin<br />
|New<br />
|-<br />
|OmpC promoter+pucBA<br />
|[[media:OmpC_promoter_+_puc_BA.txt|sequence]]<br />
[http://partsregistry.org/Part:BBa_K227008 BBa_K227008]<br />
|492/539<br />
|pSB1k3<br />
|Kanamycin<br />
|synthesized<br />
|-<br />
|Green Fluorescent Protein<br />
|[http://partsregistry.org/Part:BBa_E0240 BBa_E0240]<br />
|876<br />
|pSB1A2<br />
|Ampicillin<br />
|plate 1, 12M<br />
|}<br />
<br />
<br />
Plasmids used to create and characterize our project<br />
{|cellpadding="9" cellspacing="0" style="background-color:#FFEFD5;" border=1<br />
|'''Plasmid'''<br />
|'''Base Pairs'''<br />
|'''Resistance'''<br />
|'''Copy Number'''<br />
|-<br />
|[http://partsregistry.org/Part:pSB1A2 pSB1A2]<br />
|2,079<br />
|Ampicillin<br />
|high<br />
|-<br />
|[http://partsregistry.org/Part:pSB1K3 pSB1K3]<br />
|2,206<br />
|Kanamycin<br />
|high<br />
|-<br />
|[http://partsregistry.org/Part:pSB1A3 pSB1A3]<br />
|2,157<br />
|Ampicillin<br />
|high<br />
|-<br />
|[http://partsregistry.org/Part:pSB2K3 pSB2K3]<br />
|4,425<br />
|Kanamycin<br />
|variable<br />
|-<br />
|[http://partsregistry.org/Part:pSB1T3 pSB1T3]<br />
|2,463<br />
|Tetracycline<br />
|high<br />
|-<br />
|pRKCBC3<br />
|~11.5kb<br />
|Tetracycline<br />
|4<br />
|}<font size="2"><br />
<br />
<br />
<br />
Parts submitted to the Registry of Standard Biological Parts<br />
{|cellpadding="9" cellspacing="0" style="background-color:#FFEFD5;" border=1<br />
|'''Part/Accession #Component'''<br />
|'''Component'''<br />
|'''Type'''<br />
|'''Base Pairs'''<br />
|'''Plasmid'''<br />
|'''Resistance'''<br />
|-<br />
|[http://partsregistry.org/Part:BBa_I15010 BBa_I15010]<br />
|Cph8 (planned resubmission)<br />
|Coding<br />
|2,238<br />
|plasmid<br />
|Resistance<br />
|-<br />
|[http://partsregistry.org/Part:BBa_K227004 BBa_K227004]<br />
|puc A<br />
|Coding<br />
|165<br />
|pSB1K3<br />
|Kanamycin<br />
|-<br />
|[http://partsregistry.org/Part:BBa_K227005 BBa_K227005]<br />
|puc B<br />
|Coding<br />
|156<br />
|pSB1K3<br />
|Kanamycin<br />
|-<br />
|[http://partsregistry.org/Part:BBa_K227006 BBa_K227006]<br />
|puc BA<br />
|Coding<br />
|336<br />
|pSB1K3<br />
|Kanamycin<br />
|-<br />
|[http://partsregistry.org/Part:BBa_K227007 BBa_K227007]<br />
|puc promoter<br />
|Regulatory<br />
|651<br />
|pSB1K3<br />
|Kanamycin<br />
|-<br />
|[http://partsregistry.org/Part:BBa_K227008 BBa_K227008]<br />
|ompC+PucBA (synthesized)<br />
|Composite<br />
|492<br />
|pSB1AT3<br />
|Ampicilin,Tetracycline<br />
|-<br />
|[http://partsregistry.org/Part:BBa_K227009 BBa_K227009]<br />
|PucPromotor+GFP<br />
|Composite<br />
|1377<br />
|pSB1A2<br />
|Ampicilin<br />
|-<br />
|[http://partsregistry.org/Part:BBa_K227011 BBa_K227011]<br />
|RBS34+OmpR(sph)+Term (synthesized)<br />
|Composite<br />
|875<br />
|pSB1K3<br />
|Kanamycin<br />
|-<br />
|[http://partsregistry.org/Part:BBa_K227012 BBa_K227012]<br />
|RBS34+OmpR(sph)+Term+OmpC+PucB/A<br />
|Composite<br />
|1375<br />
|pSB1K3<br />
|Kanamycin<br />
|-<br />
|[http://partsregistry.org/Part:BBa_K227013 BBa_K227013]<br />
|ompCpro + GFP<br />
|Composite<br />
|992<br />
|pSB1A2<br />
|Ampicilin<br />
|-<br />
|[http://partsregistry.org/Part:BBa_K227014 BBa_K227014]<br />
|pucpro+pucBA<br />
|Composite<br />
|1035<br />
|pSB1K3<br />
|Kanamycin<br />
|-<br />
|[http://partsregistry.org/Part:BBa_K227015 BBa_K227015]<br />
|RBS34+OmpR(sph)+Term+OmpC+GFP<br />
|Composite<br />
|1875<br />
|pSB1A2<br />
|Ampicilin<br />
|-<br />
<br />
|}<br />
<div style="text-align: left;"><br />
[https://2009.igem.org/Team:Wash_U/Biological_Parts Back To Top]<font size="4"><br />
<br />
=='''Characterization'''==<br />
<font size="2"> <br><br />
'''pucB/A'''<br><br />
pucB/A are the genes that code for the beta and alpha subunits of the light harvesting complex LH2. Here, we measured the expression level of pucB/A from the puc promoter in the genome vs. on a low copy plasmid, pRKCBC3. This data allows us to utilize pucB/A as a reporter gene for expression from promoters, in complement to its role in cellular growth. <br> <br />
'''Method'''<br><br />
Relative LH2 expression in R. sphaeroides 2.4.1, R. sphaeroides DBCΩ and R. sphaeroides DBCΩ pRKCBC3 grown anaerobically in the dark<br><br />
<br />
Growth conditions:<br />
-Cultures grown in the dark at 34° C, shaking at 160 rpm<br />
- R. sphaeroides 2.4.1<br />
-15 ml culture in polystyrene tube<br />
-15ml M22 was inoculated with 1ml inoculant with OD600nm = 0.270<br />
- OD600nm (Volume) = 0.270<br />
-Culture was allowed to grow 19hrs<br />
- R. sphaeroides DBCΩ<br />
-15 ml culture in polystyrene tube<br />
-15ml M22 was inoculated with 865ul inoculant with OD600nm = 0.312<br />
- OD600nm (Volume) = 0.270<br />
-Culture was allowed to grow 19hrs<br />
- R. sphaeroides DBCΩ pRKCBC3<br />
-10 ml culture in polystyrene tube<br />
-10 ml M22 tet 5ug/ml was inoculated with loop of R. sphaeroides DBCΩ pRKCBC3 and capped with rubber stopper<br />
-Culture was allowed to grow for 3 days so that density was high enough to get a good reading<br />
<br />
Analysis:<br />
Cultures were removed from the incubtor and placed on ice to slow changes in cellular composition. 1 ml was extracted from each culture and a UV-vis absorption spectra of the culture was taken from 300-950 nm. <br />
The optical density of the cultures at 600nm was used to normalize the absorption spectrum by division by this value. Background subtraction of spectrophotometer data was performed in Origin 6.1 Software. A ten-point baseline was created by a "positive peak" algorithm then modified to approximate the scattering curve that falls as the inverse fourth power of wavelength. <br><br />
'''Results'''<br><br />
[[Image:puc.jpg|420px|]]<br><br />
'''Conclusion'''<br><br />
This data indicates that pucB/A can be utilized as a reporter gene as the LH2 absorption bands at 800 and 850 nm are not present in the LH2 deficient mutant DBComega. Furthermore, changes in the expression conditions of pucB/A are reflected on the absorption spectrum. In this case, expression is higher when the genes are expressed from the puc promoter within genomic DNA than on plasmid pRKCBC3, despite the fact that pRKCBC3 is maintained at 4-5 copies in a cell. <br><br />
<br />
<br />
<br />
<br />
<br />
'''Puc Promoter'''<br><br />
[[Image:Puc Promoter slide.jpg|480px|left]] The puc promoterpromotes transcription of the LH2 pucB/A genes naturally in ''Rhodobacter sphaeroides''. It is important that we are able to compare the transcription rate of the puc promoter in the natural system vs. our mutant system so that we can determine exactly how much efficiency is gained by adding a red light sensor. The absorption spectra of a DBCOmega mutant (LH2 deficient) transformed with pRKCBC3 containing the puc promoter and pucB/A genes will allow us to characterize the puc promoter under high and low oxygen conditions. <br />
More absorption of light at the LH2 spectra peaks normalized to culture OD corresponds with more transcription and vis versa.<br />
<br><br />
'''Method'''<br><br />
Cultures were grown in the dark at 34° C, shaking at 160 rpm. The anaerobic test condition was established by inoculating a 10 ml culture tube with 10 ml M22 tet 5ug/ml with a loop of R. sphaeroides DBCΩ pRKCBC3 and capped with a rubber stopper. The aerobic test condition was established by innoculating a 10 ml culture tube with 5ml M22 tet 5ug/ml with a loop of R. sphaeroides DBCΩ pRKCBC3 and covered with vented cap- the vented cap and relatively low culture volume left significant headroom in the culture for oxygen exchange. Oxygen tension was not able to be quantitatively measured due to the nature of the experiment<br><br><br />
For measurement of pucB/A expression from the puc promoter: <br><br />
Cultures were removed from the incubtor and placed on ice to slow changes in cellular composition. 1 ml was extracted from each culture and a UV-vis absorption spectra of the culture was taken from 300-950 nm. <br />
The optical density of the cultures at 600nm was used to normalize the absorption spectrum by division by this value. Background subtraction of spectrophotometer data was performed in Origin 6.1 Software. A ten-point baseline was created by a "positive peak" algorithm then modified to approximate the scattering curve that falls as the inverse fourth power of wavelength. <br><br />
'''Results'''<br><br />
[[Image:02 puc pro characterization graph a.png|700px|]]<br><br />
'''Conclusion'''<br><br />
This data matches the literature for expression from the puc promoter at different oxygen tensions and as such confirms the assumptions that we have made in modeling our system. <br><br />
See: Braatsch et al. 2002<br />
<br />
=='''Future Characterization'''==<br />
<br>[[Image:Slidex.jpg|480px|left]] The next step in our characterization of our synthetic red light response system is to analyze changes in phosphorylation of ompR in ''Rhodobacter sphaeroides''. In our final system, we only want puc genes to be transcribed and expressed via halting the autophosphorylation of ompR, not simply the puc promoter as it naturally occurs. By placing the ompR coding region downstream of the red light sensor and upstream of a terminator our modified system controls expression of the puc genes by the red light sensor. It should be impossible for the puc promoter to directly cause the transcription of puc genes due to the terminator, but instead, transcription of the puc genes must be activated via decreasing the presence of phosphorylated ompR. The end target of ompR transcription is the ompC promoter, located directly upstream of the puc genes. Placing GFP on the ompC transcript will show how often the promoter is transcribed and how often ompR is phoshorylated.<br />
<br><br><br><br><br><br><br><br><br>[[Image:Characterization 1.jpg|480px|left]] Part three of our characterization measures the effectiveness of the red light sensor in downregulating the phosphorylation of ompR. This setup is identical to that of part two except we have introduced the red light sensor. Now, the rate of ompR autophosphorylation will be halted by binding to a domain on the light-activated EnvZ kinase analogue. GFP is still attached to the end product, the ompC promoter. By comparing the fluorescence of GFP in this scenario compared with the second scenario the decrease in rate of phosphorylation should be apparent due to the activity of the red light sensor.<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br>[[Image:Slidez.jpg|480px|left]] This is the final construct that will be our actual functioning model in ''Rhodobacter sphaeroides''. This finished product will be compared to the wild type over various intensities of light and cell culture densities can be compared to see which strain, the wild type or the modified strain with the red light sensor was more efficient in harvesting light with varying intensities.<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><font size="2"><br />
[https://2009.igem.org/Team:Wash_U/Biological_Parts Back To Top]<br />
<br />
<br />
<font size="4"><br />
<br />
=='''Modeling'''==<br />
<br />
[[Image:pucBAModelDiagram.jpg|thumb|200px|pucBA Expression Model Diagram]]<br />
[[Image:pucBAModelEq.jpg|thumb|200px|pucBA Model Equations]]<br />
[[Image:pucBAModelRxn.jpg|thumb|200px|pucBA Model Reactions]]<br />
[[Image:pucBAModelTestSim.jpg|thumb|200px|Test Simulation Output]]<br />
'''Modeling the Gene Regulatory Network'''<br />
<font size="2"><br />
:Our group seeks to assess the optimality of the synthetic system that modulates pucB/A gene expression and LH2 complex assembly in ''Rhodobacter sphaeroides''. Here we employ a mathematical model of this system to generate predictions about the behavior of the active system in response to light input. Features of the system that the model may help investigate include the time scale of response to light signals, the robustness of the system in response to fluctuations in light intensity, and the translation between changes in gene expression and the absorbance spectrum of the engineered cells.<br />
<br><br><br><br />
<br />
:Though the context of the model can extend back to the transcription of PrrA/B genes involved in integration oxygen and light signals, a preliminary testing model was developed using assumptions of certain initial conditions to isolate the light signal's effect. Since Cph8 and OmpR are located on the same transcript downstream of the puc promoter region, it was assumed that their associated protein and mRNA had already reached steady state concentrations, and the phosphorylation reaction had already reached steady state. Moreover, the concentrations of the factors were assumed to be equal at this state. The model whose diagram was constructed in the Simbiology Toolbox distributed by MathWorks details key reactions leading to the translation of the pucB/A genes. The reaction rate equation used for the lack of phosphorylation of OmpR when the light signal reaches Cph8 bound to OmpR is captured in a modified form of Michaelis-Menten kinetics. A logic function that corresponds to light ON/OFF (1/0) multiplies the maximum reaction rate in the numerator of the phosphorylation equation. Thus, the model assumes that no phosphorylation occurs by this mechanism in the presence of light. The OmpC promoter binding equation was based on the Hill Equation for an Activator(1). <br />
<br><br><br><br />
<br />
:Component characterization steps and literature searches are underway in order to obtain quantitative parameters for the reaction rates. In order to simulate behavior of the system, putative values were included that exaggerate true concentrations and time scales. OmpR was given an initial concentration normalized to one, and all other components were assumed insignificant initially to this value. An ideal light pulse was introduced at an instant and removed thirty simulation seconds later. From this rudimentary simulation it can be drawn that the nonlinearities of the phosphorylation and transcription factor binding kinetics effectively smooth the sharp light input. By design, the light switch ON yields no phosphorylation of OmpR and repression of the pucB/A genes which would give rise to LH2. Conversely, when left OFF, the concentration of pucB/A recovers and increases until the steady state determined by its translation and degradation rates.<br />
<br><br><br><br />
<br />
'''References'''<br />
<br />
1. Alon, Uri. <u>Introduction to systems biology and the design principles of biological networks</u>. Boca Raton, FL: Chapman & Hall, 2006.<br />
<br><br />
2. Bower, James M. <u>Computational Modeling of Genetic and Biochemical Networks (Computational Molecular Biology).</u> New York: M.I.T. PRESS, 2001.<br />
<br><br />
3. <u>System modeling in cellular biology from concepts to nuts and bolts.</u> Cambridge, MA: MIT P, 2006.<br />
<br />
<br><br />
<br><br><br><br />
=='''Simulating A Bioreactor'''==<br />
Simulation Equations <br><br />
[[image:simulationequations.png|200px|left]]<br><br><br />
<br />
Nonlinear least-squares estimation of LH2 saturation curve <br><br><br><br><br><br><br><br />
[[Image: saturation for WT as inferred.png|450px|left]] <br><br />
<br />
Exponential response curve for mutant LH2 saturation coefficients <br><br />
[[image:lh2adaption.jpg|450px|left|]] <br><br />
<br />
'''Figure ?'''<br />
<br />
Relative growth of DBComega to WT in Flask 2 at OD 600<br />
<br />
[[Image:relative growth.png| 600px | center]]<br />
This data illustrates the contribution of LH1 to growth relative to that of LH2 <br><br />
<div style="text-align: left;"><br />
<br />
<font size="2"><br />
'''Problem:''' In a typical reactor, cells at the surface absorb more than enough light to saturate their photosynthetic apparatus, transmitting less energy to deeper layers. For wild type cells, the “saturation curve” is approximately the same for all cells, regardless of their incident light intensity.<br><br />
'''Simulating our Mutants advantage in a Bioreactor'''<br />
For our mutant cells, this curve scales as a function of light intensity, due to negative regulation of LH2 complex production<br />
<br />
<div style="text-align: left;"><br />
Saturation curve: Absorbance as a function of incident light intensity. <br>The coefficient changes with intensity in the mutant only.<br><br><br />
Assumptions:<br />
- Light intensity at next layer is given by transmittance from previous layer (assume no backscattering).<br><br />
- Total energy funneled to photosynthetic pathways is estimated as the sum of light absorbed by each layer.<br />
<br />
<br><br />
<br><br />
<font size="2"><br />
[[Image:wtvsmutsim1.jpg|450px|left]][[Image:wtvsmutsim2.jpg|450px|right]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
type here1<br />
<br />
<br />
[[Image:wtvsmutsim3.jpg|450px|left]][[Image:wtvsmutsim4.jpg|450px|right]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
type here2<br />
<br />
[[Image:wtvsmutsim5.jpg|600px|center]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
type here3<br />
<br />
<div style="text-align: left;"><br />
<br><br><br />
<br />
'''References'''<br />
<br />
#Steven, Spilatro R. "Photosynthesis Investigation Study." Photosynthesis Investigation Study. Department of Biology, Marietta College, Marietta, Ohio, 1998. Web. 21 Oct. 2009. <http://www.marietta.edu/~spilatrs/biol103/photolab/photosyn.html>.<br />
<br />
<div style="text-align: left;"><br />
[https://2009.igem.org/Team:Wash_U/Biological_Parts Back To Top]<br />
<br />
<br />
<br />
{{WashUbottom}}</div>Brendan1http://2009.igem.org/Team:Wash_U/PicturesTeam:Wash U/Pictures2009-10-22T03:06:45Z<p>Brendan1: /* Bacteria */</p>
<hr />
<div>{{WashUback}}<br />
<br />
<div align=left><br />
<font size="4"><br />
=='''Slideshow'''==<br />
<html><center><br />
<iframe align="center" src="http://www.flickr.com/slideShow/index.gne?user_id=39966176@N07" width="500" height="500" frameBorder="0" scrolling="no"></iframe><br /></a><br />
</html><br />
<div align=left><br />
<br />
<br />
<br />
=='''Planning'''==<br />
<br />
[[image:Left Board 1.jpg|315px|left]] [[image:Right Board 1.jpg|315px|right]] [[image:Left Board 2.jpg|315px|center]]<br><br />
[[image:Right Board 2.jpg|315px|left]] [[image:June 10 1.jpg|315px|right]] [[image:June 10 2.jpg|315px|center]]<br><br />
[[image:DSC00838.jpg|315px|left]] [[image:BioBrick Assembly Manual.jpg|315px|right]] [[image:DSC00859.jpg|315px|center]]<br><br><br><br />
<br />
=='''Lab'''==<br />
<br />
[[image:DSC00861.jpg|315px|left]] [[image:Setup2.jpg|315px|right]] [[image:setup1.jpg|315px|center]]<br><br />
[[image:DSC00844.JPG|315px|left]] [[image:DSC00843.JPG|315px|right]] [[image:DSC00841.JPG|315px|center]]<br><br />
[[image:DSC00855.JPG|315px|left]] [[image:DSC00849.JPG|315px|right]] [[image:DSC00854.JPG|315px|center]]<br><br />
[[image:DSC00857.JPG|315px|left]] [[image:DSC00862.jpg|315px|right]] [[image:DSC00845.jpg|315px|center]]<br><br />
[[image:setup3.JPG|315px]]<br><br />
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<br />
<br />
=='''People'''==<br />
[[image:DSC00839.jpg|315px]] [[image:DSC00864.jpg|315px]]<br><br />
<br />
<br />
=='''Bacteria'''==<br />
[[image:DSC00856.JPG|315px|left]] [[image:DSC00837.JPG|315px|right]] [[image:DSC00851.JPG|315px|center]]<br><br />
[[image:DSC00852.JPG|315px|left]] [[image:LB Media.jpg|315px|right]] [[image:sphaeroides tissue flask.jpg|315px|center]]<br><br><br><br><br><br><br><br><br><br><br><br />
<br />
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<div style="text-align: center;"><br />
{{WashUbottom}}</div>Brendan1http://2009.igem.org/Team:Wash_U/PicturesTeam:Wash U/Pictures2009-10-22T03:06:33Z<p>Brendan1: /* Bacteria */</p>
<hr />
<div>{{WashUback}}<br />
<br />
<div align=left><br />
<font size="4"><br />
=='''Slideshow'''==<br />
<html><center><br />
<iframe align="center" src="http://www.flickr.com/slideShow/index.gne?user_id=39966176@N07" width="500" height="500" frameBorder="0" scrolling="no"></iframe><br /></a><br />
</html><br />
<div align=left><br />
<br />
<br />
<br />
=='''Planning'''==<br />
<br />
[[image:Left Board 1.jpg|315px|left]] [[image:Right Board 1.jpg|315px|right]] [[image:Left Board 2.jpg|315px|center]]<br><br />
[[image:Right Board 2.jpg|315px|left]] [[image:June 10 1.jpg|315px|right]] [[image:June 10 2.jpg|315px|center]]<br><br />
[[image:DSC00838.jpg|315px|left]] [[image:BioBrick Assembly Manual.jpg|315px|right]] [[image:DSC00859.jpg|315px|center]]<br><br><br><br />
<br />
=='''Lab'''==<br />
<br />
[[image:DSC00861.jpg|315px|left]] [[image:Setup2.jpg|315px|right]] [[image:setup1.jpg|315px|center]]<br><br />
[[image:DSC00844.JPG|315px|left]] [[image:DSC00843.JPG|315px|right]] [[image:DSC00841.JPG|315px|center]]<br><br />
[[image:DSC00855.JPG|315px|left]] [[image:DSC00849.JPG|315px|right]] [[image:DSC00854.JPG|315px|center]]<br><br />
[[image:DSC00857.JPG|315px|left]] [[image:DSC00862.jpg|315px|right]] [[image:DSC00845.jpg|315px|center]]<br><br />
[[image:setup3.JPG|315px]]<br><br />
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=='''People'''==<br />
[[image:DSC00839.jpg|315px]] [[image:DSC00864.jpg|315px]]<br><br />
<br />
<br />
=='''Bacteria'''==<br />
[[image:DSC00856.JPG|315px|left]] [[image:DSC00837.JPG|315px|right]] [[image:DSC00851.JPG|315px|center]]<br><br />
[[image:DSC00852.JPG|315px|left]] [[image:LB Media.jpg|315px|right]] [[image:sphaeroides tissue flask.jpg|315px|center]]<br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
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<div style="text-align: center;"><br />
{{WashUbottom}}</div>Brendan1http://2009.igem.org/Team:Wash_U/PicturesTeam:Wash U/Pictures2009-10-22T03:06:20Z<p>Brendan1: /* Bacteria */</p>
<hr />
<div>{{WashUback}}<br />
<br />
<div align=left><br />
<font size="4"><br />
=='''Slideshow'''==<br />
<html><center><br />
<iframe align="center" src="http://www.flickr.com/slideShow/index.gne?user_id=39966176@N07" width="500" height="500" frameBorder="0" scrolling="no"></iframe><br /></a><br />
</html><br />
<div align=left><br />
<br />
<br />
<br />
=='''Planning'''==<br />
<br />
[[image:Left Board 1.jpg|315px|left]] [[image:Right Board 1.jpg|315px|right]] [[image:Left Board 2.jpg|315px|center]]<br><br />
[[image:Right Board 2.jpg|315px|left]] [[image:June 10 1.jpg|315px|right]] [[image:June 10 2.jpg|315px|center]]<br><br />
[[image:DSC00838.jpg|315px|left]] [[image:BioBrick Assembly Manual.jpg|315px|right]] [[image:DSC00859.jpg|315px|center]]<br><br><br><br />
<br />
=='''Lab'''==<br />
<br />
[[image:DSC00861.jpg|315px|left]] [[image:Setup2.jpg|315px|right]] [[image:setup1.jpg|315px|center]]<br><br />
[[image:DSC00844.JPG|315px|left]] [[image:DSC00843.JPG|315px|right]] [[image:DSC00841.JPG|315px|center]]<br><br />
[[image:DSC00855.JPG|315px|left]] [[image:DSC00849.JPG|315px|right]] [[image:DSC00854.JPG|315px|center]]<br><br />
[[image:DSC00857.JPG|315px|left]] [[image:DSC00862.jpg|315px|right]] [[image:DSC00845.jpg|315px|center]]<br><br />
[[image:setup3.JPG|315px]]<br><br />
<br />
<br />
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=='''People'''==<br />
[[image:DSC00839.jpg|315px]] [[image:DSC00864.jpg|315px]]<br><br />
<br />
<br />
=='''Bacteria'''==<br />
[[image:DSC00856.JPG|315px|left]] [[image:DSC00837.JPG|315px|right]] [[image:DSC00851.JPG|315px|center]]<br><br />
[[image:DSC00852.JPG|315px|left]] [[image:LB Media.jpg|315px|right]] [[image:sphaeroides tissue flask.jpg|315px|center]]<br><br><br><br><br><br><br><br><br><br><br><br><br><br />
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<div style="text-align: center;"><br />
{{WashUbottom}}</div>Brendan1http://2009.igem.org/Team:Wash_U/PicturesTeam:Wash U/Pictures2009-10-22T03:06:04Z<p>Brendan1: /* Bacteria */</p>
<hr />
<div>{{WashUback}}<br />
<br />
<div align=left><br />
<font size="4"><br />
=='''Slideshow'''==<br />
<html><center><br />
<iframe align="center" src="http://www.flickr.com/slideShow/index.gne?user_id=39966176@N07" width="500" height="500" frameBorder="0" scrolling="no"></iframe><br /></a><br />
</html><br />
<div align=left><br />
<br />
<br />
<br />
=='''Planning'''==<br />
<br />
[[image:Left Board 1.jpg|315px|left]] [[image:Right Board 1.jpg|315px|right]] [[image:Left Board 2.jpg|315px|center]]<br><br />
[[image:Right Board 2.jpg|315px|left]] [[image:June 10 1.jpg|315px|right]] [[image:June 10 2.jpg|315px|center]]<br><br />
[[image:DSC00838.jpg|315px|left]] [[image:BioBrick Assembly Manual.jpg|315px|right]] [[image:DSC00859.jpg|315px|center]]<br><br><br><br />
<br />
=='''Lab'''==<br />
<br />
[[image:DSC00861.jpg|315px|left]] [[image:Setup2.jpg|315px|right]] [[image:setup1.jpg|315px|center]]<br><br />
[[image:DSC00844.JPG|315px|left]] [[image:DSC00843.JPG|315px|right]] [[image:DSC00841.JPG|315px|center]]<br><br />
[[image:DSC00855.JPG|315px|left]] [[image:DSC00849.JPG|315px|right]] [[image:DSC00854.JPG|315px|center]]<br><br />
[[image:DSC00857.JPG|315px|left]] [[image:DSC00862.jpg|315px|right]] [[image:DSC00845.jpg|315px|center]]<br><br />
[[image:setup3.JPG|315px]]<br><br />
<br />
<br />
<br />
=='''People'''==<br />
[[image:DSC00839.jpg|315px]] [[image:DSC00864.jpg|315px]]<br><br />
<br />
<br />
=='''Bacteria'''==<br />
[[image:DSC00856.JPG|315px|left]] [[image:DSC00837.JPG|315px|right]] [[image:DSC00851.JPG|315px|center]]<br><br />
[[image:DSC00852.JPG|315px|left]] [[image:LB Media.jpg|315px|right]] [[image:sphaeroides tissue flask.jpg|315px|center]]<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
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{{WashUbottom}}</div>Brendan1http://2009.igem.org/Team:Wash_U/PicturesTeam:Wash U/Pictures2009-10-22T03:05:46Z<p>Brendan1: /* Bacteria */</p>
<hr />
<div>{{WashUback}}<br />
<br />
<div align=left><br />
<font size="4"><br />
=='''Slideshow'''==<br />
<html><center><br />
<iframe align="center" src="http://www.flickr.com/slideShow/index.gne?user_id=39966176@N07" width="500" height="500" frameBorder="0" scrolling="no"></iframe><br /></a><br />
</html><br />
<div align=left><br />
<br />
<br />
<br />
=='''Planning'''==<br />
<br />
[[image:Left Board 1.jpg|315px|left]] [[image:Right Board 1.jpg|315px|right]] [[image:Left Board 2.jpg|315px|center]]<br><br />
[[image:Right Board 2.jpg|315px|left]] [[image:June 10 1.jpg|315px|right]] [[image:June 10 2.jpg|315px|center]]<br><br />
[[image:DSC00838.jpg|315px|left]] [[image:BioBrick Assembly Manual.jpg|315px|right]] [[image:DSC00859.jpg|315px|center]]<br><br><br><br />
<br />
=='''Lab'''==<br />
<br />
[[image:DSC00861.jpg|315px|left]] [[image:Setup2.jpg|315px|right]] [[image:setup1.jpg|315px|center]]<br><br />
[[image:DSC00844.JPG|315px|left]] [[image:DSC00843.JPG|315px|right]] [[image:DSC00841.JPG|315px|center]]<br><br />
[[image:DSC00855.JPG|315px|left]] [[image:DSC00849.JPG|315px|right]] [[image:DSC00854.JPG|315px|center]]<br><br />
[[image:DSC00857.JPG|315px|left]] [[image:DSC00862.jpg|315px|right]] [[image:DSC00845.jpg|315px|center]]<br><br />
[[image:setup3.JPG|315px]]<br><br />
<br />
<br />
<br />
=='''People'''==<br />
[[image:DSC00839.jpg|315px]] [[image:DSC00864.jpg|315px]]<br><br />
<br />
<br />
=='''Bacteria'''==<br />
[[image:DSC00856.JPG|315px|left]] [[image:DSC00837.JPG|315px|right]] [[image:DSC00851.JPG|315px|center]]<br><br />
[[image:DSC00852.JPG|315px|left]] [[image:LB Media.jpg|315px|right]] [[image:sphaeroides tissue flask.jpg|315px|center]]<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
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<div style="text-align: center;"><br />
{{WashUbottom}}</div>Brendan1http://2009.igem.org/Team:Wash_U/PicturesTeam:Wash U/Pictures2009-10-22T03:04:26Z<p>Brendan1: /* Bacteria */</p>
<hr />
<div>{{WashUback}}<br />
<br />
<div align=left><br />
<font size="4"><br />
=='''Slideshow'''==<br />
<html><center><br />
<iframe align="center" src="http://www.flickr.com/slideShow/index.gne?user_id=39966176@N07" width="500" height="500" frameBorder="0" scrolling="no"></iframe><br /></a><br />
</html><br />
<div align=left><br />
<br />
<br />
<br />
=='''Planning'''==<br />
<br />
[[image:Left Board 1.jpg|315px|left]] [[image:Right Board 1.jpg|315px|right]] [[image:Left Board 2.jpg|315px|center]]<br><br />
[[image:Right Board 2.jpg|315px|left]] [[image:June 10 1.jpg|315px|right]] [[image:June 10 2.jpg|315px|center]]<br><br />
[[image:DSC00838.jpg|315px|left]] [[image:BioBrick Assembly Manual.jpg|315px|right]] [[image:DSC00859.jpg|315px|center]]<br><br><br><br />
<br />
=='''Lab'''==<br />
<br />
[[image:DSC00861.jpg|315px|left]] [[image:Setup2.jpg|315px|right]] [[image:setup1.jpg|315px|center]]<br><br />
[[image:DSC00844.JPG|315px|left]] [[image:DSC00843.JPG|315px|right]] [[image:DSC00841.JPG|315px|center]]<br><br />
[[image:DSC00855.JPG|315px|left]] [[image:DSC00849.JPG|315px|right]] [[image:DSC00854.JPG|315px|center]]<br><br />
[[image:DSC00857.JPG|315px|left]] [[image:DSC00862.jpg|315px|right]] [[image:DSC00845.jpg|315px|center]]<br><br />
[[image:setup3.JPG|315px]]<br><br />
<br />
<br />
<br />
=='''People'''==<br />
[[image:DSC00839.jpg|315px]] [[image:DSC00864.jpg|315px]]<br><br />
<br />
<br />
=='''Bacteria'''==<br />
[[image:DSC00856.JPG|315px|left]] [[image:DSC00837.JPG|315px|right]] [[image:DSC00851.JPG|315px|center]]<br><br />
[[image:DSC00852.JPG|315px|left]] [[image:LB Media.jpg|315px|right]] [[image:sphaeroides tissue flask.jpg|315px|left]]<br>[[image:dbc tissue flask.jpg|315px|right]]<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
<br />
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<div style="text-align: center;"><br />
{{WashUbottom}}</div>Brendan1http://2009.igem.org/Team:Wash_U/PicturesTeam:Wash U/Pictures2009-10-22T03:03:38Z<p>Brendan1: /* Bacteria */</p>
<hr />
<div>{{WashUback}}<br />
<br />
<div align=left><br />
<font size="4"><br />
=='''Slideshow'''==<br />
<html><center><br />
<iframe align="center" src="http://www.flickr.com/slideShow/index.gne?user_id=39966176@N07" width="500" height="500" frameBorder="0" scrolling="no"></iframe><br /></a><br />
</html><br />
<div align=left><br />
<br />
<br />
<br />
=='''Planning'''==<br />
<br />
[[image:Left Board 1.jpg|315px|left]] [[image:Right Board 1.jpg|315px|right]] [[image:Left Board 2.jpg|315px|center]]<br><br />
[[image:Right Board 2.jpg|315px|left]] [[image:June 10 1.jpg|315px|right]] [[image:June 10 2.jpg|315px|center]]<br><br />
[[image:DSC00838.jpg|315px|left]] [[image:BioBrick Assembly Manual.jpg|315px|right]] [[image:DSC00859.jpg|315px|center]]<br><br><br><br />
<br />
=='''Lab'''==<br />
<br />
[[image:DSC00861.jpg|315px|left]] [[image:Setup2.jpg|315px|right]] [[image:setup1.jpg|315px|center]]<br><br />
[[image:DSC00844.JPG|315px|left]] [[image:DSC00843.JPG|315px|right]] [[image:DSC00841.JPG|315px|center]]<br><br />
[[image:DSC00855.JPG|315px|left]] [[image:DSC00849.JPG|315px|right]] [[image:DSC00854.JPG|315px|center]]<br><br />
[[image:DSC00857.JPG|315px|left]] [[image:DSC00862.jpg|315px|right]] [[image:DSC00845.jpg|315px|center]]<br><br />
[[image:setup3.JPG|315px]]<br><br />
<br />
<br />
<br />
=='''People'''==<br />
[[image:DSC00839.jpg|315px]] [[image:DSC00864.jpg|315px]]<br><br />
<br />
<br />
=='''Bacteria'''==<br />
[[image:DSC00856.JPG|315px|left]] [[image:DSC00837.JPG|315px|right]] [[image:DSC00851.JPG|315px|center]]<br><br />
[[image:DSC00852.JPG|315px|left]] [[image:LB Media.jpg|315px|right]] [[image:sphaeroides tissue flask.jpg|315px|left]]<br>[[image:dbc tissue flask.jpg|315px|right]]<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<br />
{{WashUbottom}}</div>Brendan1http://2009.igem.org/Team:Wash_U/ProjectTeam:Wash U/Project2009-10-22T03:01:29Z<p>Brendan1: /* Results */</p>
<hr />
<div>{{WashUback}}<br />
__NOTOC__<br />
<br />
<br />
<div style="text-align: left;"> <font size="4" style="color:black"><br />
== '''Introduction''' == <br />
<font size="2"><br />
Our project goal is to maximize the photosynthetic productivity for a series of photobioreactors containing ''Rhodobacter sphaeroides'' under both high and low light intensities by synthetically regulating the size of the light-harvesting antenna complex LH2. We chose to undertake such a project in ''R. sphaeroides'' due to its well-characterized photosynthetic and genetic system. <br />
<br />
The antenna system functions to expand the spectrum of light available for photosynthesis by absorbing different wavelengths than that of the reaction center. Essentially, it is like the dish around the main receiver of any antenna. Marine bacteria, such as ''R. sphaeroides'', evolved very large antenna complexes to absorb light in a natural environment where there is great competition for photons. As a result, the photosynthetic machinery is saturated at low light intensities in a synthetic non-competitive environment, such as a photobioreactor. This causes up to 95% of incidental photons to be dissipated as heat or fluorescence by bacteria through a process called Non-Photochemical Quenching (NPQ) (Mussgnug et al., 2007). In essence, these photons are being wasted as NPQ reduces light penetration into and across bioreactors and starves the shaded cells for for photons. One method that has been shown to improve photosynthetic productivity in photobioreactors is the reduction of light-harvesting antenna sizes (Polle et al., 2002, Mussgnug et al., 2007). Though, current approaches to this end are difficult to precisely control from the perspective of biological engineering and synthetic biology. <br />
<br />
Our intention is to create a dynamic system to vary antenna size that is dependent on incident light intensity and that can be readily optimized using bioengineering principles. This synthetic regulation of the LH2 complex should result in the bacteria grown under high light intensities expressing fewer LH2 complexes than the cells that are more shaded, leading to a reduction of wasted incident photons while maintaining a high overall absorbance across subsequent bioreactors. Consequently, we expect to see an increase in the total photosynthetic productivity for our mutant ''Rhodobacter sphaeroides'' when compared to the wild type grown under similar conditions. <br />
<br />
<div style="text-align: center;"><br />
{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
|<br />
|<br />
|<br />
|<font size="2" style="color:black;">'''Wild Type Energy Production in a series of Bioreactors'''<br />
|<br />
|<br />
| <br />
|<font size="2" style="color:black;">'''Synthetically Regulated Antenna Energy Production in Bioreactors'''<br />
|<br />
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|}<br />
<br />
[[Image:Slide09b.jpg| 450px | left]][[Image:Slide10 a.jpg| 450px | right]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
<div style="text-align: left;"><br />
<br />
[https://2009.igem.org/Team:Wash_U/Project Back To Top]<br />
<br />
<br />
<font size="4"><br />
<br />
== '''Organism''' ==<br />
<font size="2"><br />
''Rhodobacter sphaeroides'' is a purple Alphaproteobacteria. It is flexible metabolically and can grow heterotrophically via aerobic and anaerobic respiration, as well as phototrophically under anaerobic conditions with light. <br />
<br> <br><br />
''R. sphaeroides'' is one of the best understood photosynthetic organisms. The photosystem is located in intracytoplasmic membrane invaginations and the main components are the Light Haresting Complex 2 (LH2), Light Harvesting Complex 1 (LH1), and the Reaction Center (RC). These pigment-protein complexes non-covalently bind bacteriochlorophylls and carotenoids. LH1 and RC make up the core complex and are often found in the ratio 1:1. LH2 is more peripheral and naturally ranges in the ratio to LH1/RC of 3.0:1 to 6.7:1 under varying light conditions (Scheuring et al., 2005). LH2 absorbs photons maximally at the wavelengths of 850 and 800 nm and funnels its energy to LH1 and the reaction center for photochemistry. (Scheuring et al., 2005). <br />
<br><br><br />
The two subunits of LH2 are coded for by the pucB/A genes and are naturally promoted by the puc promoter. <br />
<br />
<br><div style="text-align: center;">'''ICM of ''R. sphaeroides'' '''[[Image:R sphaeroides photosystem.png| 500 px | center]]<center>source:Sener et al.2007<br />
<br><br />
<br />
<div style="text-align: center;"><br />
{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
|<br />
|<br />
|<br />
|<font size="2" style="color:black;">'''LH2 Absorption Spectra'''<br />
|<br />
|<br />
| <br />
|<font size="2" style="color:black;">'''Plate of ''R. sphaeroides'' 2.4.1'''<br />
|<br />
|<br />
|<br />
|}<br />
<br />
[[Image:R sphaeroides aborbance.png| 450px | left]][[Image:R sphaeroides 0986.png| 450px | right]]<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br>source: Walz et al. 1998<br><br />
<div style="text-align: left;"><br />
<br />
<font size="2"><br />
<br />
====Online Resources for ''R. sphaeroides''====<br />
[http://www.kazusa.or.jp/codon/cgi-bin/showcodon.cgi?species=272943 Codon Usage Table for ''R. sphaeroides'' 2.41]<br />
<br> [http://www.ncbi.nlm.nih.gov/sites/entrez?db=genome&cmd=Retrieve&dopt=Overview&list_uids=18843 ''R. sphaeroides'' 2.41 NCBI Genome Tools]<br />
<br> [http://archaea.ucsc.edu/cgi-bin/hgPcr?wp_target=&db=0&org=Rhodobacter+sphaeroides+2+4+1&wp_f=&wp_r=&wp_size=2000&wp_perfect=15&wp_good=15&wp_showPage=true&hgsid=159191 ''In silico'' PCR of ''R. sphaeroides'' 2.41 genome]<br />
<br />
<br />
[https://2009.igem.org/Team:Wash_U/Project Back To Top]<br />
<br />
<br />
<font size="4"><br />
<br />
=='''Regulation'''==<br />
<font size="2"><br />
[[Image:pucregulation-fullpanels.png|950px]]<br />
<br />
<br />
'''The Natural System (Left)''' Under high oxygen tension, PpsR prevents transcription from the puc promoter of the pucBA genes, and thus prevents the expression of LH2(1st panel). Under low oxygen tension, this repression is removed by activated AppA, thus allowing expression of LH2 from pucBA(2nd panel).<br><br />
'''Synthetic Regulation System (Right)''' Our system takes advantage of constitutive protein expression from the puc promoter at low oxygen tension, and replaces pucBA with Cph8 at this site. Placing pucBA expression under the control of the ompC promoter allows for Cph8/OmpR control of LH2 expression under varying light conditions. Under high intensity of incident light, Cph8 will not be autophosphorylated, preventing expression of pucBA (3rd panel). When light intensity is lower, Cph8 will autophosphorylate, thus phosphorylating and activating OmpR and promoting expression of LH2 from the ompC promoter.<br><br />
This regulation system was designed such that light is able to penetrate deeper into and through photobioreactors where light is plentiful.<br />
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<br>[https://2009.igem.org/Team:Wash_U/Project Back To Top]<br />
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== '''Results''' ==<br />
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For methods, materials, and procedures regarding these results, please see [https://2009.igem.org/Team:Wash_U/Protocol#Tissue_Flask_Experiment Tissue Flask Experiment] or [https://2009.igem.org/Team:Wash_U/Biological_Parts#Modeling Modeling]<br />
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<div style="text-align: center;"> <br />
'''Figure 1'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:#black;">'''a''' Tissue Flask Position 1 Optical Density of Wild Type and DCBomega<br />
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|<font size="2" style="color:#black;">'''b''' Tissue Flask Position 2 Optical Density of Wild Type and DCBomega<br />
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[[Image:Tissue Flask 1b.png| 450 px| left]][[Image:Tissue Flask 2b.png| 450px | right]]<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:#black;">'''c''' Tissue Flask Position 3 Optical Density of Wild Type and DCBomega<br />
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|<font size="2" style="color:#black;">'''d''' Tissue Flask Position 4 Optical Density of Wild Type and DCBomega<br />
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[[Image:Tissue Flask 3b.png| 450px | left]][[Image:Tissue Flask 4b.png| 450px | right]]<br />
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'''e''' Tissue Flask Position 5 Optical Density of Wild Type and DBComega<br />
[[Image:Tissue Flask 5b.png| 450px | center]]<br><br />
Figure 1a,b,c,d,e are the optical density of a tissue flask, as assessed at 600 nm, over the course of the 6 days of the experiment.<br />
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'''Figure 2'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:#black;">'''a''' The Change in the Optical Density of the Tissue Flasks for the WT<br />
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|<font size="2" style="color:#black;">'''b''' The Change in the Optical Density of the Tissue Flasks for the WT<br />
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[[Image:3D 241.png| 450 px | left]][[Image:3D DBCOmega2.png| 450 px | right]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
Figure 2a and 2b display the growth of the tissue flasks as measured by OD assessed as absorbance at 600 nm. The graph is organized such that those closest to the "days" axis were closest to the light source (i.e. flask 1 is closer than flask 2).<br />
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'''Figure 3'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:#black;">'''a''' Cumulative Growth of Wild Type Tissue Flasks<br />
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|<font size="2" style="color:#black;">'''b''' Cumulative Growth of the DBComega Tissue Flasks<br />
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<div style="text-align: left;"><br />
[[Image:Bar 241 b.png| 450px | left]][[Image:Bar DBCOmega b.png| 450px | right]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
Figure 3a and 3b display the cumulative growth of the tissue flasks as measured by the sum of the optical density of the respective cell type's tissue flasks at a given day. Furthermore, the contribution of a given tissue flask to cumulative growth is displayed.<br><br />
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'''Figure 4'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:#black;">'''a''' Wild Type Tissue Flasks<br />
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|<font size="2" style="color:#black;">'''b''' The DBComega Tissue Flasks<br />
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<div style="text-align: left;"><br />
[[image:sphaeroides tissue flask.jpg|450 px|left|]][[image:dbc tissue flask.jpg|450px|right]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
Figure 4A and 4B display the appearance of the tissue flasks for both strains on day 6. They are arranged from left to right with left being closed to the light source (flask 1) and right furthest (flask 5)<br />
<div style="text-align: center;"> <br />
'''Figure 5'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:#black;">'''a''' DBComega Spectrum by Flask Distance from Source<br />
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|<font size="2" style="color:#black;">'''b''' R. sphaeroides Spectrum by Flask Distance from Source<br />
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[[Image: WT irradiance 3d.png| 450 px | left]][[Image: DBComega irradiance 3d.png| 450 px | right]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
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Contour of Absolute Irradiance by Flash distance from Source<br />
{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:#black;">'''c''' R. Sphaeroides 2.4.1<br />
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|<font size="2" style="color:#black;">'''d''' DBComega<br />
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<div style="text-align: left;"><br />
[[Image: r sphaeroides day 2 a.jpg| 450px | left]][[Image: dbc day 4b.jpg| 450px | right]]<br />
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<div style="text-align: center;"><br />
'''e''' Wild Type Absolute Irradiance Behind Flask 1: Day 6<br />
[[Image: irradiance WT flask 1 day6.png| 450px | center]]<br />
Caption for 5e<br><br />
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'''Figure 6'''<br><br />
Nonlinear least-squares estimation of WT LH2 saturation curve<br><br />
[[Image: saturation for WT as inferred.png| 450 px]]<br />
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Caption for figure 6<br><br><br />
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'''Figure 7'''<br><br />
Simulated Optical Density of Mutant and Wild Type Bioreactors<br><br />
Layers One and Two . Layers Three, Four and Five<br />
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<div style="text-align: center;"><br />
[[Image: OD after flask 1 and 2.jpg| 200px ]][[Image: OD after 3,4, and 5.jpg| 200px ]]<br><br />
Caption for Figure 7<br />
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[https://2009.igem.org/Team:Wash_U/Project Back To Top]<br />
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== '''Analysis''' ==<br />
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The tissue flask experiment was an interesting way to observe the effects of a full knockout of the Light Harvesting Antenna Complex 2 (LH2) vs the Wild Type on an array of conseuctive photobioreactors. A couple of conclusions can be drawn from this data that can be used in modeling our synthetic regulation system for LH2 and in the future design of systems for the optimization of Light Harvesting Antenna size for photobioreactors . <br><br><br />
The most striking difference between the Wild Type Rhodobacter Sphaeroides 2.41 and DBCOmega (LH2 knockout) growth patterns is that the first flask in the WT (closest to the light source) grew less than the second flask, while the converse was true in DBCOmega (figure 2a and 2b). This can be attributed to the photosystem saturation curves for the respective cultures. The Wild Type has an LH2 complex, meaning that their antenna size is inherently larger and that their photosystem will be saturated at lower light intensities than the LH2 deficient mutant. We observed that the incident light intensity used in this experiment led to oversaturation for the first WT tissue flask and resulted in growth-inhibiting photodamage, as is evidenced by its lesser growth relative to the second culture. In contrast, photodamage was not observed in DBCOmega as is evidenced by the first tissue flask that grew at the fastest respective rate. Furthermore, it appears that this photodamage slowed the growth of the first WT tissue flask to the point that the OD of this tissue flask was nearly equal to that of DBCOmega after 5 days of growth(.987 vs. .967) (figure 1a). As such, the contribution of LH2 to the growth of the WT in flask 1 can be assessed to be little after day 3 when the growth curve slows- though, this light (at 800 and 850 nm) was still greatly absorbed by flask 1 (Figure 5e). It is likely that much of this light was then wasted through Non-Photochemical Quenching (NPQ), depleting the flasks behind flask 1 of photons at the LH2 absorbtion peaks and decreasing the cumulative productivity of flasks 1-5. This effect of wasting photons though NPQ under high light intensities is one that we sought to minimize in the design of our synthetic regulation for the pucB/A genes. <br><br><br />
<br />
A further a comparison of the WT and DBCOmega experiments reveals that the 3rd tissue flask in each had almost identical growth rates (figure 1c), while the DBCOmega 4th and 5th tissue flask outperformed that of the WT (figure 1d and 1e). This can simply be attributed to the higher density of cells in the 2nd flask of the WT that led to an overall lesser quantity of photons passing through to the 4th and 5th flasks in the WT experiment and likely resulting in heterotrophic growth (though this will need to be confirmed with a "dark control" experiment that should be completed in time for the Jamboree). This decrease in available photons for the WT can be seen by viewing the absolute irradiance data (incident light intensity at a given wavelength) after the third culture for the WT (figure 5a and 5c). This decrease in irradiance is the most pronounced for the LH2 absorption bands at 800 and 850 nm, as was expected. Our synthetic regulation system for pucB/A was designed with the intention of allowing greater penetration of light through the first couple of flasks at the LH2 wavelengths, as expression of pucB/A is inversely correlated to incident light intensity.<br><br><br />
'''Thomas, add in some further comments about the sprectroradiometer stuff. Specifically about how it changes in the WT vs. that of DBCOmega using some specific examples on the graphs'''<br><br><br />
Overall, the cumulative culture growth of the WT exceeded that of DBCOmega (figure 3) due to the performance of the second tissue flask of the WT relative to that of DBCOmega (figure 1b) despite the photodamage that occurred in tissue flask 1 for the WT. This cumulative culture growth can be considered photosynthetic productivty and is proportional to the amount of energy/desired product that can be obtained from these cultures. <br><br><br />
'''It is an interesting exercise to model how our mutant system would perform under these same conditions: Thomas add in some comments about referencing that diagram and what we took from this tissue flask experiment to construct that (and upload it when you are done with it).'''<br />
<br><br><br />
'''add stuff here about the second flasks and approaching 20% contribution of LH1 to growth'''<br />
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=='''Conclusion and Future Work'''==<br />
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Based on model, should have had feedback mechanism- should be something that sets a minimum expression level for LH2, that is...<br><br />
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-tissue flask w prkcbc3 before jamboree and compare/interpret using characterization data<br><br />
-tissue flask w. full mutant in future- if this doesn't reveal cph8 to responding in an exponentially decaying fashion to light intensity, try w. a system that would<br />
-characterize puc promoter under light conditions and additional oxygen tensions<br />
-complete other characterization work<br />
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[https://2009.igem.org/Team:Wash_U/Project Back To Top]<br />
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== '''References''' ==<br />
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# "2007-2008 Catalog & Technical Reference." <u>New England BioLabs Inc.</u> (2007).<br />
# Alon, Uri. <u>Introduction to systems biology and the design principles of biological networks</u>. Boca Raton, FL: Chapman & Hall, 2006.<br />
# Ausubel, Fred et al. <u>Short Protocols in Molecular Biology: Third Edition.</u> Canada: John Wiley and Sons, Inc., 1999.<br />
# "BioBrick Assembly Help." <u>Registry of Standard Biological Parts.</u> 2009. 6 July 2009. <http://partsregistry.org/Main_Page>.<br />
# Bower, James M. <u>Computational Modeling of Genetic and Biochemical Networks (Computational Molecular Biology).</u> New York: M.I.T. PRESS, 2001.<br />
# Braatsch, Stephan et al. "A single Flavoprotein AppA, Integrates Both Redox and Light Signals in ''Rhodobacter sphaeroides.''" <u>Molecular Microbiology.</u> Vol. 45.3 (2002): 827-836.<br />
# Brown, Stanley B. et al. "Bile Pigment Synthesis in Plants: Incorporation of Haem into Phycocyanobilin and Phycocyanobiliproteins in Cyanidium Caldarium." <u>Biochemistry.</u> Vol. 194 (1981): 137-147.<br />
# Dragnea, Vladimira et al. "Time-Resolved Spectroscopic Studies of the AppA Blue-Light Receptor BLUF Domain from ''Rhodobacter sphaeroides'' <u>Biochemistry.</u> Vol. 44 (2005): 15,978-15,985.<br />
# "Entrez Nucleotide Search." <u>National Center for Biotechnology Information.</u> 2009. 6 June 2009 <http://www.ncbi.nlm.nih.gov/nuccore/49175990?from=3533887&to=3534606&report=gbwithparts>.<br />
# Fowler, Gregory J. S. and C. Neil Hunter. "The Synthesis and Assembly of Functional High and Low Light LH2 Antenna Complexes from ''Rhodopseudomonas palustris'' in ''Rhodobacter sphaeroides.''" <u>The Journal of Biological Chemistry.</u> Vol. 271.23 (1996): 13,356-13,361.<br />
# Gabrielsen, Mads et al. "Peripheral Complexes of Purple Bacteria." <u>The Purple Phototropic Bacteria.</u> (2009): 135-153.<br />
# "GenElute HP Plasmid Miniprep Kit: User Guide." <u>Sigma-Aldrich</u>. (2008).<br />
# Harwood, Caroline S. "Nitrogenase-Catalyzed Hydrogen Production by Purple Nonsulfur Photosynthetic Bacteria." <u>Bioenergy.</u> (2008): 259-271.<br />
# Jager, Andreas et al. "The AppA and PpsR Proteins from ''Rhodobacter sphaeroides'' Can Establish a Redox-Dependent Signal Chain but Fail to Transmit Blue-Light Signals in Other Bacteria." <u>Journal of Bacteriology.</u> Vol. 189.6 (2007): 2,274-2,282.<br />
# Jones, M. R. et al. "Mutants of ''Rhodobacter sphaeroides'' Lacking One or More Pigment-Protein Complexes and Complementation with Reaction-Centre, LH1, and LH2 Genes." <u>Molecular Microbiology.</u> Vol. 6.9 (1992): 1,173-1,184.<br />
# Kondo, Toshihiko et al. "Enhancement of Hydrogen Production by a Photosynthetic Bacterium Mutant with Reduced Pigment." <u>Journal of Bioscience and Bioengineering.</u> Vol. 93.2 (2002): 145-150.<br />
# Lagarias, J. Clark. <u>PCB from Spirulina</u>. Personal Communication. June 2009.<br />
# Laible, Philip D. "Foreign Gene Expression in Photosynthetic Bacteria." <u>The Purple Phototropic Bacteria.</u> (2009): 839-860.<br />
# Lee, Jeong K. and Samuel Kaplan. "Transcriptional Regulation of ''puc'' Operon Expression in ''Rhodobacter sphaeroides.''" <u>Journal of Biological Chemistry.</u> Vol. 270.35 (1995): 20,453-20,458.<br />
# Levskaya, Anselmetal et al. "Engineering ''Escherichia coli'' to See Light." <u>Nature.</u> Vol. 438 (2005): 441-442.<br />
# "Life Sciences Catalog 2009-2010." <u>National Diagnostics.</u> (2009).<br />
# Melis, Anastasios. "Solar Energy Conversion Efficiencies in Photosynthesis: Minimizing the Chlorophyll Antennae to Maximize Efficiency." <u>Plant Science.</u> (2009).<br />
# Mizuno, Takeshi and Shoji Mizushima. "Characterization by Deletion and Localized Mutagenesis in Vitro of the Promoter Region of the ''Escherichia coli'' OmpC Gene and Importance of the Upstream DNA Domain in Positive Regulation by the OmpR Protein." <u>Journal of Bacteriology.</u> Vol. 168.1 (1986): 86-95.<br />
# Moskvin, Oleg V. et al. "Transcriptome Analysis of the ''Rhodobacter sphaeroides'' PspR Regulon: PspR as a Master Regulator of Photosystem Development." <u>Journal of Bacteriology.</u> Vol. 187.6 (2005): 2,148-2,156.<br />
# Mussgnug, Jan H. et al. "Engineering photosynthetic light capture: impacts on improved solar energy to biomass conversion." <u> Plant Biotechnology Journal</u> Vol. 5 Issue 6. (2007) pp. 802-814<br />
# "NEBCutter V2.0." <u>New England BioLabs.</u> 2009. 6 June 2009 <http://tools.neb.com/NEBcutter2/index.php>.<br />
# "OligoAnalyzer 3.1." <u>Integrated DNA Technologies.</u> 2009. 6 June 2009. <http://www.idtdna.com/analyzer/Applications/OligoAnalyzer/><br />
# Sambrook, Joseph and David W. Russell. <u>Molecular Cloning: A Laboratory Manual.</u> Vol. 1-3. New York: Cold Springs Harbor Laboratory Press, 2001.<br />
# Scheuring, Simon and Sturgis, James N. "Chromatic Adaptation of Photosynthetic Membranes" <u>Science</u> Vol. 309 (2005) pp.484-487<br />
# Sener, Melih K. et al. "Atomic-level structural and functional model of a bacterial photosynthetic membrane vesicle" <u>Proceedings of the National Academy of Sciences of the United States of America.</u> Vol. 104, Number 40,pp. 15723-15728<br />
# Shimizu-Sato, Sae et al. "A Light-Switchable Gene Promoter System." <u>Nature Publishing Group.</u> Vol. 20 (2002): 1,041-1044.<br />
# Smith, Harry and M. Geoffrey Holmes. <u>Techniquies in Photomorphogenesis.</u> London: Academic Press, Inc., 1984.<br />
# <u>System modeling in cellular biology from concepts to nuts and bolts.</u> Cambridge, MA: MIT P, 2006.<br />
# Terry, Matthew J. "Biosynthesis and Analysis of Bilins." <u>Heme, Chlorophyll, and Bilins: Methods and Protocols.</u> (2002) 273-291. <br />
# Wang, Wanneng et al. "Heterologous Synthesis and Assembly of Functional LHII Antenna Complexes from ''Rhodovulum sulfidophilum'' in ''Rhodobacter sphaeroides'' Mutant." <u>Molecular Biology Reports.</u> Vol. 12.3 (2008).<br />
# Walz, Thomas et al. "Projection Structures of Three Photosynthetic Complexes from Rhodobacter sphaeroides: LH2 at 6 A, LH1 and RC-LH1 at 25 A" <u>Journal of Molecular Biology</u> Vol. 282 pp. 833-845<br />
# Yakovlev, A. G. et al. "Light Control Over the Size of an Antenna Unit Building Block as an Efficient Strategy for Light Harvesting in Photosynthesis." <u>Federation of European Biochemical Societies.</u> (2002):129-132.<br />
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{{WashUbottom}}</div>Brendan1http://2009.igem.org/Team:Wash_U/ProjectTeam:Wash U/Project2009-10-22T03:00:19Z<p>Brendan1: /* Results */</p>
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__NOTOC__<br />
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== '''Introduction''' == <br />
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Our project goal is to maximize the photosynthetic productivity for a series of photobioreactors containing ''Rhodobacter sphaeroides'' under both high and low light intensities by synthetically regulating the size of the light-harvesting antenna complex LH2. We chose to undertake such a project in ''R. sphaeroides'' due to its well-characterized photosynthetic and genetic system. <br />
<br />
The antenna system functions to expand the spectrum of light available for photosynthesis by absorbing different wavelengths than that of the reaction center. Essentially, it is like the dish around the main receiver of any antenna. Marine bacteria, such as ''R. sphaeroides'', evolved very large antenna complexes to absorb light in a natural environment where there is great competition for photons. As a result, the photosynthetic machinery is saturated at low light intensities in a synthetic non-competitive environment, such as a photobioreactor. This causes up to 95% of incidental photons to be dissipated as heat or fluorescence by bacteria through a process called Non-Photochemical Quenching (NPQ) (Mussgnug et al., 2007). In essence, these photons are being wasted as NPQ reduces light penetration into and across bioreactors and starves the shaded cells for for photons. One method that has been shown to improve photosynthetic productivity in photobioreactors is the reduction of light-harvesting antenna sizes (Polle et al., 2002, Mussgnug et al., 2007). Though, current approaches to this end are difficult to precisely control from the perspective of biological engineering and synthetic biology. <br />
<br />
Our intention is to create a dynamic system to vary antenna size that is dependent on incident light intensity and that can be readily optimized using bioengineering principles. This synthetic regulation of the LH2 complex should result in the bacteria grown under high light intensities expressing fewer LH2 complexes than the cells that are more shaded, leading to a reduction of wasted incident photons while maintaining a high overall absorbance across subsequent bioreactors. Consequently, we expect to see an increase in the total photosynthetic productivity for our mutant ''Rhodobacter sphaeroides'' when compared to the wild type grown under similar conditions. <br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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[[Image:Slide09b.jpg| 450px | left]][[Image:Slide10 a.jpg| 450px | right]]<br />
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== '''Organism''' ==<br />
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''Rhodobacter sphaeroides'' is a purple Alphaproteobacteria. It is flexible metabolically and can grow heterotrophically via aerobic and anaerobic respiration, as well as phototrophically under anaerobic conditions with light. <br />
<br> <br><br />
''R. sphaeroides'' is one of the best understood photosynthetic organisms. The photosystem is located in intracytoplasmic membrane invaginations and the main components are the Light Haresting Complex 2 (LH2), Light Harvesting Complex 1 (LH1), and the Reaction Center (RC). These pigment-protein complexes non-covalently bind bacteriochlorophylls and carotenoids. LH1 and RC make up the core complex and are often found in the ratio 1:1. LH2 is more peripheral and naturally ranges in the ratio to LH1/RC of 3.0:1 to 6.7:1 under varying light conditions (Scheuring et al., 2005). LH2 absorbs photons maximally at the wavelengths of 850 and 800 nm and funnels its energy to LH1 and the reaction center for photochemistry. (Scheuring et al., 2005). <br />
<br><br><br />
The two subunits of LH2 are coded for by the pucB/A genes and are naturally promoted by the puc promoter. <br />
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<br><div style="text-align: center;">'''ICM of ''R. sphaeroides'' '''[[Image:R sphaeroides photosystem.png| 500 px | center]]<center>source:Sener et al.2007<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:black;">'''Plate of ''R. sphaeroides'' 2.4.1'''<br />
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[[Image:R sphaeroides aborbance.png| 450px | left]][[Image:R sphaeroides 0986.png| 450px | right]]<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br>source: Walz et al. 1998<br><br />
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====Online Resources for ''R. sphaeroides''====<br />
[http://www.kazusa.or.jp/codon/cgi-bin/showcodon.cgi?species=272943 Codon Usage Table for ''R. sphaeroides'' 2.41]<br />
<br> [http://www.ncbi.nlm.nih.gov/sites/entrez?db=genome&cmd=Retrieve&dopt=Overview&list_uids=18843 ''R. sphaeroides'' 2.41 NCBI Genome Tools]<br />
<br> [http://archaea.ucsc.edu/cgi-bin/hgPcr?wp_target=&db=0&org=Rhodobacter+sphaeroides+2+4+1&wp_f=&wp_r=&wp_size=2000&wp_perfect=15&wp_good=15&wp_showPage=true&hgsid=159191 ''In silico'' PCR of ''R. sphaeroides'' 2.41 genome]<br />
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[https://2009.igem.org/Team:Wash_U/Project Back To Top]<br />
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=='''Regulation'''==<br />
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[[Image:pucregulation-fullpanels.png|950px]]<br />
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'''The Natural System (Left)''' Under high oxygen tension, PpsR prevents transcription from the puc promoter of the pucBA genes, and thus prevents the expression of LH2(1st panel). Under low oxygen tension, this repression is removed by activated AppA, thus allowing expression of LH2 from pucBA(2nd panel).<br><br />
'''Synthetic Regulation System (Right)''' Our system takes advantage of constitutive protein expression from the puc promoter at low oxygen tension, and replaces pucBA with Cph8 at this site. Placing pucBA expression under the control of the ompC promoter allows for Cph8/OmpR control of LH2 expression under varying light conditions. Under high intensity of incident light, Cph8 will not be autophosphorylated, preventing expression of pucBA (3rd panel). When light intensity is lower, Cph8 will autophosphorylate, thus phosphorylating and activating OmpR and promoting expression of LH2 from the ompC promoter.<br><br />
This regulation system was designed such that light is able to penetrate deeper into and through photobioreactors where light is plentiful.<br />
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<br>[https://2009.igem.org/Team:Wash_U/Project Back To Top]<br />
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== '''Results''' ==<br />
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For methods, materials, and procedures regarding these results, please see [https://2009.igem.org/Team:Wash_U/Protocol#Tissue_Flask_Experiment Tissue Flask Experiment] or [https://2009.igem.org/Team:Wash_U/Biological_Parts#Modeling Modeling]<br />
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<div style="text-align: center;"> <br />
'''Figure 1'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:#black;">'''a''' Tissue Flask Position 1 Optical Density of Wild Type and DCBomega<br />
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|<font size="2" style="color:#black;">'''b''' Tissue Flask Position 2 Optical Density of Wild Type and DCBomega<br />
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[[Image:Tissue Flask 1b.png| 450 px| left]][[Image:Tissue Flask 2b.png| 450px | right]]<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:#black;">'''c''' Tissue Flask Position 3 Optical Density of Wild Type and DCBomega<br />
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|<font size="2" style="color:#black;">'''d''' Tissue Flask Position 4 Optical Density of Wild Type and DCBomega<br />
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[[Image:Tissue Flask 3b.png| 450px | left]][[Image:Tissue Flask 4b.png| 450px | right]]<br />
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'''e''' Tissue Flask Position 5 Optical Density of Wild Type and DBComega<br />
[[Image:Tissue Flask 5b.png| 450px | center]]<br><br />
Figure 1a,b,c,d,e are the optical density of a tissue flask, as assessed at 600 nm, over the course of the 6 days of the experiment.<br />
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'''Figure 2'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:#black;">'''a''' The Change in the Optical Density of the Tissue Flasks for the WT<br />
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|<font size="2" style="color:#black;">'''b''' The Change in the Optical Density of the Tissue Flasks for the WT<br />
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[[Image:3D 241.png| 450 px | left]][[Image:3D DBCOmega2.png| 450 px | right]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
Figure 2a and 2b display the growth of the tissue flasks as measured by OD assessed as absorbance at 600 nm. The graph is organized such that those closest to the "days" axis were closest to the light source (i.e. flask 1 is closer than flask 2).<br />
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'''Figure 3'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:#black;">'''a''' Cumulative Growth of Wild Type Tissue Flasks<br />
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|<font size="2" style="color:#black;">'''b''' Cumulative Growth of the DBComega Tissue Flasks<br />
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<div style="text-align: left;"><br />
[[Image:Bar 241 b.png| 450px | left]][[Image:Bar DBCOmega b.png| 450px | right]]<br />
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Figure 3a and 3b display the cumulative growth of the tissue flasks as measured by the sum of the optical density of the respective cell type's tissue flasks at a given day. Furthermore, the contribution of a given tissue flask to cumulative growth is displayed.<br><br />
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'''Figure 4'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:#black;">'''a''' Wild Type Tissue Flasks<br />
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|<font size="2" style="color:#black;">'''b''' The DBComega Tissue Flasks<br />
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<div style="text-align: left;"><br />
[[image:sphaeroides tissue flask.jpg|450 px|left|]][[image:dbc tissue flask.jpg|450px|right]]<br />
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Figure 4A and 4B display the appearance of the tissue flasks for both strains on day 6. They are arranged from left to right with left being closed to the light source (flask 1) and right furthest (flask 5)<br />
<div style="text-align: center;"> <br />
'''Figure 5'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:#black;">'''a''' DBComega Spectrum by Flask Distance from Source<br />
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|<font size="2" style="color:#black;">'''b''' R. sphaeroides Spectrum by Flask Distance from Source<br />
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[[Image: WT irradiance 3d.png| 450 px | left]][[Image: DBComega irradiance 3d.png| 450 px | right]]<br />
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Contour of Absolute Irradiance by Flash distance from Source<br />
{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:#black;">'''c''' R. Sphaeroides 2.4.1<br />
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|<font size="2" style="color:#black;">'''d''' DBComega<br />
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<div style="text-align: left;"><br />
[[Image: r sphaeroides day 2 a.jpg| 450px | left]][[Image: dbc day 4b.jpg| 450px | right]]<br />
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<div style="text-align: center;"><br />
'''e''' Wild Type Absolute Irradiance Behind Flask 1: Day 6<br />
[[Image: irradiance WT flask 1 day6.png| 450px | center]]<br />
Caption for 5e<br><br />
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'''Figure 6'''<br><br />
Nonlinear least-squares estimation of WT LH2 saturation curve<br><br />
[[Image: saturation for WT as inferred.png| 450 px]]<br />
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Caption for figure 6<br><br><br />
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<div style="text-align: center;"> <br />
'''Figure 7'''<br><br />
Simulated Optical Density of Mutant and Wild Type Bioreactors<br><br />
Layers One and Two . Layers Three, Four and Five<br />
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[[Image: OD after flask 1 and 2.jpg| 200px ]][[Image: OD after 3,4, and 5.jpg| 200px ]]<br />
Caption for Figure 7<br />
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[https://2009.igem.org/Team:Wash_U/Project Back To Top]<br />
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== '''Analysis''' ==<br />
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The tissue flask experiment was an interesting way to observe the effects of a full knockout of the Light Harvesting Antenna Complex 2 (LH2) vs the Wild Type on an array of conseuctive photobioreactors. A couple of conclusions can be drawn from this data that can be used in modeling our synthetic regulation system for LH2 and in the future design of systems for the optimization of Light Harvesting Antenna size for photobioreactors . <br><br><br />
The most striking difference between the Wild Type Rhodobacter Sphaeroides 2.41 and DBCOmega (LH2 knockout) growth patterns is that the first flask in the WT (closest to the light source) grew less than the second flask, while the converse was true in DBCOmega (figure 2a and 2b). This can be attributed to the photosystem saturation curves for the respective cultures. The Wild Type has an LH2 complex, meaning that their antenna size is inherently larger and that their photosystem will be saturated at lower light intensities than the LH2 deficient mutant. We observed that the incident light intensity used in this experiment led to oversaturation for the first WT tissue flask and resulted in growth-inhibiting photodamage, as is evidenced by its lesser growth relative to the second culture. In contrast, photodamage was not observed in DBCOmega as is evidenced by the first tissue flask that grew at the fastest respective rate. Furthermore, it appears that this photodamage slowed the growth of the first WT tissue flask to the point that the OD of this tissue flask was nearly equal to that of DBCOmega after 5 days of growth(.987 vs. .967) (figure 1a). As such, the contribution of LH2 to the growth of the WT in flask 1 can be assessed to be little after day 3 when the growth curve slows- though, this light (at 800 and 850 nm) was still greatly absorbed by flask 1 (Figure 5e). It is likely that much of this light was then wasted through Non-Photochemical Quenching (NPQ), depleting the flasks behind flask 1 of photons at the LH2 absorbtion peaks and decreasing the cumulative productivity of flasks 1-5. This effect of wasting photons though NPQ under high light intensities is one that we sought to minimize in the design of our synthetic regulation for the pucB/A genes. <br><br><br />
<br />
A further a comparison of the WT and DBCOmega experiments reveals that the 3rd tissue flask in each had almost identical growth rates (figure 1c), while the DBCOmega 4th and 5th tissue flask outperformed that of the WT (figure 1d and 1e). This can simply be attributed to the higher density of cells in the 2nd flask of the WT that led to an overall lesser quantity of photons passing through to the 4th and 5th flasks in the WT experiment and likely resulting in heterotrophic growth (though this will need to be confirmed with a "dark control" experiment that should be completed in time for the Jamboree). This decrease in available photons for the WT can be seen by viewing the absolute irradiance data (incident light intensity at a given wavelength) after the third culture for the WT (figure 5a and 5c). This decrease in irradiance is the most pronounced for the LH2 absorption bands at 800 and 850 nm, as was expected. Our synthetic regulation system for pucB/A was designed with the intention of allowing greater penetration of light through the first couple of flasks at the LH2 wavelengths, as expression of pucB/A is inversely correlated to incident light intensity.<br><br><br />
'''Thomas, add in some further comments about the sprectroradiometer stuff. Specifically about how it changes in the WT vs. that of DBCOmega using some specific examples on the graphs'''<br><br><br />
Overall, the cumulative culture growth of the WT exceeded that of DBCOmega (figure 3) due to the performance of the second tissue flask of the WT relative to that of DBCOmega (figure 1b) despite the photodamage that occurred in tissue flask 1 for the WT. This cumulative culture growth can be considered photosynthetic productivty and is proportional to the amount of energy/desired product that can be obtained from these cultures. <br><br><br />
'''It is an interesting exercise to model how our mutant system would perform under these same conditions: Thomas add in some comments about referencing that diagram and what we took from this tissue flask experiment to construct that (and upload it when you are done with it).'''<br />
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'''add stuff here about the second flasks and approaching 20% contribution of LH1 to growth'''<br />
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=='''Conclusion and Future Work'''==<br />
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Based on model, should have had feedback mechanism- should be something that sets a minimum expression level for LH2, that is...<br><br />
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-tissue flask w prkcbc3 before jamboree and compare/interpret using characterization data<br><br />
-tissue flask w. full mutant in future- if this doesn't reveal cph8 to responding in an exponentially decaying fashion to light intensity, try w. a system that would<br />
-characterize puc promoter under light conditions and additional oxygen tensions<br />
-complete other characterization work<br />
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[https://2009.igem.org/Team:Wash_U/Project Back To Top]<br />
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== '''References''' ==<br />
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# "2007-2008 Catalog & Technical Reference." <u>New England BioLabs Inc.</u> (2007).<br />
# Alon, Uri. <u>Introduction to systems biology and the design principles of biological networks</u>. Boca Raton, FL: Chapman & Hall, 2006.<br />
# Ausubel, Fred et al. <u>Short Protocols in Molecular Biology: Third Edition.</u> Canada: John Wiley and Sons, Inc., 1999.<br />
# "BioBrick Assembly Help." <u>Registry of Standard Biological Parts.</u> 2009. 6 July 2009. <http://partsregistry.org/Main_Page>.<br />
# Bower, James M. <u>Computational Modeling of Genetic and Biochemical Networks (Computational Molecular Biology).</u> New York: M.I.T. PRESS, 2001.<br />
# Braatsch, Stephan et al. "A single Flavoprotein AppA, Integrates Both Redox and Light Signals in ''Rhodobacter sphaeroides.''" <u>Molecular Microbiology.</u> Vol. 45.3 (2002): 827-836.<br />
# Brown, Stanley B. et al. "Bile Pigment Synthesis in Plants: Incorporation of Haem into Phycocyanobilin and Phycocyanobiliproteins in Cyanidium Caldarium." <u>Biochemistry.</u> Vol. 194 (1981): 137-147.<br />
# Dragnea, Vladimira et al. "Time-Resolved Spectroscopic Studies of the AppA Blue-Light Receptor BLUF Domain from ''Rhodobacter sphaeroides'' <u>Biochemistry.</u> Vol. 44 (2005): 15,978-15,985.<br />
# "Entrez Nucleotide Search." <u>National Center for Biotechnology Information.</u> 2009. 6 June 2009 <http://www.ncbi.nlm.nih.gov/nuccore/49175990?from=3533887&to=3534606&report=gbwithparts>.<br />
# Fowler, Gregory J. S. and C. Neil Hunter. "The Synthesis and Assembly of Functional High and Low Light LH2 Antenna Complexes from ''Rhodopseudomonas palustris'' in ''Rhodobacter sphaeroides.''" <u>The Journal of Biological Chemistry.</u> Vol. 271.23 (1996): 13,356-13,361.<br />
# Gabrielsen, Mads et al. "Peripheral Complexes of Purple Bacteria." <u>The Purple Phototropic Bacteria.</u> (2009): 135-153.<br />
# "GenElute HP Plasmid Miniprep Kit: User Guide." <u>Sigma-Aldrich</u>. (2008).<br />
# Harwood, Caroline S. "Nitrogenase-Catalyzed Hydrogen Production by Purple Nonsulfur Photosynthetic Bacteria." <u>Bioenergy.</u> (2008): 259-271.<br />
# Jager, Andreas et al. "The AppA and PpsR Proteins from ''Rhodobacter sphaeroides'' Can Establish a Redox-Dependent Signal Chain but Fail to Transmit Blue-Light Signals in Other Bacteria." <u>Journal of Bacteriology.</u> Vol. 189.6 (2007): 2,274-2,282.<br />
# Jones, M. R. et al. "Mutants of ''Rhodobacter sphaeroides'' Lacking One or More Pigment-Protein Complexes and Complementation with Reaction-Centre, LH1, and LH2 Genes." <u>Molecular Microbiology.</u> Vol. 6.9 (1992): 1,173-1,184.<br />
# Kondo, Toshihiko et al. "Enhancement of Hydrogen Production by a Photosynthetic Bacterium Mutant with Reduced Pigment." <u>Journal of Bioscience and Bioengineering.</u> Vol. 93.2 (2002): 145-150.<br />
# Lagarias, J. Clark. <u>PCB from Spirulina</u>. Personal Communication. June 2009.<br />
# Laible, Philip D. "Foreign Gene Expression in Photosynthetic Bacteria." <u>The Purple Phototropic Bacteria.</u> (2009): 839-860.<br />
# Lee, Jeong K. and Samuel Kaplan. "Transcriptional Regulation of ''puc'' Operon Expression in ''Rhodobacter sphaeroides.''" <u>Journal of Biological Chemistry.</u> Vol. 270.35 (1995): 20,453-20,458.<br />
# Levskaya, Anselmetal et al. "Engineering ''Escherichia coli'' to See Light." <u>Nature.</u> Vol. 438 (2005): 441-442.<br />
# "Life Sciences Catalog 2009-2010." <u>National Diagnostics.</u> (2009).<br />
# Melis, Anastasios. "Solar Energy Conversion Efficiencies in Photosynthesis: Minimizing the Chlorophyll Antennae to Maximize Efficiency." <u>Plant Science.</u> (2009).<br />
# Mizuno, Takeshi and Shoji Mizushima. "Characterization by Deletion and Localized Mutagenesis in Vitro of the Promoter Region of the ''Escherichia coli'' OmpC Gene and Importance of the Upstream DNA Domain in Positive Regulation by the OmpR Protein." <u>Journal of Bacteriology.</u> Vol. 168.1 (1986): 86-95.<br />
# Moskvin, Oleg V. et al. "Transcriptome Analysis of the ''Rhodobacter sphaeroides'' PspR Regulon: PspR as a Master Regulator of Photosystem Development." <u>Journal of Bacteriology.</u> Vol. 187.6 (2005): 2,148-2,156.<br />
# Mussgnug, Jan H. et al. "Engineering photosynthetic light capture: impacts on improved solar energy to biomass conversion." <u> Plant Biotechnology Journal</u> Vol. 5 Issue 6. (2007) pp. 802-814<br />
# "NEBCutter V2.0." <u>New England BioLabs.</u> 2009. 6 June 2009 <http://tools.neb.com/NEBcutter2/index.php>.<br />
# "OligoAnalyzer 3.1." <u>Integrated DNA Technologies.</u> 2009. 6 June 2009. <http://www.idtdna.com/analyzer/Applications/OligoAnalyzer/><br />
# Sambrook, Joseph and David W. Russell. <u>Molecular Cloning: A Laboratory Manual.</u> Vol. 1-3. New York: Cold Springs Harbor Laboratory Press, 2001.<br />
# Scheuring, Simon and Sturgis, James N. "Chromatic Adaptation of Photosynthetic Membranes" <u>Science</u> Vol. 309 (2005) pp.484-487<br />
# Sener, Melih K. et al. "Atomic-level structural and functional model of a bacterial photosynthetic membrane vesicle" <u>Proceedings of the National Academy of Sciences of the United States of America.</u> Vol. 104, Number 40,pp. 15723-15728<br />
# Shimizu-Sato, Sae et al. "A Light-Switchable Gene Promoter System." <u>Nature Publishing Group.</u> Vol. 20 (2002): 1,041-1044.<br />
# Smith, Harry and M. Geoffrey Holmes. <u>Techniquies in Photomorphogenesis.</u> London: Academic Press, Inc., 1984.<br />
# <u>System modeling in cellular biology from concepts to nuts and bolts.</u> Cambridge, MA: MIT P, 2006.<br />
# Terry, Matthew J. "Biosynthesis and Analysis of Bilins." <u>Heme, Chlorophyll, and Bilins: Methods and Protocols.</u> (2002) 273-291. <br />
# Wang, Wanneng et al. "Heterologous Synthesis and Assembly of Functional LHII Antenna Complexes from ''Rhodovulum sulfidophilum'' in ''Rhodobacter sphaeroides'' Mutant." <u>Molecular Biology Reports.</u> Vol. 12.3 (2008).<br />
# Walz, Thomas et al. "Projection Structures of Three Photosynthetic Complexes from Rhodobacter sphaeroides: LH2 at 6 A, LH1 and RC-LH1 at 25 A" <u>Journal of Molecular Biology</u> Vol. 282 pp. 833-845<br />
# Yakovlev, A. G. et al. "Light Control Over the Size of an Antenna Unit Building Block as an Efficient Strategy for Light Harvesting in Photosynthesis." <u>Federation of European Biochemical Societies.</u> (2002):129-132.<br />
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{{WashUbottom}}</div>Brendan1http://2009.igem.org/Team:Wash_U/ProjectTeam:Wash U/Project2009-10-22T02:46:12Z<p>Brendan1: /* Results */</p>
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__NOTOC__<br />
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== '''Introduction''' == <br />
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Our project goal is to maximize the photosynthetic productivity for a series of photobioreactors containing ''Rhodobacter sphaeroides'' under both high and low light intensities by synthetically regulating the size of the light-harvesting antenna complex LH2. We chose to undertake such a project in ''R. sphaeroides'' due to its well-characterized photosynthetic and genetic system. <br />
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The antenna system functions to expand the spectrum of light available for photosynthesis by absorbing different wavelengths than that of the reaction center. Essentially, it is like the dish around the main receiver of any antenna. Marine bacteria, such as ''R. sphaeroides'', evolved very large antenna complexes to absorb light in a natural environment where there is great competition for photons. As a result, the photosynthetic machinery is saturated at low light intensities in a synthetic non-competitive environment, such as a photobioreactor. This causes up to 95% of incidental photons to be dissipated as heat or fluorescence by bacteria through a process called Non-Photochemical Quenching (NPQ) (Mussgnug et al., 2007). In essence, these photons are being wasted as NPQ reduces light penetration into and across bioreactors and starves the shaded cells for for photons. One method that has been shown to improve photosynthetic productivity in photobioreactors is the reduction of light-harvesting antenna sizes (Polle et al., 2002, Mussgnug et al., 2007). Though, current approaches to this end are difficult to precisely control from the perspective of biological engineering and synthetic biology. <br />
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Our intention is to create a dynamic system to vary antenna size that is dependent on incident light intensity and that can be readily optimized using bioengineering principles. This synthetic regulation of the LH2 complex should result in the bacteria grown under high light intensities expressing fewer LH2 complexes than the cells that are more shaded, leading to a reduction of wasted incident photons while maintaining a high overall absorbance across subsequent bioreactors. Consequently, we expect to see an increase in the total photosynthetic productivity for our mutant ''Rhodobacter sphaeroides'' when compared to the wild type grown under similar conditions. <br />
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[[Image:Slide09b.jpg| 450px | left]][[Image:Slide10 a.jpg| 450px | right]]<br />
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== '''Organism''' ==<br />
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''Rhodobacter sphaeroides'' is a purple Alphaproteobacteria. It is flexible metabolically and can grow heterotrophically via aerobic and anaerobic respiration, as well as phototrophically under anaerobic conditions with light. <br />
<br> <br><br />
''R. sphaeroides'' is one of the best understood photosynthetic organisms. The photosystem is located in intracytoplasmic membrane invaginations and the main components are the Light Haresting Complex 2 (LH2), Light Harvesting Complex 1 (LH1), and the Reaction Center (RC). These pigment-protein complexes non-covalently bind bacteriochlorophylls and carotenoids. LH1 and RC make up the core complex and are often found in the ratio 1:1. LH2 is more peripheral and naturally ranges in the ratio to LH1/RC of 3.0:1 to 6.7:1 under varying light conditions (Scheuring et al., 2005). LH2 absorbs photons maximally at the wavelengths of 850 and 800 nm and funnels its energy to LH1 and the reaction center for photochemistry. (Scheuring et al., 2005). <br />
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The two subunits of LH2 are coded for by the pucB/A genes and are naturally promoted by the puc promoter. <br />
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<br><div style="text-align: center;">'''ICM of ''R. sphaeroides'' '''[[Image:R sphaeroides photosystem.png| 500 px | center]]<center>source:Sener et al.2007<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:black;">'''Plate of ''R. sphaeroides'' 2.4.1'''<br />
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[[Image:R sphaeroides aborbance.png| 450px | left]][[Image:R sphaeroides 0986.png| 450px | right]]<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br>source: Walz et al. 1998<br><br />
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====Online Resources for ''R. sphaeroides''====<br />
[http://www.kazusa.or.jp/codon/cgi-bin/showcodon.cgi?species=272943 Codon Usage Table for ''R. sphaeroides'' 2.41]<br />
<br> [http://www.ncbi.nlm.nih.gov/sites/entrez?db=genome&cmd=Retrieve&dopt=Overview&list_uids=18843 ''R. sphaeroides'' 2.41 NCBI Genome Tools]<br />
<br> [http://archaea.ucsc.edu/cgi-bin/hgPcr?wp_target=&db=0&org=Rhodobacter+sphaeroides+2+4+1&wp_f=&wp_r=&wp_size=2000&wp_perfect=15&wp_good=15&wp_showPage=true&hgsid=159191 ''In silico'' PCR of ''R. sphaeroides'' 2.41 genome]<br />
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[https://2009.igem.org/Team:Wash_U/Project Back To Top]<br />
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=='''Regulation'''==<br />
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[[Image:pucregulation-fullpanels.png|950px]]<br />
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'''The Natural System (Left)''' Under high oxygen tension, PpsR prevents transcription from the puc promoter of the pucBA genes, and thus prevents the expression of LH2(1st panel). Under low oxygen tension, this repression is removed by activated AppA, thus allowing expression of LH2 from pucBA(2nd panel).<br><br />
'''Synthetic Regulation System (Right)''' Our system takes advantage of constitutive protein expression from the puc promoter at low oxygen tension, and replaces pucBA with Cph8 at this site. Placing pucBA expression under the control of the ompC promoter allows for Cph8/OmpR control of LH2 expression under varying light conditions. Under high intensity of incident light, Cph8 will not be autophosphorylated, preventing expression of pucBA (3rd panel). When light intensity is lower, Cph8 will autophosphorylate, thus phosphorylating and activating OmpR and promoting expression of LH2 from the ompC promoter.<br><br />
This regulation system was designed such that light is able to penetrate deeper into and through photobioreactors where light is plentiful.<br />
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<br>[https://2009.igem.org/Team:Wash_U/Project Back To Top]<br />
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== '''Results''' ==<br />
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For methods, materials, and procedures regarding these results, please see [https://2009.igem.org/Team:Wash_U/Protocol#Tissue_Flask_Experiment Tissue Flask Experiment] or [https://2009.igem.org/Team:Wash_U/Biological_Parts#Modeling Modeling]<br />
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'''Figure 1'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:#black;">'''a''' Tissue Flask Position 1 Optical Density of Wild Type and DCBomega<br />
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|<font size="2" style="color:#black;">'''b''' Tissue Flask Position 2 Optical Density of Wild Type and DCBomega<br />
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[[Image:Tissue Flask 1b.png| 450 px| left]][[Image:Tissue Flask 2b.png| 450px | right]]<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
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|<font size="2" style="color:#black;">'''c''' Tissue Flask Position 3 Optical Density of Wild Type and DCBomega<br />
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|<font size="2" style="color:#black;">'''d''' Tissue Flask Position 4 Optical Density of Wild Type and DCBomega<br />
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[[Image:Tissue Flask 3b.png| 450px | left]][[Image:Tissue Flask 4b.png| 450px | right]]<br />
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'''e''' Tissue Flask Position 5 Optical Density of Wild Type and DBComega<br />
[[Image:Tissue Flask 5b.png| 450px | center]]<br><br />
Figure 1a,b,c,d,e are the optical density of a tissue flask, as assessed at 600 nm, over the course of the 6 days of the experiment.<br />
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'''Figure 2'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:#black;">'''a''' The Change in the Optical Density of the Tissue Flasks for the WT<br />
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|<font size="2" style="color:#black;">'''b''' The Change in the Optical Density of the Tissue Flasks for the WT<br />
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[[Image:3D 241.png| 450 px | left]][[Image:3D DBCOmega2.png| 450 px | right]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
Figure 2a and 2b display the growth of the tissue flasks as measured by OD assessed as absorbance at 600 nm. The graph is organized such that those closest to the "days" axis were closest to the light source (i.e. flask 1 is closer than flask 2).<br />
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'''Figure 3'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:#black;">'''a''' Cumulative Growth of Wild Type Tissue Flasks<br />
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|<font size="2" style="color:#black;">'''b''' Cumulative Growth of the DBComega Tissue Flasks<br />
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[[Image:Bar 241 b.png| 450px | left]][[Image:Bar DBCOmega b.png| 450px | right]]<br />
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Figure 3a and 3b display the cumulative growth of the tissue flasks as measured by the sum of the optical density of the respective cell type's tissue flasks at a given day. Furthermore, the contribution of a given tissue flask to cumulative growth is displayed.<br><br />
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'''Figure 4'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:#black;">'''a''' Wild Type Tissue Flasks<br />
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|<font size="2" style="color:#black;">'''b''' The DBComega Tissue Flasks<br />
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[[image:sphaeroides tissue flask.jpg|450 px|left|]][[image:dbc tissue flask.jpg|450px|right]]<br />
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Figure 4A and 4B display the appearance of the tissue flasks for both strains on day 6. They are arranged from left to right with left being closed to the light source (flask 1) and right furthest (flask 5)<br />
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'''Figure 5'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:#black;">'''a''' DBComega Spectrum by Flask Distance from Source<br />
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|<font size="2" style="color:#black;">'''b''' R. sphaeroides Spectrum by Flask Distance from Source<br />
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[[Image: WT irradiance 3d.png| 450 px | left]][[Image: DBComega irradiance 3d.png| 450 px | right]]<br />
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Contour of Absolute Irradiance by Flash distance from Source<br />
{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:#black;">'''c''' R. Sphaeroides 2.4.1<br />
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|<font size="2" style="color:#black;">'''d''' DBComega<br />
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[[Image: r sphaeroides day 2 a.jpg| 450px | left]][[Image: dbc day 4b.jpg| 450px | right]]<br />
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<div style="text-align: center;"><br />
'''e''' Wild Type Absolute Irradiance Behind Flask 1: Day 6<br />
[[Image: irradiance WT flask 1 day6.png| 450px | center]]<br />
Caption for 5e<br><br />
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'''Figure 6'''<br><br />
Nonlinear least-squares estimation of WT LH2 saturation curve<br><br />
[[Image: saturation for WT as inferred.png| 450 px]]<br />
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Caption for figure 6<br><br><br />
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'''Figure 7'''<br><br />
Simulated Optical Density of Mutant and Wild Type Bioreactors<br><br />
Layers One and Two . Layers Three, Four and Five<br />
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[[Image: OD after flask 1 and 2.jpg| 200px ]][[Image: OD after 3,4, and 5.jpg| 200px ]]<br />
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[https://2009.igem.org/Team:Wash_U/Project Back To Top]<br />
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== '''Analysis''' ==<br />
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The tissue flask experiment was an interesting way to observe the effects of a full knockout of the Light Harvesting Antenna Complex 2 (LH2) vs the Wild Type on an array of conseuctive photobioreactors. A couple of conclusions can be drawn from this data that can be used in modeling our synthetic regulation system for LH2 and in the future design of systems for the optimization of Light Harvesting Antenna size for photobioreactors . <br><br><br />
The most striking difference between the Wild Type Rhodobacter Sphaeroides 2.41 and DBCOmega (LH2 knockout) growth patterns is that the first flask in the WT (closest to the light source) grew less than the second flask, while the converse was true in DBCOmega (figure 2a and 2b). This can be attributed to the photosystem saturation curves for the respective cultures. The Wild Type has an LH2 complex, meaning that their antenna size is inherently larger and that their photosystem will be saturated at lower light intensities than the LH2 deficient mutant. We observed that the incident light intensity used in this experiment led to oversaturation for the first WT tissue flask and resulted in growth-inhibiting photodamage, as is evidenced by its lesser growth relative to the second culture. In contrast, photodamage was not observed in DBCOmega as is evidenced by the first tissue flask that grew at the fastest respective rate. Furthermore, it appears that this photodamage slowed the growth of the first WT tissue flask to the point that the OD of this tissue flask was nearly equal to that of DBCOmega after 5 days of growth(.987 vs. .967) (figure 1a). As such, the contribution of LH2 to the growth of the WT in flask 1 can be assessed to be little after day 3 when the growth curve slows- though, this light (at 800 and 850 nm) was still greatly absorbed by flask 1 (Figure 5e). It is likely that much of this light was then wasted through Non-Photochemical Quenching (NPQ), depleting the flasks behind flask 1 of photons at the LH2 absorbtion peaks and decreasing the cumulative productivity of flasks 1-5. This effect of wasting photons though NPQ under high light intensities is one that we sought to minimize in the design of our synthetic regulation for the pucB/A genes. <br><br><br />
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A further a comparison of the WT and DBCOmega experiments reveals that the 3rd tissue flask in each had almost identical growth rates (figure 1c), while the DBCOmega 4th and 5th tissue flask outperformed that of the WT (figure 1d and 1e). This can simply be attributed to the higher density of cells in the 2nd flask of the WT that led to an overall lesser quantity of photons passing through to the 4th and 5th flasks in the WT experiment and likely resulting in heterotrophic growth (though this will need to be confirmed with a "dark control" experiment that should be completed in time for the Jamboree). This decrease in available photons for the WT can be seen by viewing the absolute irradiance data (incident light intensity at a given wavelength) after the third culture for the WT (figure 5a and 5c). This decrease in irradiance is the most pronounced for the LH2 absorption bands at 800 and 850 nm, as was expected. Our synthetic regulation system for pucB/A was designed with the intention of allowing greater penetration of light through the first couple of flasks at the LH2 wavelengths, as expression of pucB/A is inversely correlated to incident light intensity.<br><br><br />
'''Thomas, add in some further comments about the sprectroradiometer stuff. Specifically about how it changes in the WT vs. that of DBCOmega using some specific examples on the graphs'''<br><br><br />
Overall, the cumulative culture growth of the WT exceeded that of DBCOmega (figure 3) due to the performance of the second tissue flask of the WT relative to that of DBCOmega (figure 1b) despite the photodamage that occurred in tissue flask 1 for the WT. This cumulative culture growth can be considered photosynthetic productivty and is proportional to the amount of energy/desired product that can be obtained from these cultures. <br><br><br />
'''It is an interesting exercise to model how our mutant system would perform under these same conditions: Thomas add in some comments about referencing that diagram and what we took from this tissue flask experiment to construct that (and upload it when you are done with it).'''<br />
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'''add stuff here about the second flasks and approaching 20% contribution of LH1 to growth'''<br />
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=='''Conclusion and Future Work'''==<br />
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Based on model, should have had feedback mechanism- should be something that sets a minimum expression level for LH2, that is...<br><br />
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-tissue flask w prkcbc3 before jamboree and compare/interpret using characterization data<br><br />
-tissue flask w. full mutant in future- if this doesn't reveal cph8 to responding in an exponentially decaying fashion to light intensity, try w. a system that would<br />
-characterize puc promoter under light conditions and additional oxygen tensions<br />
-complete other characterization work<br />
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[https://2009.igem.org/Team:Wash_U/Project Back To Top]<br />
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== '''References''' ==<br />
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# "2007-2008 Catalog & Technical Reference." <u>New England BioLabs Inc.</u> (2007).<br />
# Alon, Uri. <u>Introduction to systems biology and the design principles of biological networks</u>. Boca Raton, FL: Chapman & Hall, 2006.<br />
# Ausubel, Fred et al. <u>Short Protocols in Molecular Biology: Third Edition.</u> Canada: John Wiley and Sons, Inc., 1999.<br />
# "BioBrick Assembly Help." <u>Registry of Standard Biological Parts.</u> 2009. 6 July 2009. <http://partsregistry.org/Main_Page>.<br />
# Bower, James M. <u>Computational Modeling of Genetic and Biochemical Networks (Computational Molecular Biology).</u> New York: M.I.T. PRESS, 2001.<br />
# Braatsch, Stephan et al. "A single Flavoprotein AppA, Integrates Both Redox and Light Signals in ''Rhodobacter sphaeroides.''" <u>Molecular Microbiology.</u> Vol. 45.3 (2002): 827-836.<br />
# Brown, Stanley B. et al. "Bile Pigment Synthesis in Plants: Incorporation of Haem into Phycocyanobilin and Phycocyanobiliproteins in Cyanidium Caldarium." <u>Biochemistry.</u> Vol. 194 (1981): 137-147.<br />
# Dragnea, Vladimira et al. "Time-Resolved Spectroscopic Studies of the AppA Blue-Light Receptor BLUF Domain from ''Rhodobacter sphaeroides'' <u>Biochemistry.</u> Vol. 44 (2005): 15,978-15,985.<br />
# "Entrez Nucleotide Search." <u>National Center for Biotechnology Information.</u> 2009. 6 June 2009 <http://www.ncbi.nlm.nih.gov/nuccore/49175990?from=3533887&to=3534606&report=gbwithparts>.<br />
# Fowler, Gregory J. S. and C. Neil Hunter. "The Synthesis and Assembly of Functional High and Low Light LH2 Antenna Complexes from ''Rhodopseudomonas palustris'' in ''Rhodobacter sphaeroides.''" <u>The Journal of Biological Chemistry.</u> Vol. 271.23 (1996): 13,356-13,361.<br />
# Gabrielsen, Mads et al. "Peripheral Complexes of Purple Bacteria." <u>The Purple Phototropic Bacteria.</u> (2009): 135-153.<br />
# "GenElute HP Plasmid Miniprep Kit: User Guide." <u>Sigma-Aldrich</u>. (2008).<br />
# Harwood, Caroline S. "Nitrogenase-Catalyzed Hydrogen Production by Purple Nonsulfur Photosynthetic Bacteria." <u>Bioenergy.</u> (2008): 259-271.<br />
# Jager, Andreas et al. "The AppA and PpsR Proteins from ''Rhodobacter sphaeroides'' Can Establish a Redox-Dependent Signal Chain but Fail to Transmit Blue-Light Signals in Other Bacteria." <u>Journal of Bacteriology.</u> Vol. 189.6 (2007): 2,274-2,282.<br />
# Jones, M. R. et al. "Mutants of ''Rhodobacter sphaeroides'' Lacking One or More Pigment-Protein Complexes and Complementation with Reaction-Centre, LH1, and LH2 Genes." <u>Molecular Microbiology.</u> Vol. 6.9 (1992): 1,173-1,184.<br />
# Kondo, Toshihiko et al. "Enhancement of Hydrogen Production by a Photosynthetic Bacterium Mutant with Reduced Pigment." <u>Journal of Bioscience and Bioengineering.</u> Vol. 93.2 (2002): 145-150.<br />
# Lagarias, J. Clark. <u>PCB from Spirulina</u>. Personal Communication. June 2009.<br />
# Laible, Philip D. "Foreign Gene Expression in Photosynthetic Bacteria." <u>The Purple Phototropic Bacteria.</u> (2009): 839-860.<br />
# Lee, Jeong K. and Samuel Kaplan. "Transcriptional Regulation of ''puc'' Operon Expression in ''Rhodobacter sphaeroides.''" <u>Journal of Biological Chemistry.</u> Vol. 270.35 (1995): 20,453-20,458.<br />
# Levskaya, Anselmetal et al. "Engineering ''Escherichia coli'' to See Light." <u>Nature.</u> Vol. 438 (2005): 441-442.<br />
# "Life Sciences Catalog 2009-2010." <u>National Diagnostics.</u> (2009).<br />
# Melis, Anastasios. "Solar Energy Conversion Efficiencies in Photosynthesis: Minimizing the Chlorophyll Antennae to Maximize Efficiency." <u>Plant Science.</u> (2009).<br />
# Mizuno, Takeshi and Shoji Mizushima. "Characterization by Deletion and Localized Mutagenesis in Vitro of the Promoter Region of the ''Escherichia coli'' OmpC Gene and Importance of the Upstream DNA Domain in Positive Regulation by the OmpR Protein." <u>Journal of Bacteriology.</u> Vol. 168.1 (1986): 86-95.<br />
# Moskvin, Oleg V. et al. "Transcriptome Analysis of the ''Rhodobacter sphaeroides'' PspR Regulon: PspR as a Master Regulator of Photosystem Development." <u>Journal of Bacteriology.</u> Vol. 187.6 (2005): 2,148-2,156.<br />
# Mussgnug, Jan H. et al. "Engineering photosynthetic light capture: impacts on improved solar energy to biomass conversion." <u> Plant Biotechnology Journal</u> Vol. 5 Issue 6. (2007) pp. 802-814<br />
# "NEBCutter V2.0." <u>New England BioLabs.</u> 2009. 6 June 2009 <http://tools.neb.com/NEBcutter2/index.php>.<br />
# "OligoAnalyzer 3.1." <u>Integrated DNA Technologies.</u> 2009. 6 June 2009. <http://www.idtdna.com/analyzer/Applications/OligoAnalyzer/><br />
# Sambrook, Joseph and David W. Russell. <u>Molecular Cloning: A Laboratory Manual.</u> Vol. 1-3. New York: Cold Springs Harbor Laboratory Press, 2001.<br />
# Scheuring, Simon and Sturgis, James N. "Chromatic Adaptation of Photosynthetic Membranes" <u>Science</u> Vol. 309 (2005) pp.484-487<br />
# Sener, Melih K. et al. "Atomic-level structural and functional model of a bacterial photosynthetic membrane vesicle" <u>Proceedings of the National Academy of Sciences of the United States of America.</u> Vol. 104, Number 40,pp. 15723-15728<br />
# Shimizu-Sato, Sae et al. "A Light-Switchable Gene Promoter System." <u>Nature Publishing Group.</u> Vol. 20 (2002): 1,041-1044.<br />
# Smith, Harry and M. Geoffrey Holmes. <u>Techniquies in Photomorphogenesis.</u> London: Academic Press, Inc., 1984.<br />
# <u>System modeling in cellular biology from concepts to nuts and bolts.</u> Cambridge, MA: MIT P, 2006.<br />
# Terry, Matthew J. "Biosynthesis and Analysis of Bilins." <u>Heme, Chlorophyll, and Bilins: Methods and Protocols.</u> (2002) 273-291. <br />
# Wang, Wanneng et al. "Heterologous Synthesis and Assembly of Functional LHII Antenna Complexes from ''Rhodovulum sulfidophilum'' in ''Rhodobacter sphaeroides'' Mutant." <u>Molecular Biology Reports.</u> Vol. 12.3 (2008).<br />
# Walz, Thomas et al. "Projection Structures of Three Photosynthetic Complexes from Rhodobacter sphaeroides: LH2 at 6 A, LH1 and RC-LH1 at 25 A" <u>Journal of Molecular Biology</u> Vol. 282 pp. 833-845<br />
# Yakovlev, A. G. et al. "Light Control Over the Size of an Antenna Unit Building Block as an Efficient Strategy for Light Harvesting in Photosynthesis." <u>Federation of European Biochemical Societies.</u> (2002):129-132.<br />
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{{WashUbottom}}</div>Brendan1http://2009.igem.org/Team:Wash_U/ProjectTeam:Wash U/Project2009-10-22T02:45:48Z<p>Brendan1: /* Results */</p>
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__NOTOC__<br />
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== '''Introduction''' == <br />
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Our project goal is to maximize the photosynthetic productivity for a series of photobioreactors containing ''Rhodobacter sphaeroides'' under both high and low light intensities by synthetically regulating the size of the light-harvesting antenna complex LH2. We chose to undertake such a project in ''R. sphaeroides'' due to its well-characterized photosynthetic and genetic system. <br />
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The antenna system functions to expand the spectrum of light available for photosynthesis by absorbing different wavelengths than that of the reaction center. Essentially, it is like the dish around the main receiver of any antenna. Marine bacteria, such as ''R. sphaeroides'', evolved very large antenna complexes to absorb light in a natural environment where there is great competition for photons. As a result, the photosynthetic machinery is saturated at low light intensities in a synthetic non-competitive environment, such as a photobioreactor. This causes up to 95% of incidental photons to be dissipated as heat or fluorescence by bacteria through a process called Non-Photochemical Quenching (NPQ) (Mussgnug et al., 2007). In essence, these photons are being wasted as NPQ reduces light penetration into and across bioreactors and starves the shaded cells for for photons. One method that has been shown to improve photosynthetic productivity in photobioreactors is the reduction of light-harvesting antenna sizes (Polle et al., 2002, Mussgnug et al., 2007). Though, current approaches to this end are difficult to precisely control from the perspective of biological engineering and synthetic biology. <br />
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Our intention is to create a dynamic system to vary antenna size that is dependent on incident light intensity and that can be readily optimized using bioengineering principles. This synthetic regulation of the LH2 complex should result in the bacteria grown under high light intensities expressing fewer LH2 complexes than the cells that are more shaded, leading to a reduction of wasted incident photons while maintaining a high overall absorbance across subsequent bioreactors. Consequently, we expect to see an increase in the total photosynthetic productivity for our mutant ''Rhodobacter sphaeroides'' when compared to the wild type grown under similar conditions. <br />
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[[Image:Slide09b.jpg| 450px | left]][[Image:Slide10 a.jpg| 450px | right]]<br />
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== '''Organism''' ==<br />
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''Rhodobacter sphaeroides'' is a purple Alphaproteobacteria. It is flexible metabolically and can grow heterotrophically via aerobic and anaerobic respiration, as well as phototrophically under anaerobic conditions with light. <br />
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''R. sphaeroides'' is one of the best understood photosynthetic organisms. The photosystem is located in intracytoplasmic membrane invaginations and the main components are the Light Haresting Complex 2 (LH2), Light Harvesting Complex 1 (LH1), and the Reaction Center (RC). These pigment-protein complexes non-covalently bind bacteriochlorophylls and carotenoids. LH1 and RC make up the core complex and are often found in the ratio 1:1. LH2 is more peripheral and naturally ranges in the ratio to LH1/RC of 3.0:1 to 6.7:1 under varying light conditions (Scheuring et al., 2005). LH2 absorbs photons maximally at the wavelengths of 850 and 800 nm and funnels its energy to LH1 and the reaction center for photochemistry. (Scheuring et al., 2005). <br />
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The two subunits of LH2 are coded for by the pucB/A genes and are naturally promoted by the puc promoter. <br />
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<br><div style="text-align: center;">'''ICM of ''R. sphaeroides'' '''[[Image:R sphaeroides photosystem.png| 500 px | center]]<center>source:Sener et al.2007<br />
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|<font size="2" style="color:black;">'''LH2 Absorption Spectra'''<br />
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|<font size="2" style="color:black;">'''Plate of ''R. sphaeroides'' 2.4.1'''<br />
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[[Image:R sphaeroides aborbance.png| 450px | left]][[Image:R sphaeroides 0986.png| 450px | right]]<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br>source: Walz et al. 1998<br><br />
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====Online Resources for ''R. sphaeroides''====<br />
[http://www.kazusa.or.jp/codon/cgi-bin/showcodon.cgi?species=272943 Codon Usage Table for ''R. sphaeroides'' 2.41]<br />
<br> [http://www.ncbi.nlm.nih.gov/sites/entrez?db=genome&cmd=Retrieve&dopt=Overview&list_uids=18843 ''R. sphaeroides'' 2.41 NCBI Genome Tools]<br />
<br> [http://archaea.ucsc.edu/cgi-bin/hgPcr?wp_target=&db=0&org=Rhodobacter+sphaeroides+2+4+1&wp_f=&wp_r=&wp_size=2000&wp_perfect=15&wp_good=15&wp_showPage=true&hgsid=159191 ''In silico'' PCR of ''R. sphaeroides'' 2.41 genome]<br />
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[https://2009.igem.org/Team:Wash_U/Project Back To Top]<br />
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=='''Regulation'''==<br />
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[[Image:pucregulation-fullpanels.png|950px]]<br />
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'''The Natural System (Left)''' Under high oxygen tension, PpsR prevents transcription from the puc promoter of the pucBA genes, and thus prevents the expression of LH2(1st panel). Under low oxygen tension, this repression is removed by activated AppA, thus allowing expression of LH2 from pucBA(2nd panel).<br><br />
'''Synthetic Regulation System (Right)''' Our system takes advantage of constitutive protein expression from the puc promoter at low oxygen tension, and replaces pucBA with Cph8 at this site. Placing pucBA expression under the control of the ompC promoter allows for Cph8/OmpR control of LH2 expression under varying light conditions. Under high intensity of incident light, Cph8 will not be autophosphorylated, preventing expression of pucBA (3rd panel). When light intensity is lower, Cph8 will autophosphorylate, thus phosphorylating and activating OmpR and promoting expression of LH2 from the ompC promoter.<br><br />
This regulation system was designed such that light is able to penetrate deeper into and through photobioreactors where light is plentiful.<br />
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<br>[https://2009.igem.org/Team:Wash_U/Project Back To Top]<br />
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== '''Results''' ==<br />
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For methods, materials, and procedures regarding these results, please see [https://2009.igem.org/Team:Wash_U/Protocol#Tissue_Flask_Experiment Tissue Flask Experiment] or [https://2009.igem.org/Team:Wash_U/Biological_Parts#Modeling Modeling]<br />
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'''Figure 1'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:#black;">'''a''' Tissue Flask Position 1 Optical Density of Wild Type and DCBomega<br />
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|<font size="2" style="color:#black;">'''b''' Tissue Flask Position 2 Optical Density of Wild Type and DCBomega<br />
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[[Image:Tissue Flask 1b.png| 450 px| left]][[Image:Tissue Flask 2b.png| 450px | right]]<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:#black;">'''c''' Tissue Flask Position 3 Optical Density of Wild Type and DCBomega<br />
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|<font size="2" style="color:#black;">'''d''' Tissue Flask Position 4 Optical Density of Wild Type and DCBomega<br />
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[[Image:Tissue Flask 3b.png| 450px | left]][[Image:Tissue Flask 4b.png| 450px | right]]<br />
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'''e''' Tissue Flask Position 5 Optical Density of Wild Type and DBComega<br />
[[Image:Tissue Flask 5b.png| 450px | center]]<br />
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Figure 1a,b,c,d,e are the optical density of a tissue flask, as assessed at 600 nm, over the course of the 6 days of the experiment.<br />
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'''Figure 2'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:#black;">'''a''' The Change in the Optical Density of the Tissue Flasks for the WT<br />
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|<font size="2" style="color:#black;">'''b''' The Change in the Optical Density of the Tissue Flasks for the WT<br />
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[[Image:3D 241.png| 450 px | left]][[Image:3D DBCOmega2.png| 450 px | right]]<br />
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Figure 2a and 2b display the growth of the tissue flasks as measured by OD assessed as absorbance at 600 nm. The graph is organized such that those closest to the "days" axis were closest to the light source (i.e. flask 1 is closer than flask 2).<br />
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'''Figure 3'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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[[Image:Bar 241 b.png| 450px | left]][[Image:Bar DBCOmega b.png| 450px | right]]<br />
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Figure 3a and 3b display the cumulative growth of the tissue flasks as measured by the sum of the optical density of the respective cell type's tissue flasks at a given day. Furthermore, the contribution of a given tissue flask to cumulative growth is displayed.<br><br />
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'''Figure 4'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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[[image:sphaeroides tissue flask.jpg|450 px|left|]][[image:dbc tissue flask.jpg|450px|right]]<br />
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Figure 4A and 4B display the appearance of the tissue flasks for both strains on day 6. They are arranged from left to right with left being closed to the light source (flask 1) and right furthest (flask 5)<br />
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'''Figure 5'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:#black;">'''a''' DBComega Spectrum by Flask Distance from Source<br />
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|<font size="2" style="color:#black;">'''b''' R. sphaeroides Spectrum by Flask Distance from Source<br />
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[[Image: WT irradiance 3d.png| 450 px | left]][[Image: DBComega irradiance 3d.png| 450 px | right]]<br />
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Contour of Absolute Irradiance by Flash distance from Source<br />
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|<font size="2" style="color:#black;">'''c''' R. Sphaeroides 2.4.1<br />
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[[Image: r sphaeroides day 2 a.jpg| 450px | left]][[Image: dbc day 4b.jpg| 450px | right]]<br />
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'''e''' Wild Type Absolute Irradiance Behind Flask 1: Day 6<br />
[[Image: irradiance WT flask 1 day6.png| 450px | center]]<br />
Caption for 5e<br><br />
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'''Figure 6'''<br><br />
Nonlinear least-squares estimation of WT LH2 saturation curve<br><br />
[[Image: saturation for WT as inferred.png| 450 px]]<br />
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Caption for figure 6<br><br><br />
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'''Figure 7'''<br><br />
Simulated Optical Density of Mutant and Wild Type Bioreactors<br><br />
Layers One and Two . Layers Three, Four and Five<br />
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[[Image: OD after flask 1 and 2.jpg| 200px ]][[Image: OD after 3,4, and 5.jpg| 200px ]]<br />
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[https://2009.igem.org/Team:Wash_U/Project Back To Top]<br />
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== '''Analysis''' ==<br />
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The tissue flask experiment was an interesting way to observe the effects of a full knockout of the Light Harvesting Antenna Complex 2 (LH2) vs the Wild Type on an array of conseuctive photobioreactors. A couple of conclusions can be drawn from this data that can be used in modeling our synthetic regulation system for LH2 and in the future design of systems for the optimization of Light Harvesting Antenna size for photobioreactors . <br><br><br />
The most striking difference between the Wild Type Rhodobacter Sphaeroides 2.41 and DBCOmega (LH2 knockout) growth patterns is that the first flask in the WT (closest to the light source) grew less than the second flask, while the converse was true in DBCOmega (figure 2a and 2b). This can be attributed to the photosystem saturation curves for the respective cultures. The Wild Type has an LH2 complex, meaning that their antenna size is inherently larger and that their photosystem will be saturated at lower light intensities than the LH2 deficient mutant. We observed that the incident light intensity used in this experiment led to oversaturation for the first WT tissue flask and resulted in growth-inhibiting photodamage, as is evidenced by its lesser growth relative to the second culture. In contrast, photodamage was not observed in DBCOmega as is evidenced by the first tissue flask that grew at the fastest respective rate. Furthermore, it appears that this photodamage slowed the growth of the first WT tissue flask to the point that the OD of this tissue flask was nearly equal to that of DBCOmega after 5 days of growth(.987 vs. .967) (figure 1a). As such, the contribution of LH2 to the growth of the WT in flask 1 can be assessed to be little after day 3 when the growth curve slows- though, this light (at 800 and 850 nm) was still greatly absorbed by flask 1 (Figure 5e). It is likely that much of this light was then wasted through Non-Photochemical Quenching (NPQ), depleting the flasks behind flask 1 of photons at the LH2 absorbtion peaks and decreasing the cumulative productivity of flasks 1-5. This effect of wasting photons though NPQ under high light intensities is one that we sought to minimize in the design of our synthetic regulation for the pucB/A genes. <br><br><br />
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A further a comparison of the WT and DBCOmega experiments reveals that the 3rd tissue flask in each had almost identical growth rates (figure 1c), while the DBCOmega 4th and 5th tissue flask outperformed that of the WT (figure 1d and 1e). This can simply be attributed to the higher density of cells in the 2nd flask of the WT that led to an overall lesser quantity of photons passing through to the 4th and 5th flasks in the WT experiment and likely resulting in heterotrophic growth (though this will need to be confirmed with a "dark control" experiment that should be completed in time for the Jamboree). This decrease in available photons for the WT can be seen by viewing the absolute irradiance data (incident light intensity at a given wavelength) after the third culture for the WT (figure 5a and 5c). This decrease in irradiance is the most pronounced for the LH2 absorption bands at 800 and 850 nm, as was expected. Our synthetic regulation system for pucB/A was designed with the intention of allowing greater penetration of light through the first couple of flasks at the LH2 wavelengths, as expression of pucB/A is inversely correlated to incident light intensity.<br><br><br />
'''Thomas, add in some further comments about the sprectroradiometer stuff. Specifically about how it changes in the WT vs. that of DBCOmega using some specific examples on the graphs'''<br><br><br />
Overall, the cumulative culture growth of the WT exceeded that of DBCOmega (figure 3) due to the performance of the second tissue flask of the WT relative to that of DBCOmega (figure 1b) despite the photodamage that occurred in tissue flask 1 for the WT. This cumulative culture growth can be considered photosynthetic productivty and is proportional to the amount of energy/desired product that can be obtained from these cultures. <br><br><br />
'''It is an interesting exercise to model how our mutant system would perform under these same conditions: Thomas add in some comments about referencing that diagram and what we took from this tissue flask experiment to construct that (and upload it when you are done with it).'''<br />
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'''add stuff here about the second flasks and approaching 20% contribution of LH1 to growth'''<br />
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=='''Conclusion and Future Work'''==<br />
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Based on model, should have had feedback mechanism- should be something that sets a minimum expression level for LH2, that is...<br><br />
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-tissue flask w prkcbc3 before jamboree and compare/interpret using characterization data<br><br />
-tissue flask w. full mutant in future- if this doesn't reveal cph8 to responding in an exponentially decaying fashion to light intensity, try w. a system that would<br />
-characterize puc promoter under light conditions and additional oxygen tensions<br />
-complete other characterization work<br />
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[https://2009.igem.org/Team:Wash_U/Project Back To Top]<br />
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== '''References''' ==<br />
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# "2007-2008 Catalog & Technical Reference." <u>New England BioLabs Inc.</u> (2007).<br />
# Alon, Uri. <u>Introduction to systems biology and the design principles of biological networks</u>. Boca Raton, FL: Chapman & Hall, 2006.<br />
# Ausubel, Fred et al. <u>Short Protocols in Molecular Biology: Third Edition.</u> Canada: John Wiley and Sons, Inc., 1999.<br />
# "BioBrick Assembly Help." <u>Registry of Standard Biological Parts.</u> 2009. 6 July 2009. <http://partsregistry.org/Main_Page>.<br />
# Bower, James M. <u>Computational Modeling of Genetic and Biochemical Networks (Computational Molecular Biology).</u> New York: M.I.T. PRESS, 2001.<br />
# Braatsch, Stephan et al. "A single Flavoprotein AppA, Integrates Both Redox and Light Signals in ''Rhodobacter sphaeroides.''" <u>Molecular Microbiology.</u> Vol. 45.3 (2002): 827-836.<br />
# Brown, Stanley B. et al. "Bile Pigment Synthesis in Plants: Incorporation of Haem into Phycocyanobilin and Phycocyanobiliproteins in Cyanidium Caldarium." <u>Biochemistry.</u> Vol. 194 (1981): 137-147.<br />
# Dragnea, Vladimira et al. "Time-Resolved Spectroscopic Studies of the AppA Blue-Light Receptor BLUF Domain from ''Rhodobacter sphaeroides'' <u>Biochemistry.</u> Vol. 44 (2005): 15,978-15,985.<br />
# "Entrez Nucleotide Search." <u>National Center for Biotechnology Information.</u> 2009. 6 June 2009 <http://www.ncbi.nlm.nih.gov/nuccore/49175990?from=3533887&to=3534606&report=gbwithparts>.<br />
# Fowler, Gregory J. S. and C. Neil Hunter. "The Synthesis and Assembly of Functional High and Low Light LH2 Antenna Complexes from ''Rhodopseudomonas palustris'' in ''Rhodobacter sphaeroides.''" <u>The Journal of Biological Chemistry.</u> Vol. 271.23 (1996): 13,356-13,361.<br />
# Gabrielsen, Mads et al. "Peripheral Complexes of Purple Bacteria." <u>The Purple Phototropic Bacteria.</u> (2009): 135-153.<br />
# "GenElute HP Plasmid Miniprep Kit: User Guide." <u>Sigma-Aldrich</u>. (2008).<br />
# Harwood, Caroline S. "Nitrogenase-Catalyzed Hydrogen Production by Purple Nonsulfur Photosynthetic Bacteria." <u>Bioenergy.</u> (2008): 259-271.<br />
# Jager, Andreas et al. "The AppA and PpsR Proteins from ''Rhodobacter sphaeroides'' Can Establish a Redox-Dependent Signal Chain but Fail to Transmit Blue-Light Signals in Other Bacteria." <u>Journal of Bacteriology.</u> Vol. 189.6 (2007): 2,274-2,282.<br />
# Jones, M. R. et al. "Mutants of ''Rhodobacter sphaeroides'' Lacking One or More Pigment-Protein Complexes and Complementation with Reaction-Centre, LH1, and LH2 Genes." <u>Molecular Microbiology.</u> Vol. 6.9 (1992): 1,173-1,184.<br />
# Kondo, Toshihiko et al. "Enhancement of Hydrogen Production by a Photosynthetic Bacterium Mutant with Reduced Pigment." <u>Journal of Bioscience and Bioengineering.</u> Vol. 93.2 (2002): 145-150.<br />
# Lagarias, J. Clark. <u>PCB from Spirulina</u>. Personal Communication. June 2009.<br />
# Laible, Philip D. "Foreign Gene Expression in Photosynthetic Bacteria." <u>The Purple Phototropic Bacteria.</u> (2009): 839-860.<br />
# Lee, Jeong K. and Samuel Kaplan. "Transcriptional Regulation of ''puc'' Operon Expression in ''Rhodobacter sphaeroides.''" <u>Journal of Biological Chemistry.</u> Vol. 270.35 (1995): 20,453-20,458.<br />
# Levskaya, Anselmetal et al. "Engineering ''Escherichia coli'' to See Light." <u>Nature.</u> Vol. 438 (2005): 441-442.<br />
# "Life Sciences Catalog 2009-2010." <u>National Diagnostics.</u> (2009).<br />
# Melis, Anastasios. "Solar Energy Conversion Efficiencies in Photosynthesis: Minimizing the Chlorophyll Antennae to Maximize Efficiency." <u>Plant Science.</u> (2009).<br />
# Mizuno, Takeshi and Shoji Mizushima. "Characterization by Deletion and Localized Mutagenesis in Vitro of the Promoter Region of the ''Escherichia coli'' OmpC Gene and Importance of the Upstream DNA Domain in Positive Regulation by the OmpR Protein." <u>Journal of Bacteriology.</u> Vol. 168.1 (1986): 86-95.<br />
# Moskvin, Oleg V. et al. "Transcriptome Analysis of the ''Rhodobacter sphaeroides'' PspR Regulon: PspR as a Master Regulator of Photosystem Development." <u>Journal of Bacteriology.</u> Vol. 187.6 (2005): 2,148-2,156.<br />
# Mussgnug, Jan H. et al. "Engineering photosynthetic light capture: impacts on improved solar energy to biomass conversion." <u> Plant Biotechnology Journal</u> Vol. 5 Issue 6. (2007) pp. 802-814<br />
# "NEBCutter V2.0." <u>New England BioLabs.</u> 2009. 6 June 2009 <http://tools.neb.com/NEBcutter2/index.php>.<br />
# "OligoAnalyzer 3.1." <u>Integrated DNA Technologies.</u> 2009. 6 June 2009. <http://www.idtdna.com/analyzer/Applications/OligoAnalyzer/><br />
# Sambrook, Joseph and David W. Russell. <u>Molecular Cloning: A Laboratory Manual.</u> Vol. 1-3. New York: Cold Springs Harbor Laboratory Press, 2001.<br />
# Scheuring, Simon and Sturgis, James N. "Chromatic Adaptation of Photosynthetic Membranes" <u>Science</u> Vol. 309 (2005) pp.484-487<br />
# Sener, Melih K. et al. "Atomic-level structural and functional model of a bacterial photosynthetic membrane vesicle" <u>Proceedings of the National Academy of Sciences of the United States of America.</u> Vol. 104, Number 40,pp. 15723-15728<br />
# Shimizu-Sato, Sae et al. "A Light-Switchable Gene Promoter System." <u>Nature Publishing Group.</u> Vol. 20 (2002): 1,041-1044.<br />
# Smith, Harry and M. Geoffrey Holmes. <u>Techniquies in Photomorphogenesis.</u> London: Academic Press, Inc., 1984.<br />
# <u>System modeling in cellular biology from concepts to nuts and bolts.</u> Cambridge, MA: MIT P, 2006.<br />
# Terry, Matthew J. "Biosynthesis and Analysis of Bilins." <u>Heme, Chlorophyll, and Bilins: Methods and Protocols.</u> (2002) 273-291. <br />
# Wang, Wanneng et al. "Heterologous Synthesis and Assembly of Functional LHII Antenna Complexes from ''Rhodovulum sulfidophilum'' in ''Rhodobacter sphaeroides'' Mutant." <u>Molecular Biology Reports.</u> Vol. 12.3 (2008).<br />
# Walz, Thomas et al. "Projection Structures of Three Photosynthetic Complexes from Rhodobacter sphaeroides: LH2 at 6 A, LH1 and RC-LH1 at 25 A" <u>Journal of Molecular Biology</u> Vol. 282 pp. 833-845<br />
# Yakovlev, A. G. et al. "Light Control Over the Size of an Antenna Unit Building Block as an Efficient Strategy for Light Harvesting in Photosynthesis." <u>Federation of European Biochemical Societies.</u> (2002):129-132.<br />
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{{WashUbottom}}</div>Brendan1http://2009.igem.org/Team:Wash_U/ProjectTeam:Wash U/Project2009-10-22T02:45:31Z<p>Brendan1: /* Results */</p>
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__NOTOC__<br />
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== '''Introduction''' == <br />
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Our project goal is to maximize the photosynthetic productivity for a series of photobioreactors containing ''Rhodobacter sphaeroides'' under both high and low light intensities by synthetically regulating the size of the light-harvesting antenna complex LH2. We chose to undertake such a project in ''R. sphaeroides'' due to its well-characterized photosynthetic and genetic system. <br />
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The antenna system functions to expand the spectrum of light available for photosynthesis by absorbing different wavelengths than that of the reaction center. Essentially, it is like the dish around the main receiver of any antenna. Marine bacteria, such as ''R. sphaeroides'', evolved very large antenna complexes to absorb light in a natural environment where there is great competition for photons. As a result, the photosynthetic machinery is saturated at low light intensities in a synthetic non-competitive environment, such as a photobioreactor. This causes up to 95% of incidental photons to be dissipated as heat or fluorescence by bacteria through a process called Non-Photochemical Quenching (NPQ) (Mussgnug et al., 2007). In essence, these photons are being wasted as NPQ reduces light penetration into and across bioreactors and starves the shaded cells for for photons. One method that has been shown to improve photosynthetic productivity in photobioreactors is the reduction of light-harvesting antenna sizes (Polle et al., 2002, Mussgnug et al., 2007). Though, current approaches to this end are difficult to precisely control from the perspective of biological engineering and synthetic biology. <br />
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Our intention is to create a dynamic system to vary antenna size that is dependent on incident light intensity and that can be readily optimized using bioengineering principles. This synthetic regulation of the LH2 complex should result in the bacteria grown under high light intensities expressing fewer LH2 complexes than the cells that are more shaded, leading to a reduction of wasted incident photons while maintaining a high overall absorbance across subsequent bioreactors. Consequently, we expect to see an increase in the total photosynthetic productivity for our mutant ''Rhodobacter sphaeroides'' when compared to the wild type grown under similar conditions. <br />
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[https://2009.igem.org/Team:Wash_U/Project Back To Top]<br />
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== '''Organism''' ==<br />
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''Rhodobacter sphaeroides'' is a purple Alphaproteobacteria. It is flexible metabolically and can grow heterotrophically via aerobic and anaerobic respiration, as well as phototrophically under anaerobic conditions with light. <br />
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''R. sphaeroides'' is one of the best understood photosynthetic organisms. The photosystem is located in intracytoplasmic membrane invaginations and the main components are the Light Haresting Complex 2 (LH2), Light Harvesting Complex 1 (LH1), and the Reaction Center (RC). These pigment-protein complexes non-covalently bind bacteriochlorophylls and carotenoids. LH1 and RC make up the core complex and are often found in the ratio 1:1. LH2 is more peripheral and naturally ranges in the ratio to LH1/RC of 3.0:1 to 6.7:1 under varying light conditions (Scheuring et al., 2005). LH2 absorbs photons maximally at the wavelengths of 850 and 800 nm and funnels its energy to LH1 and the reaction center for photochemistry. (Scheuring et al., 2005). <br />
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The two subunits of LH2 are coded for by the pucB/A genes and are naturally promoted by the puc promoter. <br />
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<br><div style="text-align: center;">'''ICM of ''R. sphaeroides'' '''[[Image:R sphaeroides photosystem.png| 500 px | center]]<center>source:Sener et al.2007<br />
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|<font size="2" style="color:black;">'''Plate of ''R. sphaeroides'' 2.4.1'''<br />
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[[Image:R sphaeroides aborbance.png| 450px | left]][[Image:R sphaeroides 0986.png| 450px | right]]<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br>source: Walz et al. 1998<br><br />
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====Online Resources for ''R. sphaeroides''====<br />
[http://www.kazusa.or.jp/codon/cgi-bin/showcodon.cgi?species=272943 Codon Usage Table for ''R. sphaeroides'' 2.41]<br />
<br> [http://www.ncbi.nlm.nih.gov/sites/entrez?db=genome&cmd=Retrieve&dopt=Overview&list_uids=18843 ''R. sphaeroides'' 2.41 NCBI Genome Tools]<br />
<br> [http://archaea.ucsc.edu/cgi-bin/hgPcr?wp_target=&db=0&org=Rhodobacter+sphaeroides+2+4+1&wp_f=&wp_r=&wp_size=2000&wp_perfect=15&wp_good=15&wp_showPage=true&hgsid=159191 ''In silico'' PCR of ''R. sphaeroides'' 2.41 genome]<br />
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[https://2009.igem.org/Team:Wash_U/Project Back To Top]<br />
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=='''Regulation'''==<br />
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[[Image:pucregulation-fullpanels.png|950px]]<br />
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'''The Natural System (Left)''' Under high oxygen tension, PpsR prevents transcription from the puc promoter of the pucBA genes, and thus prevents the expression of LH2(1st panel). Under low oxygen tension, this repression is removed by activated AppA, thus allowing expression of LH2 from pucBA(2nd panel).<br><br />
'''Synthetic Regulation System (Right)''' Our system takes advantage of constitutive protein expression from the puc promoter at low oxygen tension, and replaces pucBA with Cph8 at this site. Placing pucBA expression under the control of the ompC promoter allows for Cph8/OmpR control of LH2 expression under varying light conditions. Under high intensity of incident light, Cph8 will not be autophosphorylated, preventing expression of pucBA (3rd panel). When light intensity is lower, Cph8 will autophosphorylate, thus phosphorylating and activating OmpR and promoting expression of LH2 from the ompC promoter.<br><br />
This regulation system was designed such that light is able to penetrate deeper into and through photobioreactors where light is plentiful.<br />
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<br>[https://2009.igem.org/Team:Wash_U/Project Back To Top]<br />
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== '''Results''' ==<br />
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For methods, materials, and procedures regarding these results, please see [https://2009.igem.org/Team:Wash_U/Protocol#Tissue_Flask_Experiment Tissue Flask Experiment] or [https://2009.igem.org/Team:Wash_U/Biological_Parts#Modeling Modeling]<br />
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'''Figure 1'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:#black;">'''a''' Tissue Flask Position 1 Optical Density of Wild Type and DCBomega<br />
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|<font size="2" style="color:#black;">'''b''' Tissue Flask Position 2 Optical Density of Wild Type and DCBomega<br />
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[[Image:Tissue Flask 1b.png| 450 px| left]][[Image:Tissue Flask 2b.png| 450px | right]]<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:#black;">'''c''' Tissue Flask Position 3 Optical Density of Wild Type and DCBomega<br />
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|<font size="2" style="color:#black;">'''d''' Tissue Flask Position 4 Optical Density of Wild Type and DCBomega<br />
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[[Image:Tissue Flask 3b.png| 450px | left]][[Image:Tissue Flask 4b.png| 450px | right]]<br />
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'''e''' Tissue Flask Position 5 Optical Density of Wild Type and DBComega<br />
[[Image:Tissue Flask 5b.png| 450px | center]]<br />
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Figure 1a,b,c,d,e are the optical density of a tissue flask, as assessed at 600 nm, over the course of the 6 days of the experiment.<br />
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'''Figure 2'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:#black;">'''a''' The Change in the Optical Density of the Tissue Flasks for the WT<br />
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|<font size="2" style="color:#black;">'''b''' The Change in the Optical Density of the Tissue Flasks for the WT<br />
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[[Image:3D 241.png| 450 px | left]][[Image:3D DBCOmega2.png| 450 px | right]]<br />
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Figure 2a and 2b display the growth of the tissue flasks as measured by OD assessed as absorbance at 600 nm. The graph is organized such that those closest to the "days" axis were closest to the light source (i.e. flask 1 is closer than flask 2).<br />
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'''Figure 3'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:#black;">'''a''' Cumulative Growth of Wild Type Tissue Flasks<br />
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[[Image:Bar 241 b.png| 450px | left]][[Image:Bar DBCOmega b.png| 450px | right]]<br />
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Figure 3a and 3b display the cumulative growth of the tissue flasks as measured by the sum of the optical density of the respective cell type's tissue flasks at a given day. Furthermore, the contribution of a given tissue flask to cumulative growth is displayed.<br><br />
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'''Figure 4'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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[[image:sphaeroides tissue flask.jpg|450 px|left|]][[image:dbc tissue flask.jpg|450px|right]]<br />
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Figure 4A and 4B display the appearance of the tissue flasks for both strains on day 6. They are arranged from left to right with left being closed to the light source (flask 1) and right furthest (flask 5)<br />
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'''Figure 5'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:#black;">'''a''' DBComega Spectrum by Flask Distance from Source<br />
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|<font size="2" style="color:#black;">'''b''' R. sphaeroides Spectrum by Flask Distance from Source<br />
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[[Image: WT irradiance 3d.png| 450 px | left]][[Image: DBComega irradiance 3d.png| 450 px | right]]<br />
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Contour of Absolute Irradiance by Flash distance from Source<br />
{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:#black;">'''c''' R. Sphaeroides 2.4.1<br />
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|<font size="2" style="color:#black;">'''d''' DBComega<br />
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[[Image: r sphaeroides day 2 a.jpg| 450px | left]][[Image: dbc day 4b.jpg| 450px | right]]<br />
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'''e''' Wild Type Absolute Irradiance Behind Flask 1: Day 6<br />
[[Image: irradiance WT flask 1 day6.png| 450px | center]]<br />
Caption for 5e<br><br />
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'''Figure 6'''<br><br />
Nonlinear least-squares estimation of WT LH2 saturation curve<br><br />
[[Image: saturation for WT as inferred.png| 450 px]]<br />
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Caption for figure 6<br><br><br />
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'''Figure 7'''<br><br />
Simulated Optical Density of Mutant and Wild Type Bioreactors<br><br />
Layers One and Two . Layers Three, Four and Five<br />
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[[Image: OD after flask 1 and 2.jpg| 200px ]][[Image: OD after 3,4, and 5.jpg| 200px ]]<br />
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[https://2009.igem.org/Team:Wash_U/Project Back To Top]<br />
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== '''Analysis''' ==<br />
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The tissue flask experiment was an interesting way to observe the effects of a full knockout of the Light Harvesting Antenna Complex 2 (LH2) vs the Wild Type on an array of conseuctive photobioreactors. A couple of conclusions can be drawn from this data that can be used in modeling our synthetic regulation system for LH2 and in the future design of systems for the optimization of Light Harvesting Antenna size for photobioreactors . <br><br><br />
The most striking difference between the Wild Type Rhodobacter Sphaeroides 2.41 and DBCOmega (LH2 knockout) growth patterns is that the first flask in the WT (closest to the light source) grew less than the second flask, while the converse was true in DBCOmega (figure 2a and 2b). This can be attributed to the photosystem saturation curves for the respective cultures. The Wild Type has an LH2 complex, meaning that their antenna size is inherently larger and that their photosystem will be saturated at lower light intensities than the LH2 deficient mutant. We observed that the incident light intensity used in this experiment led to oversaturation for the first WT tissue flask and resulted in growth-inhibiting photodamage, as is evidenced by its lesser growth relative to the second culture. In contrast, photodamage was not observed in DBCOmega as is evidenced by the first tissue flask that grew at the fastest respective rate. Furthermore, it appears that this photodamage slowed the growth of the first WT tissue flask to the point that the OD of this tissue flask was nearly equal to that of DBCOmega after 5 days of growth(.987 vs. .967) (figure 1a). As such, the contribution of LH2 to the growth of the WT in flask 1 can be assessed to be little after day 3 when the growth curve slows- though, this light (at 800 and 850 nm) was still greatly absorbed by flask 1 (Figure 5e). It is likely that much of this light was then wasted through Non-Photochemical Quenching (NPQ), depleting the flasks behind flask 1 of photons at the LH2 absorbtion peaks and decreasing the cumulative productivity of flasks 1-5. This effect of wasting photons though NPQ under high light intensities is one that we sought to minimize in the design of our synthetic regulation for the pucB/A genes. <br><br><br />
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A further a comparison of the WT and DBCOmega experiments reveals that the 3rd tissue flask in each had almost identical growth rates (figure 1c), while the DBCOmega 4th and 5th tissue flask outperformed that of the WT (figure 1d and 1e). This can simply be attributed to the higher density of cells in the 2nd flask of the WT that led to an overall lesser quantity of photons passing through to the 4th and 5th flasks in the WT experiment and likely resulting in heterotrophic growth (though this will need to be confirmed with a "dark control" experiment that should be completed in time for the Jamboree). This decrease in available photons for the WT can be seen by viewing the absolute irradiance data (incident light intensity at a given wavelength) after the third culture for the WT (figure 5a and 5c). This decrease in irradiance is the most pronounced for the LH2 absorption bands at 800 and 850 nm, as was expected. Our synthetic regulation system for pucB/A was designed with the intention of allowing greater penetration of light through the first couple of flasks at the LH2 wavelengths, as expression of pucB/A is inversely correlated to incident light intensity.<br><br><br />
'''Thomas, add in some further comments about the sprectroradiometer stuff. Specifically about how it changes in the WT vs. that of DBCOmega using some specific examples on the graphs'''<br><br><br />
Overall, the cumulative culture growth of the WT exceeded that of DBCOmega (figure 3) due to the performance of the second tissue flask of the WT relative to that of DBCOmega (figure 1b) despite the photodamage that occurred in tissue flask 1 for the WT. This cumulative culture growth can be considered photosynthetic productivty and is proportional to the amount of energy/desired product that can be obtained from these cultures. <br><br><br />
'''It is an interesting exercise to model how our mutant system would perform under these same conditions: Thomas add in some comments about referencing that diagram and what we took from this tissue flask experiment to construct that (and upload it when you are done with it).'''<br />
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'''add stuff here about the second flasks and approaching 20% contribution of LH1 to growth'''<br />
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=='''Conclusion and Future Work'''==<br />
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Based on model, should have had feedback mechanism- should be something that sets a minimum expression level for LH2, that is...<br><br />
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-tissue flask w prkcbc3 before jamboree and compare/interpret using characterization data<br><br />
-tissue flask w. full mutant in future- if this doesn't reveal cph8 to responding in an exponentially decaying fashion to light intensity, try w. a system that would<br />
-characterize puc promoter under light conditions and additional oxygen tensions<br />
-complete other characterization work<br />
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[https://2009.igem.org/Team:Wash_U/Project Back To Top]<br />
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== '''References''' ==<br />
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# "2007-2008 Catalog & Technical Reference." <u>New England BioLabs Inc.</u> (2007).<br />
# Alon, Uri. <u>Introduction to systems biology and the design principles of biological networks</u>. Boca Raton, FL: Chapman & Hall, 2006.<br />
# Ausubel, Fred et al. <u>Short Protocols in Molecular Biology: Third Edition.</u> Canada: John Wiley and Sons, Inc., 1999.<br />
# "BioBrick Assembly Help." <u>Registry of Standard Biological Parts.</u> 2009. 6 July 2009. <http://partsregistry.org/Main_Page>.<br />
# Bower, James M. <u>Computational Modeling of Genetic and Biochemical Networks (Computational Molecular Biology).</u> New York: M.I.T. PRESS, 2001.<br />
# Braatsch, Stephan et al. "A single Flavoprotein AppA, Integrates Both Redox and Light Signals in ''Rhodobacter sphaeroides.''" <u>Molecular Microbiology.</u> Vol. 45.3 (2002): 827-836.<br />
# Brown, Stanley B. et al. "Bile Pigment Synthesis in Plants: Incorporation of Haem into Phycocyanobilin and Phycocyanobiliproteins in Cyanidium Caldarium." <u>Biochemistry.</u> Vol. 194 (1981): 137-147.<br />
# Dragnea, Vladimira et al. "Time-Resolved Spectroscopic Studies of the AppA Blue-Light Receptor BLUF Domain from ''Rhodobacter sphaeroides'' <u>Biochemistry.</u> Vol. 44 (2005): 15,978-15,985.<br />
# "Entrez Nucleotide Search." <u>National Center for Biotechnology Information.</u> 2009. 6 June 2009 <http://www.ncbi.nlm.nih.gov/nuccore/49175990?from=3533887&to=3534606&report=gbwithparts>.<br />
# Fowler, Gregory J. S. and C. Neil Hunter. "The Synthesis and Assembly of Functional High and Low Light LH2 Antenna Complexes from ''Rhodopseudomonas palustris'' in ''Rhodobacter sphaeroides.''" <u>The Journal of Biological Chemistry.</u> Vol. 271.23 (1996): 13,356-13,361.<br />
# Gabrielsen, Mads et al. "Peripheral Complexes of Purple Bacteria." <u>The Purple Phototropic Bacteria.</u> (2009): 135-153.<br />
# "GenElute HP Plasmid Miniprep Kit: User Guide." <u>Sigma-Aldrich</u>. (2008).<br />
# Harwood, Caroline S. "Nitrogenase-Catalyzed Hydrogen Production by Purple Nonsulfur Photosynthetic Bacteria." <u>Bioenergy.</u> (2008): 259-271.<br />
# Jager, Andreas et al. "The AppA and PpsR Proteins from ''Rhodobacter sphaeroides'' Can Establish a Redox-Dependent Signal Chain but Fail to Transmit Blue-Light Signals in Other Bacteria." <u>Journal of Bacteriology.</u> Vol. 189.6 (2007): 2,274-2,282.<br />
# Jones, M. R. et al. "Mutants of ''Rhodobacter sphaeroides'' Lacking One or More Pigment-Protein Complexes and Complementation with Reaction-Centre, LH1, and LH2 Genes." <u>Molecular Microbiology.</u> Vol. 6.9 (1992): 1,173-1,184.<br />
# Kondo, Toshihiko et al. "Enhancement of Hydrogen Production by a Photosynthetic Bacterium Mutant with Reduced Pigment." <u>Journal of Bioscience and Bioengineering.</u> Vol. 93.2 (2002): 145-150.<br />
# Lagarias, J. Clark. <u>PCB from Spirulina</u>. Personal Communication. June 2009.<br />
# Laible, Philip D. "Foreign Gene Expression in Photosynthetic Bacteria." <u>The Purple Phototropic Bacteria.</u> (2009): 839-860.<br />
# Lee, Jeong K. and Samuel Kaplan. "Transcriptional Regulation of ''puc'' Operon Expression in ''Rhodobacter sphaeroides.''" <u>Journal of Biological Chemistry.</u> Vol. 270.35 (1995): 20,453-20,458.<br />
# Levskaya, Anselmetal et al. "Engineering ''Escherichia coli'' to See Light." <u>Nature.</u> Vol. 438 (2005): 441-442.<br />
# "Life Sciences Catalog 2009-2010." <u>National Diagnostics.</u> (2009).<br />
# Melis, Anastasios. "Solar Energy Conversion Efficiencies in Photosynthesis: Minimizing the Chlorophyll Antennae to Maximize Efficiency." <u>Plant Science.</u> (2009).<br />
# Mizuno, Takeshi and Shoji Mizushima. "Characterization by Deletion and Localized Mutagenesis in Vitro of the Promoter Region of the ''Escherichia coli'' OmpC Gene and Importance of the Upstream DNA Domain in Positive Regulation by the OmpR Protein." <u>Journal of Bacteriology.</u> Vol. 168.1 (1986): 86-95.<br />
# Moskvin, Oleg V. et al. "Transcriptome Analysis of the ''Rhodobacter sphaeroides'' PspR Regulon: PspR as a Master Regulator of Photosystem Development." <u>Journal of Bacteriology.</u> Vol. 187.6 (2005): 2,148-2,156.<br />
# Mussgnug, Jan H. et al. "Engineering photosynthetic light capture: impacts on improved solar energy to biomass conversion." <u> Plant Biotechnology Journal</u> Vol. 5 Issue 6. (2007) pp. 802-814<br />
# "NEBCutter V2.0." <u>New England BioLabs.</u> 2009. 6 June 2009 <http://tools.neb.com/NEBcutter2/index.php>.<br />
# "OligoAnalyzer 3.1." <u>Integrated DNA Technologies.</u> 2009. 6 June 2009. <http://www.idtdna.com/analyzer/Applications/OligoAnalyzer/><br />
# Sambrook, Joseph and David W. Russell. <u>Molecular Cloning: A Laboratory Manual.</u> Vol. 1-3. New York: Cold Springs Harbor Laboratory Press, 2001.<br />
# Scheuring, Simon and Sturgis, James N. "Chromatic Adaptation of Photosynthetic Membranes" <u>Science</u> Vol. 309 (2005) pp.484-487<br />
# Sener, Melih K. et al. "Atomic-level structural and functional model of a bacterial photosynthetic membrane vesicle" <u>Proceedings of the National Academy of Sciences of the United States of America.</u> Vol. 104, Number 40,pp. 15723-15728<br />
# Shimizu-Sato, Sae et al. "A Light-Switchable Gene Promoter System." <u>Nature Publishing Group.</u> Vol. 20 (2002): 1,041-1044.<br />
# Smith, Harry and M. Geoffrey Holmes. <u>Techniquies in Photomorphogenesis.</u> London: Academic Press, Inc., 1984.<br />
# <u>System modeling in cellular biology from concepts to nuts and bolts.</u> Cambridge, MA: MIT P, 2006.<br />
# Terry, Matthew J. "Biosynthesis and Analysis of Bilins." <u>Heme, Chlorophyll, and Bilins: Methods and Protocols.</u> (2002) 273-291. <br />
# Wang, Wanneng et al. "Heterologous Synthesis and Assembly of Functional LHII Antenna Complexes from ''Rhodovulum sulfidophilum'' in ''Rhodobacter sphaeroides'' Mutant." <u>Molecular Biology Reports.</u> Vol. 12.3 (2008).<br />
# Walz, Thomas et al. "Projection Structures of Three Photosynthetic Complexes from Rhodobacter sphaeroides: LH2 at 6 A, LH1 and RC-LH1 at 25 A" <u>Journal of Molecular Biology</u> Vol. 282 pp. 833-845<br />
# Yakovlev, A. G. et al. "Light Control Over the Size of an Antenna Unit Building Block as an Efficient Strategy for Light Harvesting in Photosynthesis." <u>Federation of European Biochemical Societies.</u> (2002):129-132.<br />
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{{WashUbottom}}</div>Brendan1http://2009.igem.org/Team:Wash_U/ProjectTeam:Wash U/Project2009-10-22T02:45:01Z<p>Brendan1: /* Results */</p>
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__NOTOC__<br />
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== '''Introduction''' == <br />
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Our project goal is to maximize the photosynthetic productivity for a series of photobioreactors containing ''Rhodobacter sphaeroides'' under both high and low light intensities by synthetically regulating the size of the light-harvesting antenna complex LH2. We chose to undertake such a project in ''R. sphaeroides'' due to its well-characterized photosynthetic and genetic system. <br />
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The antenna system functions to expand the spectrum of light available for photosynthesis by absorbing different wavelengths than that of the reaction center. Essentially, it is like the dish around the main receiver of any antenna. Marine bacteria, such as ''R. sphaeroides'', evolved very large antenna complexes to absorb light in a natural environment where there is great competition for photons. As a result, the photosynthetic machinery is saturated at low light intensities in a synthetic non-competitive environment, such as a photobioreactor. This causes up to 95% of incidental photons to be dissipated as heat or fluorescence by bacteria through a process called Non-Photochemical Quenching (NPQ) (Mussgnug et al., 2007). In essence, these photons are being wasted as NPQ reduces light penetration into and across bioreactors and starves the shaded cells for for photons. One method that has been shown to improve photosynthetic productivity in photobioreactors is the reduction of light-harvesting antenna sizes (Polle et al., 2002, Mussgnug et al., 2007). Though, current approaches to this end are difficult to precisely control from the perspective of biological engineering and synthetic biology. <br />
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Our intention is to create a dynamic system to vary antenna size that is dependent on incident light intensity and that can be readily optimized using bioengineering principles. This synthetic regulation of the LH2 complex should result in the bacteria grown under high light intensities expressing fewer LH2 complexes than the cells that are more shaded, leading to a reduction of wasted incident photons while maintaining a high overall absorbance across subsequent bioreactors. Consequently, we expect to see an increase in the total photosynthetic productivity for our mutant ''Rhodobacter sphaeroides'' when compared to the wild type grown under similar conditions. <br />
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[[Image:Slide09b.jpg| 450px | left]][[Image:Slide10 a.jpg| 450px | right]]<br />
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[https://2009.igem.org/Team:Wash_U/Project Back To Top]<br />
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== '''Organism''' ==<br />
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''Rhodobacter sphaeroides'' is a purple Alphaproteobacteria. It is flexible metabolically and can grow heterotrophically via aerobic and anaerobic respiration, as well as phototrophically under anaerobic conditions with light. <br />
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''R. sphaeroides'' is one of the best understood photosynthetic organisms. The photosystem is located in intracytoplasmic membrane invaginations and the main components are the Light Haresting Complex 2 (LH2), Light Harvesting Complex 1 (LH1), and the Reaction Center (RC). These pigment-protein complexes non-covalently bind bacteriochlorophylls and carotenoids. LH1 and RC make up the core complex and are often found in the ratio 1:1. LH2 is more peripheral and naturally ranges in the ratio to LH1/RC of 3.0:1 to 6.7:1 under varying light conditions (Scheuring et al., 2005). LH2 absorbs photons maximally at the wavelengths of 850 and 800 nm and funnels its energy to LH1 and the reaction center for photochemistry. (Scheuring et al., 2005). <br />
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The two subunits of LH2 are coded for by the pucB/A genes and are naturally promoted by the puc promoter. <br />
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<br><div style="text-align: center;">'''ICM of ''R. sphaeroides'' '''[[Image:R sphaeroides photosystem.png| 500 px | center]]<center>source:Sener et al.2007<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:black;">'''Plate of ''R. sphaeroides'' 2.4.1'''<br />
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[[Image:R sphaeroides aborbance.png| 450px | left]][[Image:R sphaeroides 0986.png| 450px | right]]<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br>source: Walz et al. 1998<br><br />
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====Online Resources for ''R. sphaeroides''====<br />
[http://www.kazusa.or.jp/codon/cgi-bin/showcodon.cgi?species=272943 Codon Usage Table for ''R. sphaeroides'' 2.41]<br />
<br> [http://www.ncbi.nlm.nih.gov/sites/entrez?db=genome&cmd=Retrieve&dopt=Overview&list_uids=18843 ''R. sphaeroides'' 2.41 NCBI Genome Tools]<br />
<br> [http://archaea.ucsc.edu/cgi-bin/hgPcr?wp_target=&db=0&org=Rhodobacter+sphaeroides+2+4+1&wp_f=&wp_r=&wp_size=2000&wp_perfect=15&wp_good=15&wp_showPage=true&hgsid=159191 ''In silico'' PCR of ''R. sphaeroides'' 2.41 genome]<br />
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[https://2009.igem.org/Team:Wash_U/Project Back To Top]<br />
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=='''Regulation'''==<br />
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[[Image:pucregulation-fullpanels.png|950px]]<br />
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'''The Natural System (Left)''' Under high oxygen tension, PpsR prevents transcription from the puc promoter of the pucBA genes, and thus prevents the expression of LH2(1st panel). Under low oxygen tension, this repression is removed by activated AppA, thus allowing expression of LH2 from pucBA(2nd panel).<br><br />
'''Synthetic Regulation System (Right)''' Our system takes advantage of constitutive protein expression from the puc promoter at low oxygen tension, and replaces pucBA with Cph8 at this site. Placing pucBA expression under the control of the ompC promoter allows for Cph8/OmpR control of LH2 expression under varying light conditions. Under high intensity of incident light, Cph8 will not be autophosphorylated, preventing expression of pucBA (3rd panel). When light intensity is lower, Cph8 will autophosphorylate, thus phosphorylating and activating OmpR and promoting expression of LH2 from the ompC promoter.<br><br />
This regulation system was designed such that light is able to penetrate deeper into and through photobioreactors where light is plentiful.<br />
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<br>[https://2009.igem.org/Team:Wash_U/Project Back To Top]<br />
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== '''Results''' ==<br />
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For methods, materials, and procedures regarding these results, please see [https://2009.igem.org/Team:Wash_U/Protocol#Tissue_Flask_Experiment Tissue Flask Experiment] or [https://2009.igem.org/Team:Wash_U/Biological_Parts#Modeling Modeling]<br />
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'''Figure 1'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:#black;">'''a''' Tissue Flask Position 1 Optical Density of Wild Type and DCBomega<br />
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|<font size="2" style="color:#black;">'''b''' Tissue Flask Position 2 Optical Density of Wild Type and DCBomega<br />
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[[Image:Tissue Flask 1b.png| 450 px| left]][[Image:Tissue Flask 2b.png| 450px | right]]<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:#black;">'''c''' Tissue Flask Position 3 Optical Density of Wild Type and DCBomega<br />
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|<font size="2" style="color:#black;">'''d''' Tissue Flask Position 4 Optical Density of Wild Type and DCBomega<br />
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[[Image:Tissue Flask 3b.png| 450px | left]][[Image:Tissue Flask 4b.png| 450px | right]]<br />
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'''e''' Tissue Flask Position 5 Optical Density of Wild Type and DBComega<br />
[[Image:Tissue Flask 5b.png| 450px | center]]<br />
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Figure 1a,b,c,d,e are the optical density of a tissue flask, as assessed at 600 nm, over the course of the 6 days of the experiment.<br />
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'''Figure 2'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:#black;">'''b''' The Change in the Optical Density of the Tissue Flasks for the WT<br />
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[[Image:3D 241.png| 450 px | left]][[Image:3D DBCOmega2.png| 450 px | right]]<br />
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Figure 2a and 2b display the growth of the tissue flasks as measured by OD assessed as absorbance at 600 nm. The graph is organized such that those closest to the "days" axis were closest to the light source (i.e. flask 1 is closer than flask 2).<br />
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'''Figure 3'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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[[Image:Bar 241 b.png| 450px | left]][[Image:Bar DBCOmega b.png| 450px | right]]<br />
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Figure 3a and 3b display the cumulative growth of the tissue flasks as measured by the sum of the optical density of the respective cell type's tissue flasks at a given day. Furthermore, the contribution of a given tissue flask to cumulative growth is displayed.<br><br />
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'''Figure 4'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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[[image:sphaeroides tissue flask.jpg|450 px|left|]][[image:dbc tissue flask.jpg|450px|right]]<br />
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Figure 4A and 4B display the appearance of the tissue flasks for both strains on day 6. They are arranged from left to right with left being closed to the light source (flask 1) and right furthest (flask 5)<br />
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'''Figure 5'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:#black;">'''b''' R. sphaeroides Spectrum by Flask Distance from Source<br />
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[[Image: WT irradiance 3d.png| 450 px | left]][[Image: DBComega irradiance 3d.png| 450 px | right]]<br />
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Contour of Absolute Irradiance by Flash distance from Source<br />
{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:#black;">'''c''' R. Sphaeroides 2.4.1<br />
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[[Image: r sphaeroides day 2 a.jpg| 450px | left]][[Image: dbc day 4b.jpg| 450px | right]]<br />
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'''e''' Wild Type Absolute Irradiance Behind Flask 1: Day 6<br />
[[Image: irradiance WT flask 1 day6.png| 450px | center]]<br />
Caption for 5e<br><br />
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'''Figure 6'''<br><br />
Nonlinear least-squares estimation of WT LH2 saturation curve<br><br />
[[Image: saturation for WT as inferred.png| 450 px]]<br />
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Caption for figure 6<br><br><br />
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'''Figure 7'''<br><br />
Simulated Optical Density of Mutant and Wild Type Bioreactors<br><br />
Layers One and Two . Layers Three, Four and Five<br />
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[[Image: OD after flask 1 and 2.jpg| 200px ]][[Image: OD after 3,4, and 5.jpg| 200px ]]<br />
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[https://2009.igem.org/Team:Wash_U/Project Back To Top]<br />
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== '''Analysis''' ==<br />
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The tissue flask experiment was an interesting way to observe the effects of a full knockout of the Light Harvesting Antenna Complex 2 (LH2) vs the Wild Type on an array of conseuctive photobioreactors. A couple of conclusions can be drawn from this data that can be used in modeling our synthetic regulation system for LH2 and in the future design of systems for the optimization of Light Harvesting Antenna size for photobioreactors . <br><br><br />
The most striking difference between the Wild Type Rhodobacter Sphaeroides 2.41 and DBCOmega (LH2 knockout) growth patterns is that the first flask in the WT (closest to the light source) grew less than the second flask, while the converse was true in DBCOmega (figure 2a and 2b). This can be attributed to the photosystem saturation curves for the respective cultures. The Wild Type has an LH2 complex, meaning that their antenna size is inherently larger and that their photosystem will be saturated at lower light intensities than the LH2 deficient mutant. We observed that the incident light intensity used in this experiment led to oversaturation for the first WT tissue flask and resulted in growth-inhibiting photodamage, as is evidenced by its lesser growth relative to the second culture. In contrast, photodamage was not observed in DBCOmega as is evidenced by the first tissue flask that grew at the fastest respective rate. Furthermore, it appears that this photodamage slowed the growth of the first WT tissue flask to the point that the OD of this tissue flask was nearly equal to that of DBCOmega after 5 days of growth(.987 vs. .967) (figure 1a). As such, the contribution of LH2 to the growth of the WT in flask 1 can be assessed to be little after day 3 when the growth curve slows- though, this light (at 800 and 850 nm) was still greatly absorbed by flask 1 (Figure 5e). It is likely that much of this light was then wasted through Non-Photochemical Quenching (NPQ), depleting the flasks behind flask 1 of photons at the LH2 absorbtion peaks and decreasing the cumulative productivity of flasks 1-5. This effect of wasting photons though NPQ under high light intensities is one that we sought to minimize in the design of our synthetic regulation for the pucB/A genes. <br><br><br />
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A further a comparison of the WT and DBCOmega experiments reveals that the 3rd tissue flask in each had almost identical growth rates (figure 1c), while the DBCOmega 4th and 5th tissue flask outperformed that of the WT (figure 1d and 1e). This can simply be attributed to the higher density of cells in the 2nd flask of the WT that led to an overall lesser quantity of photons passing through to the 4th and 5th flasks in the WT experiment and likely resulting in heterotrophic growth (though this will need to be confirmed with a "dark control" experiment that should be completed in time for the Jamboree). This decrease in available photons for the WT can be seen by viewing the absolute irradiance data (incident light intensity at a given wavelength) after the third culture for the WT (figure 5a and 5c). This decrease in irradiance is the most pronounced for the LH2 absorption bands at 800 and 850 nm, as was expected. Our synthetic regulation system for pucB/A was designed with the intention of allowing greater penetration of light through the first couple of flasks at the LH2 wavelengths, as expression of pucB/A is inversely correlated to incident light intensity.<br><br><br />
'''Thomas, add in some further comments about the sprectroradiometer stuff. Specifically about how it changes in the WT vs. that of DBCOmega using some specific examples on the graphs'''<br><br><br />
Overall, the cumulative culture growth of the WT exceeded that of DBCOmega (figure 3) due to the performance of the second tissue flask of the WT relative to that of DBCOmega (figure 1b) despite the photodamage that occurred in tissue flask 1 for the WT. This cumulative culture growth can be considered photosynthetic productivty and is proportional to the amount of energy/desired product that can be obtained from these cultures. <br><br><br />
'''It is an interesting exercise to model how our mutant system would perform under these same conditions: Thomas add in some comments about referencing that diagram and what we took from this tissue flask experiment to construct that (and upload it when you are done with it).'''<br />
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'''add stuff here about the second flasks and approaching 20% contribution of LH1 to growth'''<br />
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=='''Conclusion and Future Work'''==<br />
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Based on model, should have had feedback mechanism- should be something that sets a minimum expression level for LH2, that is...<br><br />
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-tissue flask w prkcbc3 before jamboree and compare/interpret using characterization data<br><br />
-tissue flask w. full mutant in future- if this doesn't reveal cph8 to responding in an exponentially decaying fashion to light intensity, try w. a system that would<br />
-characterize puc promoter under light conditions and additional oxygen tensions<br />
-complete other characterization work<br />
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[https://2009.igem.org/Team:Wash_U/Project Back To Top]<br />
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== '''References''' ==<br />
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# "2007-2008 Catalog & Technical Reference." <u>New England BioLabs Inc.</u> (2007).<br />
# Alon, Uri. <u>Introduction to systems biology and the design principles of biological networks</u>. Boca Raton, FL: Chapman & Hall, 2006.<br />
# Ausubel, Fred et al. <u>Short Protocols in Molecular Biology: Third Edition.</u> Canada: John Wiley and Sons, Inc., 1999.<br />
# "BioBrick Assembly Help." <u>Registry of Standard Biological Parts.</u> 2009. 6 July 2009. <http://partsregistry.org/Main_Page>.<br />
# Bower, James M. <u>Computational Modeling of Genetic and Biochemical Networks (Computational Molecular Biology).</u> New York: M.I.T. PRESS, 2001.<br />
# Braatsch, Stephan et al. "A single Flavoprotein AppA, Integrates Both Redox and Light Signals in ''Rhodobacter sphaeroides.''" <u>Molecular Microbiology.</u> Vol. 45.3 (2002): 827-836.<br />
# Brown, Stanley B. et al. "Bile Pigment Synthesis in Plants: Incorporation of Haem into Phycocyanobilin and Phycocyanobiliproteins in Cyanidium Caldarium." <u>Biochemistry.</u> Vol. 194 (1981): 137-147.<br />
# Dragnea, Vladimira et al. "Time-Resolved Spectroscopic Studies of the AppA Blue-Light Receptor BLUF Domain from ''Rhodobacter sphaeroides'' <u>Biochemistry.</u> Vol. 44 (2005): 15,978-15,985.<br />
# "Entrez Nucleotide Search." <u>National Center for Biotechnology Information.</u> 2009. 6 June 2009 <http://www.ncbi.nlm.nih.gov/nuccore/49175990?from=3533887&to=3534606&report=gbwithparts>.<br />
# Fowler, Gregory J. S. and C. Neil Hunter. "The Synthesis and Assembly of Functional High and Low Light LH2 Antenna Complexes from ''Rhodopseudomonas palustris'' in ''Rhodobacter sphaeroides.''" <u>The Journal of Biological Chemistry.</u> Vol. 271.23 (1996): 13,356-13,361.<br />
# Gabrielsen, Mads et al. "Peripheral Complexes of Purple Bacteria." <u>The Purple Phototropic Bacteria.</u> (2009): 135-153.<br />
# "GenElute HP Plasmid Miniprep Kit: User Guide." <u>Sigma-Aldrich</u>. (2008).<br />
# Harwood, Caroline S. "Nitrogenase-Catalyzed Hydrogen Production by Purple Nonsulfur Photosynthetic Bacteria." <u>Bioenergy.</u> (2008): 259-271.<br />
# Jager, Andreas et al. "The AppA and PpsR Proteins from ''Rhodobacter sphaeroides'' Can Establish a Redox-Dependent Signal Chain but Fail to Transmit Blue-Light Signals in Other Bacteria." <u>Journal of Bacteriology.</u> Vol. 189.6 (2007): 2,274-2,282.<br />
# Jones, M. R. et al. "Mutants of ''Rhodobacter sphaeroides'' Lacking One or More Pigment-Protein Complexes and Complementation with Reaction-Centre, LH1, and LH2 Genes." <u>Molecular Microbiology.</u> Vol. 6.9 (1992): 1,173-1,184.<br />
# Kondo, Toshihiko et al. "Enhancement of Hydrogen Production by a Photosynthetic Bacterium Mutant with Reduced Pigment." <u>Journal of Bioscience and Bioengineering.</u> Vol. 93.2 (2002): 145-150.<br />
# Lagarias, J. Clark. <u>PCB from Spirulina</u>. Personal Communication. June 2009.<br />
# Laible, Philip D. "Foreign Gene Expression in Photosynthetic Bacteria." <u>The Purple Phototropic Bacteria.</u> (2009): 839-860.<br />
# Lee, Jeong K. and Samuel Kaplan. "Transcriptional Regulation of ''puc'' Operon Expression in ''Rhodobacter sphaeroides.''" <u>Journal of Biological Chemistry.</u> Vol. 270.35 (1995): 20,453-20,458.<br />
# Levskaya, Anselmetal et al. "Engineering ''Escherichia coli'' to See Light." <u>Nature.</u> Vol. 438 (2005): 441-442.<br />
# "Life Sciences Catalog 2009-2010." <u>National Diagnostics.</u> (2009).<br />
# Melis, Anastasios. "Solar Energy Conversion Efficiencies in Photosynthesis: Minimizing the Chlorophyll Antennae to Maximize Efficiency." <u>Plant Science.</u> (2009).<br />
# Mizuno, Takeshi and Shoji Mizushima. "Characterization by Deletion and Localized Mutagenesis in Vitro of the Promoter Region of the ''Escherichia coli'' OmpC Gene and Importance of the Upstream DNA Domain in Positive Regulation by the OmpR Protein." <u>Journal of Bacteriology.</u> Vol. 168.1 (1986): 86-95.<br />
# Moskvin, Oleg V. et al. "Transcriptome Analysis of the ''Rhodobacter sphaeroides'' PspR Regulon: PspR as a Master Regulator of Photosystem Development." <u>Journal of Bacteriology.</u> Vol. 187.6 (2005): 2,148-2,156.<br />
# Mussgnug, Jan H. et al. "Engineering photosynthetic light capture: impacts on improved solar energy to biomass conversion." <u> Plant Biotechnology Journal</u> Vol. 5 Issue 6. (2007) pp. 802-814<br />
# "NEBCutter V2.0." <u>New England BioLabs.</u> 2009. 6 June 2009 <http://tools.neb.com/NEBcutter2/index.php>.<br />
# "OligoAnalyzer 3.1." <u>Integrated DNA Technologies.</u> 2009. 6 June 2009. <http://www.idtdna.com/analyzer/Applications/OligoAnalyzer/><br />
# Sambrook, Joseph and David W. Russell. <u>Molecular Cloning: A Laboratory Manual.</u> Vol. 1-3. New York: Cold Springs Harbor Laboratory Press, 2001.<br />
# Scheuring, Simon and Sturgis, James N. "Chromatic Adaptation of Photosynthetic Membranes" <u>Science</u> Vol. 309 (2005) pp.484-487<br />
# Sener, Melih K. et al. "Atomic-level structural and functional model of a bacterial photosynthetic membrane vesicle" <u>Proceedings of the National Academy of Sciences of the United States of America.</u> Vol. 104, Number 40,pp. 15723-15728<br />
# Shimizu-Sato, Sae et al. "A Light-Switchable Gene Promoter System." <u>Nature Publishing Group.</u> Vol. 20 (2002): 1,041-1044.<br />
# Smith, Harry and M. Geoffrey Holmes. <u>Techniquies in Photomorphogenesis.</u> London: Academic Press, Inc., 1984.<br />
# <u>System modeling in cellular biology from concepts to nuts and bolts.</u> Cambridge, MA: MIT P, 2006.<br />
# Terry, Matthew J. "Biosynthesis and Analysis of Bilins." <u>Heme, Chlorophyll, and Bilins: Methods and Protocols.</u> (2002) 273-291. <br />
# Wang, Wanneng et al. "Heterologous Synthesis and Assembly of Functional LHII Antenna Complexes from ''Rhodovulum sulfidophilum'' in ''Rhodobacter sphaeroides'' Mutant." <u>Molecular Biology Reports.</u> Vol. 12.3 (2008).<br />
# Walz, Thomas et al. "Projection Structures of Three Photosynthetic Complexes from Rhodobacter sphaeroides: LH2 at 6 A, LH1 and RC-LH1 at 25 A" <u>Journal of Molecular Biology</u> Vol. 282 pp. 833-845<br />
# Yakovlev, A. G. et al. "Light Control Over the Size of an Antenna Unit Building Block as an Efficient Strategy for Light Harvesting in Photosynthesis." <u>Federation of European Biochemical Societies.</u> (2002):129-132.<br />
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{{WashUbottom}}</div>Brendan1http://2009.igem.org/Team:Wash_U/ProjectTeam:Wash U/Project2009-10-22T02:43:23Z<p>Brendan1: /* Results */</p>
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== '''Introduction''' == <br />
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Our project goal is to maximize the photosynthetic productivity for a series of photobioreactors containing ''Rhodobacter sphaeroides'' under both high and low light intensities by synthetically regulating the size of the light-harvesting antenna complex LH2. We chose to undertake such a project in ''R. sphaeroides'' due to its well-characterized photosynthetic and genetic system. <br />
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The antenna system functions to expand the spectrum of light available for photosynthesis by absorbing different wavelengths than that of the reaction center. Essentially, it is like the dish around the main receiver of any antenna. Marine bacteria, such as ''R. sphaeroides'', evolved very large antenna complexes to absorb light in a natural environment where there is great competition for photons. As a result, the photosynthetic machinery is saturated at low light intensities in a synthetic non-competitive environment, such as a photobioreactor. This causes up to 95% of incidental photons to be dissipated as heat or fluorescence by bacteria through a process called Non-Photochemical Quenching (NPQ) (Mussgnug et al., 2007). In essence, these photons are being wasted as NPQ reduces light penetration into and across bioreactors and starves the shaded cells for for photons. One method that has been shown to improve photosynthetic productivity in photobioreactors is the reduction of light-harvesting antenna sizes (Polle et al., 2002, Mussgnug et al., 2007). Though, current approaches to this end are difficult to precisely control from the perspective of biological engineering and synthetic biology. <br />
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Our intention is to create a dynamic system to vary antenna size that is dependent on incident light intensity and that can be readily optimized using bioengineering principles. This synthetic regulation of the LH2 complex should result in the bacteria grown under high light intensities expressing fewer LH2 complexes than the cells that are more shaded, leading to a reduction of wasted incident photons while maintaining a high overall absorbance across subsequent bioreactors. Consequently, we expect to see an increase in the total photosynthetic productivity for our mutant ''Rhodobacter sphaeroides'' when compared to the wild type grown under similar conditions. <br />
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|<font size="2" style="color:black;">'''Wild Type Energy Production in a series of Bioreactors'''<br />
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|<font size="2" style="color:black;">'''Synthetically Regulated Antenna Energy Production in Bioreactors'''<br />
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[https://2009.igem.org/Team:Wash_U/Project Back To Top]<br />
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== '''Organism''' ==<br />
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''Rhodobacter sphaeroides'' is a purple Alphaproteobacteria. It is flexible metabolically and can grow heterotrophically via aerobic and anaerobic respiration, as well as phototrophically under anaerobic conditions with light. <br />
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''R. sphaeroides'' is one of the best understood photosynthetic organisms. The photosystem is located in intracytoplasmic membrane invaginations and the main components are the Light Haresting Complex 2 (LH2), Light Harvesting Complex 1 (LH1), and the Reaction Center (RC). These pigment-protein complexes non-covalently bind bacteriochlorophylls and carotenoids. LH1 and RC make up the core complex and are often found in the ratio 1:1. LH2 is more peripheral and naturally ranges in the ratio to LH1/RC of 3.0:1 to 6.7:1 under varying light conditions (Scheuring et al., 2005). LH2 absorbs photons maximally at the wavelengths of 850 and 800 nm and funnels its energy to LH1 and the reaction center for photochemistry. (Scheuring et al., 2005). <br />
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The two subunits of LH2 are coded for by the pucB/A genes and are naturally promoted by the puc promoter. <br />
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<br><div style="text-align: center;">'''ICM of ''R. sphaeroides'' '''[[Image:R sphaeroides photosystem.png| 500 px | center]]<center>source:Sener et al.2007<br />
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|<font size="2" style="color:black;">'''Plate of ''R. sphaeroides'' 2.4.1'''<br />
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[[Image:R sphaeroides aborbance.png| 450px | left]][[Image:R sphaeroides 0986.png| 450px | right]]<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br>source: Walz et al. 1998<br><br />
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====Online Resources for ''R. sphaeroides''====<br />
[http://www.kazusa.or.jp/codon/cgi-bin/showcodon.cgi?species=272943 Codon Usage Table for ''R. sphaeroides'' 2.41]<br />
<br> [http://www.ncbi.nlm.nih.gov/sites/entrez?db=genome&cmd=Retrieve&dopt=Overview&list_uids=18843 ''R. sphaeroides'' 2.41 NCBI Genome Tools]<br />
<br> [http://archaea.ucsc.edu/cgi-bin/hgPcr?wp_target=&db=0&org=Rhodobacter+sphaeroides+2+4+1&wp_f=&wp_r=&wp_size=2000&wp_perfect=15&wp_good=15&wp_showPage=true&hgsid=159191 ''In silico'' PCR of ''R. sphaeroides'' 2.41 genome]<br />
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[https://2009.igem.org/Team:Wash_U/Project Back To Top]<br />
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=='''Regulation'''==<br />
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[[Image:pucregulation-fullpanels.png|950px]]<br />
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'''The Natural System (Left)''' Under high oxygen tension, PpsR prevents transcription from the puc promoter of the pucBA genes, and thus prevents the expression of LH2(1st panel). Under low oxygen tension, this repression is removed by activated AppA, thus allowing expression of LH2 from pucBA(2nd panel).<br><br />
'''Synthetic Regulation System (Right)''' Our system takes advantage of constitutive protein expression from the puc promoter at low oxygen tension, and replaces pucBA with Cph8 at this site. Placing pucBA expression under the control of the ompC promoter allows for Cph8/OmpR control of LH2 expression under varying light conditions. Under high intensity of incident light, Cph8 will not be autophosphorylated, preventing expression of pucBA (3rd panel). When light intensity is lower, Cph8 will autophosphorylate, thus phosphorylating and activating OmpR and promoting expression of LH2 from the ompC promoter.<br><br />
This regulation system was designed such that light is able to penetrate deeper into and through photobioreactors where light is plentiful.<br />
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<br>[https://2009.igem.org/Team:Wash_U/Project Back To Top]<br />
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== '''Results''' ==<br />
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For methods, materials, and procedures regarding these results, please see [https://2009.igem.org/Team:Wash_U/Protocol#Tissue_Flask_Experiment Tissue Flask Experiment] or [https://2009.igem.org/Team:Wash_U/Biological_Parts#Modeling Modeling]<br />
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'''Figure 1'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:#black;">'''a''' Tissue Flask Position 1 Optical Density of Wild Type and DCBomega<br />
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|<font size="2" style="color:#black;">'''b''' Tissue Flask Position 2 Optical Density of Wild Type and DCBomega<br />
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[[Image:Tissue Flask 1b.png| 450 px| left]][[Image:Tissue Flask 2b.png| 450px | right]]<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:#black;">'''c''' Tissue Flask Position 3 Optical Density of Wild Type and DCBomega<br />
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|<font size="2" style="color:#black;">'''d''' Tissue Flask Position 4 Optical Density of Wild Type and DCBomega<br />
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[[Image:Tissue Flask 3b.png| 450px | left]][[Image:Tissue Flask 4b.png| 450px | right]]<br />
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'''e''' Tissue Flask Position 5 Optical Density of Wild Type and DBComega<br />
[[Image:Tissue Flask 5b.png| 450px | left]]<br />
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Figure 1a,b,c,d,e are the optical density of a tissue flask, as assessed at 600 nm, over the course of the 6 days of the experiment.<br />
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'''Figure 2'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:#black;">'''a''' The Change in the Optical Density of the Tissue Flasks for the WT<br />
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|<font size="2" style="color:#black;">'''b''' The Change in the Optical Density of the Tissue Flasks for the WT<br />
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[[Image:3D 241.png| 450 px | left]][[Image:3D DBCOmega2.png| 450 px | right]]<br />
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Figure 2a and 2b display the growth of the tissue flasks as measured by OD assessed as absorbance at 600 nm. The graph is organized such that those closest to the "days" axis were closest to the light source (i.e. flask 1 is closer than flask 2).<br />
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'''Figure 3'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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[[Image:Bar 241 b.png| 450px | left]][[Image:Bar DBCOmega b.png| 450px | right]]<br />
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Figure 3a and 3b display the cumulative growth of the tissue flasks as measured by the sum of the optical density of the respective cell type's tissue flasks at a given day. Furthermore, the contribution of a given tissue flask to cumulative growth is displayed.<br><br />
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'''Figure 4'''<br />
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[[image:sphaeroides tissue flask.jpg|450 px|left|]][[image:dbc tissue flask.jpg|450px|right]]<br />
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Figure 4A and 4B display the appearance of the tissue flasks for both strains on day 6. They are arranged from left to right with left being closed to the light source (flask 1) and right furthest (flask 5)<br />
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'''Figure 5'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:#black;">'''a''' DBComega Spectrum by Flask Distance from Source<br />
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|<font size="2" style="color:#black;">'''b''' R. sphaeroides Spectrum by Flask Distance from Source<br />
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[[Image: WT irradiance 3d.png| 450 px | left]][[Image: DBComega irradiance 3d.png| 450 px | right]]<br />
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Contour of Absolute Irradiance by Flash distance from Source<br />
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|<font size="2" style="color:#black;">'''c''' R. Sphaeroides 2.4.1<br />
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[[Image: r sphaeroides day 2 a.jpg| 450px | left]][[Image: dbc day 4b.jpg| 450px | right]]<br />
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'''e''' Wild Type Absolute Irradiance Behind Flask 1: Day 6<br />
[[Image: irradiance WT flask 1 day6.png| 450px | center]]<br />
Caption for 5e<br><br />
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'''Figure 6'''<br><br />
Nonlinear least-squares estimation of WT LH2 saturation curve<br><br />
[[Image: saturation for WT as inferred.png| 450 px]]<br />
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Caption for figure 6<br><br><br />
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'''Figure 7'''<br><br />
Simulated Optical Density of Mutant and Wild Type Bioreactors<br><br />
Layers One and Two . Layers Three, Four and Five<br />
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[[Image: OD after flask 1 and 2.jpg| 200px ]][[Image: OD after 3,4, and 5.jpg| 200px ]]<br />
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[https://2009.igem.org/Team:Wash_U/Project Back To Top]<br />
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== '''Analysis''' ==<br />
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The tissue flask experiment was an interesting way to observe the effects of a full knockout of the Light Harvesting Antenna Complex 2 (LH2) vs the Wild Type on an array of conseuctive photobioreactors. A couple of conclusions can be drawn from this data that can be used in modeling our synthetic regulation system for LH2 and in the future design of systems for the optimization of Light Harvesting Antenna size for photobioreactors . <br><br><br />
The most striking difference between the Wild Type Rhodobacter Sphaeroides 2.41 and DBCOmega (LH2 knockout) growth patterns is that the first flask in the WT (closest to the light source) grew less than the second flask, while the converse was true in DBCOmega (figure 2a and 2b). This can be attributed to the photosystem saturation curves for the respective cultures. The Wild Type has an LH2 complex, meaning that their antenna size is inherently larger and that their photosystem will be saturated at lower light intensities than the LH2 deficient mutant. We observed that the incident light intensity used in this experiment led to oversaturation for the first WT tissue flask and resulted in growth-inhibiting photodamage, as is evidenced by its lesser growth relative to the second culture. In contrast, photodamage was not observed in DBCOmega as is evidenced by the first tissue flask that grew at the fastest respective rate. Furthermore, it appears that this photodamage slowed the growth of the first WT tissue flask to the point that the OD of this tissue flask was nearly equal to that of DBCOmega after 5 days of growth(.987 vs. .967) (figure 1a). As such, the contribution of LH2 to the growth of the WT in flask 1 can be assessed to be little after day 3 when the growth curve slows- though, this light (at 800 and 850 nm) was still greatly absorbed by flask 1 (Figure 5e). It is likely that much of this light was then wasted through Non-Photochemical Quenching (NPQ), depleting the flasks behind flask 1 of photons at the LH2 absorbtion peaks and decreasing the cumulative productivity of flasks 1-5. This effect of wasting photons though NPQ under high light intensities is one that we sought to minimize in the design of our synthetic regulation for the pucB/A genes. <br><br><br />
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A further a comparison of the WT and DBCOmega experiments reveals that the 3rd tissue flask in each had almost identical growth rates (figure 1c), while the DBCOmega 4th and 5th tissue flask outperformed that of the WT (figure 1d and 1e). This can simply be attributed to the higher density of cells in the 2nd flask of the WT that led to an overall lesser quantity of photons passing through to the 4th and 5th flasks in the WT experiment and likely resulting in heterotrophic growth (though this will need to be confirmed with a "dark control" experiment that should be completed in time for the Jamboree). This decrease in available photons for the WT can be seen by viewing the absolute irradiance data (incident light intensity at a given wavelength) after the third culture for the WT (figure 5a and 5c). This decrease in irradiance is the most pronounced for the LH2 absorption bands at 800 and 850 nm, as was expected. Our synthetic regulation system for pucB/A was designed with the intention of allowing greater penetration of light through the first couple of flasks at the LH2 wavelengths, as expression of pucB/A is inversely correlated to incident light intensity.<br><br><br />
'''Thomas, add in some further comments about the sprectroradiometer stuff. Specifically about how it changes in the WT vs. that of DBCOmega using some specific examples on the graphs'''<br><br><br />
Overall, the cumulative culture growth of the WT exceeded that of DBCOmega (figure 3) due to the performance of the second tissue flask of the WT relative to that of DBCOmega (figure 1b) despite the photodamage that occurred in tissue flask 1 for the WT. This cumulative culture growth can be considered photosynthetic productivty and is proportional to the amount of energy/desired product that can be obtained from these cultures. <br><br><br />
'''It is an interesting exercise to model how our mutant system would perform under these same conditions: Thomas add in some comments about referencing that diagram and what we took from this tissue flask experiment to construct that (and upload it when you are done with it).'''<br />
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'''add stuff here about the second flasks and approaching 20% contribution of LH1 to growth'''<br />
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=='''Conclusion and Future Work'''==<br />
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Based on model, should have had feedback mechanism- should be something that sets a minimum expression level for LH2, that is...<br><br />
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-tissue flask w prkcbc3 before jamboree and compare/interpret using characterization data<br><br />
-tissue flask w. full mutant in future- if this doesn't reveal cph8 to responding in an exponentially decaying fashion to light intensity, try w. a system that would<br />
-characterize puc promoter under light conditions and additional oxygen tensions<br />
-complete other characterization work<br />
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[https://2009.igem.org/Team:Wash_U/Project Back To Top]<br />
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== '''References''' ==<br />
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# "2007-2008 Catalog & Technical Reference." <u>New England BioLabs Inc.</u> (2007).<br />
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# Braatsch, Stephan et al. "A single Flavoprotein AppA, Integrates Both Redox and Light Signals in ''Rhodobacter sphaeroides.''" <u>Molecular Microbiology.</u> Vol. 45.3 (2002): 827-836.<br />
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# Mussgnug, Jan H. et al. "Engineering photosynthetic light capture: impacts on improved solar energy to biomass conversion." <u> Plant Biotechnology Journal</u> Vol. 5 Issue 6. (2007) pp. 802-814<br />
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# <u>System modeling in cellular biology from concepts to nuts and bolts.</u> Cambridge, MA: MIT P, 2006.<br />
# Terry, Matthew J. "Biosynthesis and Analysis of Bilins." <u>Heme, Chlorophyll, and Bilins: Methods and Protocols.</u> (2002) 273-291. <br />
# Wang, Wanneng et al. "Heterologous Synthesis and Assembly of Functional LHII Antenna Complexes from ''Rhodovulum sulfidophilum'' in ''Rhodobacter sphaeroides'' Mutant." <u>Molecular Biology Reports.</u> Vol. 12.3 (2008).<br />
# Walz, Thomas et al. "Projection Structures of Three Photosynthetic Complexes from Rhodobacter sphaeroides: LH2 at 6 A, LH1 and RC-LH1 at 25 A" <u>Journal of Molecular Biology</u> Vol. 282 pp. 833-845<br />
# Yakovlev, A. G. et al. "Light Control Over the Size of an Antenna Unit Building Block as an Efficient Strategy for Light Harvesting in Photosynthesis." <u>Federation of European Biochemical Societies.</u> (2002):129-132.<br />
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{{WashUbottom}}</div>Brendan1http://2009.igem.org/Team:Wash_U/ProjectTeam:Wash U/Project2009-10-22T02:42:26Z<p>Brendan1: /* Results */</p>
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== '''Introduction''' == <br />
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Our project goal is to maximize the photosynthetic productivity for a series of photobioreactors containing ''Rhodobacter sphaeroides'' under both high and low light intensities by synthetically regulating the size of the light-harvesting antenna complex LH2. We chose to undertake such a project in ''R. sphaeroides'' due to its well-characterized photosynthetic and genetic system. <br />
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The antenna system functions to expand the spectrum of light available for photosynthesis by absorbing different wavelengths than that of the reaction center. Essentially, it is like the dish around the main receiver of any antenna. Marine bacteria, such as ''R. sphaeroides'', evolved very large antenna complexes to absorb light in a natural environment where there is great competition for photons. As a result, the photosynthetic machinery is saturated at low light intensities in a synthetic non-competitive environment, such as a photobioreactor. This causes up to 95% of incidental photons to be dissipated as heat or fluorescence by bacteria through a process called Non-Photochemical Quenching (NPQ) (Mussgnug et al., 2007). In essence, these photons are being wasted as NPQ reduces light penetration into and across bioreactors and starves the shaded cells for for photons. One method that has been shown to improve photosynthetic productivity in photobioreactors is the reduction of light-harvesting antenna sizes (Polle et al., 2002, Mussgnug et al., 2007). Though, current approaches to this end are difficult to precisely control from the perspective of biological engineering and synthetic biology. <br />
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Our intention is to create a dynamic system to vary antenna size that is dependent on incident light intensity and that can be readily optimized using bioengineering principles. This synthetic regulation of the LH2 complex should result in the bacteria grown under high light intensities expressing fewer LH2 complexes than the cells that are more shaded, leading to a reduction of wasted incident photons while maintaining a high overall absorbance across subsequent bioreactors. Consequently, we expect to see an increase in the total photosynthetic productivity for our mutant ''Rhodobacter sphaeroides'' when compared to the wild type grown under similar conditions. <br />
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|<font size="2" style="color:black;">'''Wild Type Energy Production in a series of Bioreactors'''<br />
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|<font size="2" style="color:black;">'''Synthetically Regulated Antenna Energy Production in Bioreactors'''<br />
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== '''Organism''' ==<br />
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''Rhodobacter sphaeroides'' is a purple Alphaproteobacteria. It is flexible metabolically and can grow heterotrophically via aerobic and anaerobic respiration, as well as phototrophically under anaerobic conditions with light. <br />
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''R. sphaeroides'' is one of the best understood photosynthetic organisms. The photosystem is located in intracytoplasmic membrane invaginations and the main components are the Light Haresting Complex 2 (LH2), Light Harvesting Complex 1 (LH1), and the Reaction Center (RC). These pigment-protein complexes non-covalently bind bacteriochlorophylls and carotenoids. LH1 and RC make up the core complex and are often found in the ratio 1:1. LH2 is more peripheral and naturally ranges in the ratio to LH1/RC of 3.0:1 to 6.7:1 under varying light conditions (Scheuring et al., 2005). LH2 absorbs photons maximally at the wavelengths of 850 and 800 nm and funnels its energy to LH1 and the reaction center for photochemistry. (Scheuring et al., 2005). <br />
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The two subunits of LH2 are coded for by the pucB/A genes and are naturally promoted by the puc promoter. <br />
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<br><div style="text-align: center;">'''ICM of ''R. sphaeroides'' '''[[Image:R sphaeroides photosystem.png| 500 px | center]]<center>source:Sener et al.2007<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:black;">'''Plate of ''R. sphaeroides'' 2.4.1'''<br />
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[[Image:R sphaeroides aborbance.png| 450px | left]][[Image:R sphaeroides 0986.png| 450px | right]]<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br>source: Walz et al. 1998<br><br />
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====Online Resources for ''R. sphaeroides''====<br />
[http://www.kazusa.or.jp/codon/cgi-bin/showcodon.cgi?species=272943 Codon Usage Table for ''R. sphaeroides'' 2.41]<br />
<br> [http://www.ncbi.nlm.nih.gov/sites/entrez?db=genome&cmd=Retrieve&dopt=Overview&list_uids=18843 ''R. sphaeroides'' 2.41 NCBI Genome Tools]<br />
<br> [http://archaea.ucsc.edu/cgi-bin/hgPcr?wp_target=&db=0&org=Rhodobacter+sphaeroides+2+4+1&wp_f=&wp_r=&wp_size=2000&wp_perfect=15&wp_good=15&wp_showPage=true&hgsid=159191 ''In silico'' PCR of ''R. sphaeroides'' 2.41 genome]<br />
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=='''Regulation'''==<br />
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[[Image:pucregulation-fullpanels.png|950px]]<br />
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'''The Natural System (Left)''' Under high oxygen tension, PpsR prevents transcription from the puc promoter of the pucBA genes, and thus prevents the expression of LH2(1st panel). Under low oxygen tension, this repression is removed by activated AppA, thus allowing expression of LH2 from pucBA(2nd panel).<br><br />
'''Synthetic Regulation System (Right)''' Our system takes advantage of constitutive protein expression from the puc promoter at low oxygen tension, and replaces pucBA with Cph8 at this site. Placing pucBA expression under the control of the ompC promoter allows for Cph8/OmpR control of LH2 expression under varying light conditions. Under high intensity of incident light, Cph8 will not be autophosphorylated, preventing expression of pucBA (3rd panel). When light intensity is lower, Cph8 will autophosphorylate, thus phosphorylating and activating OmpR and promoting expression of LH2 from the ompC promoter.<br><br />
This regulation system was designed such that light is able to penetrate deeper into and through photobioreactors where light is plentiful.<br />
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<br>[https://2009.igem.org/Team:Wash_U/Project Back To Top]<br />
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== '''Results''' ==<br />
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For methods, materials, and procedures regarding these results, please see [https://2009.igem.org/Team:Wash_U/Protocol#Tissue_Flask_Experiment Tissue Flask Experiment] or [https://2009.igem.org/Team:Wash_U/Biological_Parts#Modeling Modeling]<br />
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'''Figure 1'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:#black;">'''b''' Tissue Flask Position 2 Optical Density of Wild Type and DCBomega<br />
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[[Image:Tissue Flask 1b.png| 450 px| left]][[Image:Tissue Flask 2b.png| 450px | right]]<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:#black;">'''c''' Tissue Flask Position 3 Optical Density of Wild Type and DCBomega<br />
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|<font size="2" style="color:#black;">'''d''' Tissue Flask Position 4 Optical Density of Wild Type and DCBomega<br />
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[[Image:Tissue Flask 3b.png| 450px | left]][[Image:Tissue Flask 4b.png| 450px | right]]<br />
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'''e''' Tissue Flask Position 5 Optical Density of Wild Type and DBComega<br />
[[Image:Tissue Flask 5b.png| 450px | left]]<br />
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Figure 1a,b,c,d,e are the optical density of a tissue flask, as assessed at 600 nm, over the course of the 6 days of the experiment.<br />
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'''Figure 2'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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[[Image:3D 241.png| 450 px | left]][[Image:3D DBCOmega2.png| 450 px | right]]<br />
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Figure 2a and 2b display the growth of the tissue flasks as measured by OD assessed as absorbance at 600 nm. The graph is organized such that those closest to the "days" axis were closest to the light source (i.e. flask 1 is closer than flask 2).<br />
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'''Figure 3'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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[[Image:Bar 241 b.png| 450px | left]][[Image:Bar DBCOmega b.png| 450px | right]]<br />
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Figure 3a and 3b display the cumulative growth of the tissue flasks as measured by the sum of the optical density of the respective cell type's tissue flasks at a given day. Furthermore, the contribution of a given tissue flask to cumulative growth is displayed.<br><br />
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'''Figure 4'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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[[image:sphaeroides tissue flask.jpg|450 px|left|]][[image:dbc tissue flask.jpg|450px|right]]<br />
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Figure 4A and 4B display the appearance of the tissue flasks for both strains on day 6. They are arranged from left to right with left being closed to the light source (flask 1) and right furthest (flask 5)<br />
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'''Figure 5'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:#black;">'''b''' R. sphaeroides Spectrum by Flask Distance from Source<br />
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[[Image: WT irradiance 3d.png| 450 px | left]][[Image: DBComega irradiance 3d.png| 450 px | right]]<br />
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Contour of Absolute Irradiance by Flash distance from Source<br />
{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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[[Image: r sphaeroides day 2 a.jpg| 450px | left]][[Image: dbc day 4b.jpg| 450px | right]]<br />
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'''e''' Wild Type Absolute Irradiance Behind Flask 1: Day 6<br />
[[Image: irradiance WT flask 1 day6.png| 450px | center]]<br />
Caption for 5e<br><br />
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'''Figure 6'''<br><br />
Nonlinear least-squares estimation of WT LH2 saturation curve<br><br />
[[Image: saturation for WT as inferred.png| 450 px]]<br />
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Caption for figure 7<br><br><br />
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'''Figure 7'''<br><br />
Simulated Optical Density of Mutant and Wild Type Bioreactors<br><br />
Layers One and Two . Layers Three, Four and Five<br />
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[[Image: OD after flask 1 and 2.jpg| 200px ]][[Image: OD after 3,4, and 5.jpg| 200px ]]<br />
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[https://2009.igem.org/Team:Wash_U/Project Back To Top]<br />
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== '''Analysis''' ==<br />
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The tissue flask experiment was an interesting way to observe the effects of a full knockout of the Light Harvesting Antenna Complex 2 (LH2) vs the Wild Type on an array of conseuctive photobioreactors. A couple of conclusions can be drawn from this data that can be used in modeling our synthetic regulation system for LH2 and in the future design of systems for the optimization of Light Harvesting Antenna size for photobioreactors . <br><br><br />
The most striking difference between the Wild Type Rhodobacter Sphaeroides 2.41 and DBCOmega (LH2 knockout) growth patterns is that the first flask in the WT (closest to the light source) grew less than the second flask, while the converse was true in DBCOmega (figure 2a and 2b). This can be attributed to the photosystem saturation curves for the respective cultures. The Wild Type has an LH2 complex, meaning that their antenna size is inherently larger and that their photosystem will be saturated at lower light intensities than the LH2 deficient mutant. We observed that the incident light intensity used in this experiment led to oversaturation for the first WT tissue flask and resulted in growth-inhibiting photodamage, as is evidenced by its lesser growth relative to the second culture. In contrast, photodamage was not observed in DBCOmega as is evidenced by the first tissue flask that grew at the fastest respective rate. Furthermore, it appears that this photodamage slowed the growth of the first WT tissue flask to the point that the OD of this tissue flask was nearly equal to that of DBCOmega after 5 days of growth(.987 vs. .967) (figure 1a). As such, the contribution of LH2 to the growth of the WT in flask 1 can be assessed to be little after day 3 when the growth curve slows- though, this light (at 800 and 850 nm) was still greatly absorbed by flask 1 ('''see irradiance figure for wt flask 1 day 6'''). It is likely that much of this light was then wasted through Non-Photochemical Quenching (NPQ), depleting the flasks behind flask 1 of photons at the LH2 absorbtion peaks and decreasing the cumulative productivity of flasks 1-5. This effect of wasting photons though NPQ under high light intensities is one that we sought to minimize in the design of our synthetic regulation for the pucB/A genes. <br><br><br />
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A further a comparison of the WT and DBCOmega experiments reveals that the 3rd tissue flask in each had almost identical growth rates (figure 1c), while the DBCOmega 4th and 5th tissue flask outperformed that of the WT (figure 1d and 1e). This can simply be attributed to the higher density of cells in the 2nd flask of the WT that led to an overall lesser quantity of photons passing through to the 4th and 5th flasks in the WT experiment and likely resulting in heterotrophic growth (though this will need to be confirmed with a "dark control" experiment that should be completed in time for the Jamboree). This decrease in available photons for the WT can be seen by viewing the absolute irradiance data (incident light intensity at a given wavelength) after the third culture for the WT (figure 5a and 5c). This decrease in irradiance is the most pronounced for the LH2 absorption bands at 800 and 850 nm, as was expected. Our synthetic regulation system for pucB/A was designed with the intention of allowing greater penetration of light through the first couple of flasks at the LH2 wavelengths, as expression of pucB/A is inversely correlated to incident light intensity.<br><br><br />
'''Thomas, add in some further comments about the sprectroradiometer stuff. Specifically about how it changes in the WT vs. that of DBCOmega using some specific examples on the graphs'''<br><br><br />
Overall, the cumulative culture growth of the WT exceeded that of DBCOmega (figure 3) due to the performance of the second tissue flask of the WT relative to that of DBCOmega (figure 1b) despite the photodamage that occurred in tissue flask 1 for the WT. This cumulative culture growth can be considered photosynthetic productivty and is proportional to the amount of energy/desired product that can be obtained from these cultures. <br><br><br />
'''It is an interesting exercise to model how our mutant system would perform under these same conditions: Thomas add in some comments about referencing that diagram and what we took from this tissue flask experiment to construct that (and upload it when you are done with it).'''<br />
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'''add stuff here about the second flasks and approaching 20% contribution of LH1 to growth'''<br />
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=='''Conclusion and Future Work'''==<br />
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Based on model, should have had feedback mechanism- should be something that sets a minimum expression level for LH2, that is...<br><br />
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-tissue flask w prkcbc3 before jamboree and compare/interpret using characterization data<br><br />
-tissue flask w. full mutant in future- if this doesn't reveal cph8 to responding in an exponentially decaying fashion to light intensity, try w. a system that would<br />
-characterize puc promoter under light conditions and additional oxygen tensions<br />
-complete other characterization work<br />
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[https://2009.igem.org/Team:Wash_U/Project Back To Top]<br />
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== '''References''' ==<br />
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# "2007-2008 Catalog & Technical Reference." <u>New England BioLabs Inc.</u> (2007).<br />
# Alon, Uri. <u>Introduction to systems biology and the design principles of biological networks</u>. Boca Raton, FL: Chapman & Hall, 2006.<br />
# Ausubel, Fred et al. <u>Short Protocols in Molecular Biology: Third Edition.</u> Canada: John Wiley and Sons, Inc., 1999.<br />
# "BioBrick Assembly Help." <u>Registry of Standard Biological Parts.</u> 2009. 6 July 2009. <http://partsregistry.org/Main_Page>.<br />
# Bower, James M. <u>Computational Modeling of Genetic and Biochemical Networks (Computational Molecular Biology).</u> New York: M.I.T. PRESS, 2001.<br />
# Braatsch, Stephan et al. "A single Flavoprotein AppA, Integrates Both Redox and Light Signals in ''Rhodobacter sphaeroides.''" <u>Molecular Microbiology.</u> Vol. 45.3 (2002): 827-836.<br />
# Brown, Stanley B. et al. "Bile Pigment Synthesis in Plants: Incorporation of Haem into Phycocyanobilin and Phycocyanobiliproteins in Cyanidium Caldarium." <u>Biochemistry.</u> Vol. 194 (1981): 137-147.<br />
# Dragnea, Vladimira et al. "Time-Resolved Spectroscopic Studies of the AppA Blue-Light Receptor BLUF Domain from ''Rhodobacter sphaeroides'' <u>Biochemistry.</u> Vol. 44 (2005): 15,978-15,985.<br />
# "Entrez Nucleotide Search." <u>National Center for Biotechnology Information.</u> 2009. 6 June 2009 <http://www.ncbi.nlm.nih.gov/nuccore/49175990?from=3533887&to=3534606&report=gbwithparts>.<br />
# Fowler, Gregory J. S. and C. Neil Hunter. "The Synthesis and Assembly of Functional High and Low Light LH2 Antenna Complexes from ''Rhodopseudomonas palustris'' in ''Rhodobacter sphaeroides.''" <u>The Journal of Biological Chemistry.</u> Vol. 271.23 (1996): 13,356-13,361.<br />
# Gabrielsen, Mads et al. "Peripheral Complexes of Purple Bacteria." <u>The Purple Phototropic Bacteria.</u> (2009): 135-153.<br />
# "GenElute HP Plasmid Miniprep Kit: User Guide." <u>Sigma-Aldrich</u>. (2008).<br />
# Harwood, Caroline S. "Nitrogenase-Catalyzed Hydrogen Production by Purple Nonsulfur Photosynthetic Bacteria." <u>Bioenergy.</u> (2008): 259-271.<br />
# Jager, Andreas et al. "The AppA and PpsR Proteins from ''Rhodobacter sphaeroides'' Can Establish a Redox-Dependent Signal Chain but Fail to Transmit Blue-Light Signals in Other Bacteria." <u>Journal of Bacteriology.</u> Vol. 189.6 (2007): 2,274-2,282.<br />
# Jones, M. R. et al. "Mutants of ''Rhodobacter sphaeroides'' Lacking One or More Pigment-Protein Complexes and Complementation with Reaction-Centre, LH1, and LH2 Genes." <u>Molecular Microbiology.</u> Vol. 6.9 (1992): 1,173-1,184.<br />
# Kondo, Toshihiko et al. "Enhancement of Hydrogen Production by a Photosynthetic Bacterium Mutant with Reduced Pigment." <u>Journal of Bioscience and Bioengineering.</u> Vol. 93.2 (2002): 145-150.<br />
# Lagarias, J. Clark. <u>PCB from Spirulina</u>. Personal Communication. June 2009.<br />
# Laible, Philip D. "Foreign Gene Expression in Photosynthetic Bacteria." <u>The Purple Phototropic Bacteria.</u> (2009): 839-860.<br />
# Lee, Jeong K. and Samuel Kaplan. "Transcriptional Regulation of ''puc'' Operon Expression in ''Rhodobacter sphaeroides.''" <u>Journal of Biological Chemistry.</u> Vol. 270.35 (1995): 20,453-20,458.<br />
# Levskaya, Anselmetal et al. "Engineering ''Escherichia coli'' to See Light." <u>Nature.</u> Vol. 438 (2005): 441-442.<br />
# "Life Sciences Catalog 2009-2010." <u>National Diagnostics.</u> (2009).<br />
# Melis, Anastasios. "Solar Energy Conversion Efficiencies in Photosynthesis: Minimizing the Chlorophyll Antennae to Maximize Efficiency." <u>Plant Science.</u> (2009).<br />
# Mizuno, Takeshi and Shoji Mizushima. "Characterization by Deletion and Localized Mutagenesis in Vitro of the Promoter Region of the ''Escherichia coli'' OmpC Gene and Importance of the Upstream DNA Domain in Positive Regulation by the OmpR Protein." <u>Journal of Bacteriology.</u> Vol. 168.1 (1986): 86-95.<br />
# Moskvin, Oleg V. et al. "Transcriptome Analysis of the ''Rhodobacter sphaeroides'' PspR Regulon: PspR as a Master Regulator of Photosystem Development." <u>Journal of Bacteriology.</u> Vol. 187.6 (2005): 2,148-2,156.<br />
# Mussgnug, Jan H. et al. "Engineering photosynthetic light capture: impacts on improved solar energy to biomass conversion." <u> Plant Biotechnology Journal</u> Vol. 5 Issue 6. (2007) pp. 802-814<br />
# "NEBCutter V2.0." <u>New England BioLabs.</u> 2009. 6 June 2009 <http://tools.neb.com/NEBcutter2/index.php>.<br />
# "OligoAnalyzer 3.1." <u>Integrated DNA Technologies.</u> 2009. 6 June 2009. <http://www.idtdna.com/analyzer/Applications/OligoAnalyzer/><br />
# Sambrook, Joseph and David W. Russell. <u>Molecular Cloning: A Laboratory Manual.</u> Vol. 1-3. New York: Cold Springs Harbor Laboratory Press, 2001.<br />
# Scheuring, Simon and Sturgis, James N. "Chromatic Adaptation of Photosynthetic Membranes" <u>Science</u> Vol. 309 (2005) pp.484-487<br />
# Sener, Melih K. et al. "Atomic-level structural and functional model of a bacterial photosynthetic membrane vesicle" <u>Proceedings of the National Academy of Sciences of the United States of America.</u> Vol. 104, Number 40,pp. 15723-15728<br />
# Shimizu-Sato, Sae et al. "A Light-Switchable Gene Promoter System." <u>Nature Publishing Group.</u> Vol. 20 (2002): 1,041-1044.<br />
# Smith, Harry and M. Geoffrey Holmes. <u>Techniquies in Photomorphogenesis.</u> London: Academic Press, Inc., 1984.<br />
# <u>System modeling in cellular biology from concepts to nuts and bolts.</u> Cambridge, MA: MIT P, 2006.<br />
# Terry, Matthew J. "Biosynthesis and Analysis of Bilins." <u>Heme, Chlorophyll, and Bilins: Methods and Protocols.</u> (2002) 273-291. <br />
# Wang, Wanneng et al. "Heterologous Synthesis and Assembly of Functional LHII Antenna Complexes from ''Rhodovulum sulfidophilum'' in ''Rhodobacter sphaeroides'' Mutant." <u>Molecular Biology Reports.</u> Vol. 12.3 (2008).<br />
# Walz, Thomas et al. "Projection Structures of Three Photosynthetic Complexes from Rhodobacter sphaeroides: LH2 at 6 A, LH1 and RC-LH1 at 25 A" <u>Journal of Molecular Biology</u> Vol. 282 pp. 833-845<br />
# Yakovlev, A. G. et al. "Light Control Over the Size of an Antenna Unit Building Block as an Efficient Strategy for Light Harvesting in Photosynthesis." <u>Federation of European Biochemical Societies.</u> (2002):129-132.<br />
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{{WashUbottom}}</div>Brendan1http://2009.igem.org/Team:Wash_U/ProjectTeam:Wash U/Project2009-10-22T02:41:39Z<p>Brendan1: /* Results */</p>
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== '''Introduction''' == <br />
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Our project goal is to maximize the photosynthetic productivity for a series of photobioreactors containing ''Rhodobacter sphaeroides'' under both high and low light intensities by synthetically regulating the size of the light-harvesting antenna complex LH2. We chose to undertake such a project in ''R. sphaeroides'' due to its well-characterized photosynthetic and genetic system. <br />
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The antenna system functions to expand the spectrum of light available for photosynthesis by absorbing different wavelengths than that of the reaction center. Essentially, it is like the dish around the main receiver of any antenna. Marine bacteria, such as ''R. sphaeroides'', evolved very large antenna complexes to absorb light in a natural environment where there is great competition for photons. As a result, the photosynthetic machinery is saturated at low light intensities in a synthetic non-competitive environment, such as a photobioreactor. This causes up to 95% of incidental photons to be dissipated as heat or fluorescence by bacteria through a process called Non-Photochemical Quenching (NPQ) (Mussgnug et al., 2007). In essence, these photons are being wasted as NPQ reduces light penetration into and across bioreactors and starves the shaded cells for for photons. One method that has been shown to improve photosynthetic productivity in photobioreactors is the reduction of light-harvesting antenna sizes (Polle et al., 2002, Mussgnug et al., 2007). Though, current approaches to this end are difficult to precisely control from the perspective of biological engineering and synthetic biology. <br />
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Our intention is to create a dynamic system to vary antenna size that is dependent on incident light intensity and that can be readily optimized using bioengineering principles. This synthetic regulation of the LH2 complex should result in the bacteria grown under high light intensities expressing fewer LH2 complexes than the cells that are more shaded, leading to a reduction of wasted incident photons while maintaining a high overall absorbance across subsequent bioreactors. Consequently, we expect to see an increase in the total photosynthetic productivity for our mutant ''Rhodobacter sphaeroides'' when compared to the wild type grown under similar conditions. <br />
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|<font size="2" style="color:black;">'''Synthetically Regulated Antenna Energy Production in Bioreactors'''<br />
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== '''Organism''' ==<br />
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''Rhodobacter sphaeroides'' is a purple Alphaproteobacteria. It is flexible metabolically and can grow heterotrophically via aerobic and anaerobic respiration, as well as phototrophically under anaerobic conditions with light. <br />
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''R. sphaeroides'' is one of the best understood photosynthetic organisms. The photosystem is located in intracytoplasmic membrane invaginations and the main components are the Light Haresting Complex 2 (LH2), Light Harvesting Complex 1 (LH1), and the Reaction Center (RC). These pigment-protein complexes non-covalently bind bacteriochlorophylls and carotenoids. LH1 and RC make up the core complex and are often found in the ratio 1:1. LH2 is more peripheral and naturally ranges in the ratio to LH1/RC of 3.0:1 to 6.7:1 under varying light conditions (Scheuring et al., 2005). LH2 absorbs photons maximally at the wavelengths of 850 and 800 nm and funnels its energy to LH1 and the reaction center for photochemistry. (Scheuring et al., 2005). <br />
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The two subunits of LH2 are coded for by the pucB/A genes and are naturally promoted by the puc promoter. <br />
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<br><div style="text-align: center;">'''ICM of ''R. sphaeroides'' '''[[Image:R sphaeroides photosystem.png| 500 px | center]]<center>source:Sener et al.2007<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:black;">'''Plate of ''R. sphaeroides'' 2.4.1'''<br />
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[[Image:R sphaeroides aborbance.png| 450px | left]][[Image:R sphaeroides 0986.png| 450px | right]]<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br>source: Walz et al. 1998<br><br />
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====Online Resources for ''R. sphaeroides''====<br />
[http://www.kazusa.or.jp/codon/cgi-bin/showcodon.cgi?species=272943 Codon Usage Table for ''R. sphaeroides'' 2.41]<br />
<br> [http://www.ncbi.nlm.nih.gov/sites/entrez?db=genome&cmd=Retrieve&dopt=Overview&list_uids=18843 ''R. sphaeroides'' 2.41 NCBI Genome Tools]<br />
<br> [http://archaea.ucsc.edu/cgi-bin/hgPcr?wp_target=&db=0&org=Rhodobacter+sphaeroides+2+4+1&wp_f=&wp_r=&wp_size=2000&wp_perfect=15&wp_good=15&wp_showPage=true&hgsid=159191 ''In silico'' PCR of ''R. sphaeroides'' 2.41 genome]<br />
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=='''Regulation'''==<br />
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[[Image:pucregulation-fullpanels.png|950px]]<br />
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'''The Natural System (Left)''' Under high oxygen tension, PpsR prevents transcription from the puc promoter of the pucBA genes, and thus prevents the expression of LH2(1st panel). Under low oxygen tension, this repression is removed by activated AppA, thus allowing expression of LH2 from pucBA(2nd panel).<br><br />
'''Synthetic Regulation System (Right)''' Our system takes advantage of constitutive protein expression from the puc promoter at low oxygen tension, and replaces pucBA with Cph8 at this site. Placing pucBA expression under the control of the ompC promoter allows for Cph8/OmpR control of LH2 expression under varying light conditions. Under high intensity of incident light, Cph8 will not be autophosphorylated, preventing expression of pucBA (3rd panel). When light intensity is lower, Cph8 will autophosphorylate, thus phosphorylating and activating OmpR and promoting expression of LH2 from the ompC promoter.<br><br />
This regulation system was designed such that light is able to penetrate deeper into and through photobioreactors where light is plentiful.<br />
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<br>[https://2009.igem.org/Team:Wash_U/Project Back To Top]<br />
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== '''Results''' ==<br />
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For methods, materials, and procedures regarding these results, please see [https://2009.igem.org/Team:Wash_U/Protocol#Tissue_Flask_Experiment Tissue Flask Experiment] or [https://2009.igem.org/Team:Wash_U/Biological_Parts#Modeling Modeling]<br />
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'''Figure 1'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:#black;">'''b''' Tissue Flask Position 2 Optical Density of Wild Type and DCBomega<br />
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[[Image:Tissue Flask 1b.png| 450 px| left]][[Image:Tissue Flask 2b.png| 450px | right]]<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:#black;">'''c''' Tissue Flask Position 3 Optical Density of Wild Type and DCBomega<br />
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|<font size="2" style="color:#black;">'''d''' Tissue Flask Position 4 Optical Density of Wild Type and DCBomega<br />
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[[Image:Tissue Flask 3b.png| 450px | left]][[Image:Tissue Flask 4b.png| 450px | right]]<br />
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'''e''' Tissue Flask Position 5 Optical Density of Wild Type and DBComega<br />
[[Image:Tissue Flask 5b.png| 450px | left]]<br />
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Figure 1a,b,c,d,e are the optical density of a tissue flask, as assessed at 600 nm, over the course of the 6 days of the experiment.<br />
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'''Figure 2'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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[[Image:3D 241.png| 450 px | left]][[Image:3D DBCOmega2.png| 450 px | right]]<br />
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Figure 2a and 2b display the growth of the tissue flasks as measured by OD assessed as absorbance at 600 nm. The graph is organized such that those closest to the "days" axis were closest to the light source (i.e. flask 1 is closer than flask 2).<br />
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'''Figure 3'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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[[Image:Bar 241 b.png| 450px | left]][[Image:Bar DBCOmega b.png| 450px | right]]<br />
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Figure 3a and 3b display the cumulative growth of the tissue flasks as measured by the sum of the optical density of the respective cell type's tissue flasks at a given day. Furthermore, the contribution of a given tissue flask to cumulative growth is displayed.<br><br />
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'''Figure 4'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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[[image:sphaeroides tissue flask.jpg|450 px|left|]][[image:dbc tissue flask.jpg|450px|right]]<br />
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Figure 4A and 4B display the appearance of the tissue flasks for both strains on day 6. They are arranged from left to right with left being closed to the light source (flask 1) and right furthest (flask 5)<br />
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'''Figure 5'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:#black;">'''b''' R. sphaeroides Spectrum by Flask Distance from Source<br />
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[[Image: WT irradiance 3d.png| 450 px | left]][[Image: DBComega irradiance 3d.png| 450 px | right]]<br />
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Contour of Absolute Irradiance by Flash distance from Source<br />
{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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[[Image: r sphaeroides day 2 a.jpg| 450px | left]][[Image: dbc day 4b.jpg| 450px | right]]<br />
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'''e''' Wild Type Absolute Irradiance Behind Flask 1: Day 6<br />
[[Image: irradiance WT flask 1 day6.png| 450px | center]]<br><br />
Caption for 5e<br><br />
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'''Figure 6'''<br><br />
Nonlinear least-squares estimation of WT LH2 saturation curve<br><br />
[[Image: saturation for WT as inferred.png| 450 px]]<br />
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Caption for figure 7<br><br />
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'''Figure 7'''<br><br />
Simulated Optical Density of Mutant and Wild Type Bioreactors<br><br />
Layers One and Two . Layers Three, Four and Five<br />
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[[Image: OD after flask 1 and 2.jpg| 200px ]][[Image: OD after 3,4, and 5.jpg| 200px ]]<br />
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== '''Analysis''' ==<br />
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The tissue flask experiment was an interesting way to observe the effects of a full knockout of the Light Harvesting Antenna Complex 2 (LH2) vs the Wild Type on an array of conseuctive photobioreactors. A couple of conclusions can be drawn from this data that can be used in modeling our synthetic regulation system for LH2 and in the future design of systems for the optimization of Light Harvesting Antenna size for photobioreactors . <br><br><br />
The most striking difference between the Wild Type Rhodobacter Sphaeroides 2.41 and DBCOmega (LH2 knockout) growth patterns is that the first flask in the WT (closest to the light source) grew less than the second flask, while the converse was true in DBCOmega (figure 2a and 2b). This can be attributed to the photosystem saturation curves for the respective cultures. The Wild Type has an LH2 complex, meaning that their antenna size is inherently larger and that their photosystem will be saturated at lower light intensities than the LH2 deficient mutant. We observed that the incident light intensity used in this experiment led to oversaturation for the first WT tissue flask and resulted in growth-inhibiting photodamage, as is evidenced by its lesser growth relative to the second culture. In contrast, photodamage was not observed in DBCOmega as is evidenced by the first tissue flask that grew at the fastest respective rate. Furthermore, it appears that this photodamage slowed the growth of the first WT tissue flask to the point that the OD of this tissue flask was nearly equal to that of DBCOmega after 5 days of growth(.987 vs. .967) (figure 1a). As such, the contribution of LH2 to the growth of the WT in flask 1 can be assessed to be little after day 3 when the growth curve slows- though, this light (at 800 and 850 nm) was still greatly absorbed by flask 1 ('''see irradiance figure for wt flask 1 day 6'''). It is likely that much of this light was then wasted through Non-Photochemical Quenching (NPQ), depleting the flasks behind flask 1 of photons at the LH2 absorbtion peaks and decreasing the cumulative productivity of flasks 1-5. This effect of wasting photons though NPQ under high light intensities is one that we sought to minimize in the design of our synthetic regulation for the pucB/A genes. <br><br><br />
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A further a comparison of the WT and DBCOmega experiments reveals that the 3rd tissue flask in each had almost identical growth rates (figure 1c), while the DBCOmega 4th and 5th tissue flask outperformed that of the WT (figure 1d and 1e). This can simply be attributed to the higher density of cells in the 2nd flask of the WT that led to an overall lesser quantity of photons passing through to the 4th and 5th flasks in the WT experiment and likely resulting in heterotrophic growth (though this will need to be confirmed with a "dark control" experiment that should be completed in time for the Jamboree). This decrease in available photons for the WT can be seen by viewing the absolute irradiance data (incident light intensity at a given wavelength) after the third culture for the WT (figure 5a and 5c). This decrease in irradiance is the most pronounced for the LH2 absorption bands at 800 and 850 nm, as was expected. Our synthetic regulation system for pucB/A was designed with the intention of allowing greater penetration of light through the first couple of flasks at the LH2 wavelengths, as expression of pucB/A is inversely correlated to incident light intensity.<br><br><br />
'''Thomas, add in some further comments about the sprectroradiometer stuff. Specifically about how it changes in the WT vs. that of DBCOmega using some specific examples on the graphs'''<br><br><br />
Overall, the cumulative culture growth of the WT exceeded that of DBCOmega (figure 3) due to the performance of the second tissue flask of the WT relative to that of DBCOmega (figure 1b) despite the photodamage that occurred in tissue flask 1 for the WT. This cumulative culture growth can be considered photosynthetic productivty and is proportional to the amount of energy/desired product that can be obtained from these cultures. <br><br><br />
'''It is an interesting exercise to model how our mutant system would perform under these same conditions: Thomas add in some comments about referencing that diagram and what we took from this tissue flask experiment to construct that (and upload it when you are done with it).'''<br />
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'''add stuff here about the second flasks and approaching 20% contribution of LH1 to growth'''<br />
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=='''Conclusion and Future Work'''==<br />
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Based on model, should have had feedback mechanism- should be something that sets a minimum expression level for LH2, that is...<br><br />
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-tissue flask w prkcbc3 before jamboree and compare/interpret using characterization data<br><br />
-tissue flask w. full mutant in future- if this doesn't reveal cph8 to responding in an exponentially decaying fashion to light intensity, try w. a system that would<br />
-characterize puc promoter under light conditions and additional oxygen tensions<br />
-complete other characterization work<br />
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== '''References''' ==<br />
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# "2007-2008 Catalog & Technical Reference." <u>New England BioLabs Inc.</u> (2007).<br />
# Alon, Uri. <u>Introduction to systems biology and the design principles of biological networks</u>. Boca Raton, FL: Chapman & Hall, 2006.<br />
# Ausubel, Fred et al. <u>Short Protocols in Molecular Biology: Third Edition.</u> Canada: John Wiley and Sons, Inc., 1999.<br />
# "BioBrick Assembly Help." <u>Registry of Standard Biological Parts.</u> 2009. 6 July 2009. <http://partsregistry.org/Main_Page>.<br />
# Bower, James M. <u>Computational Modeling of Genetic and Biochemical Networks (Computational Molecular Biology).</u> New York: M.I.T. PRESS, 2001.<br />
# Braatsch, Stephan et al. "A single Flavoprotein AppA, Integrates Both Redox and Light Signals in ''Rhodobacter sphaeroides.''" <u>Molecular Microbiology.</u> Vol. 45.3 (2002): 827-836.<br />
# Brown, Stanley B. et al. "Bile Pigment Synthesis in Plants: Incorporation of Haem into Phycocyanobilin and Phycocyanobiliproteins in Cyanidium Caldarium." <u>Biochemistry.</u> Vol. 194 (1981): 137-147.<br />
# Dragnea, Vladimira et al. "Time-Resolved Spectroscopic Studies of the AppA Blue-Light Receptor BLUF Domain from ''Rhodobacter sphaeroides'' <u>Biochemistry.</u> Vol. 44 (2005): 15,978-15,985.<br />
# "Entrez Nucleotide Search." <u>National Center for Biotechnology Information.</u> 2009. 6 June 2009 <http://www.ncbi.nlm.nih.gov/nuccore/49175990?from=3533887&to=3534606&report=gbwithparts>.<br />
# Fowler, Gregory J. S. and C. Neil Hunter. "The Synthesis and Assembly of Functional High and Low Light LH2 Antenna Complexes from ''Rhodopseudomonas palustris'' in ''Rhodobacter sphaeroides.''" <u>The Journal of Biological Chemistry.</u> Vol. 271.23 (1996): 13,356-13,361.<br />
# Gabrielsen, Mads et al. "Peripheral Complexes of Purple Bacteria." <u>The Purple Phototropic Bacteria.</u> (2009): 135-153.<br />
# "GenElute HP Plasmid Miniprep Kit: User Guide." <u>Sigma-Aldrich</u>. (2008).<br />
# Harwood, Caroline S. "Nitrogenase-Catalyzed Hydrogen Production by Purple Nonsulfur Photosynthetic Bacteria." <u>Bioenergy.</u> (2008): 259-271.<br />
# Jager, Andreas et al. "The AppA and PpsR Proteins from ''Rhodobacter sphaeroides'' Can Establish a Redox-Dependent Signal Chain but Fail to Transmit Blue-Light Signals in Other Bacteria." <u>Journal of Bacteriology.</u> Vol. 189.6 (2007): 2,274-2,282.<br />
# Jones, M. R. et al. "Mutants of ''Rhodobacter sphaeroides'' Lacking One or More Pigment-Protein Complexes and Complementation with Reaction-Centre, LH1, and LH2 Genes." <u>Molecular Microbiology.</u> Vol. 6.9 (1992): 1,173-1,184.<br />
# Kondo, Toshihiko et al. "Enhancement of Hydrogen Production by a Photosynthetic Bacterium Mutant with Reduced Pigment." <u>Journal of Bioscience and Bioengineering.</u> Vol. 93.2 (2002): 145-150.<br />
# Lagarias, J. Clark. <u>PCB from Spirulina</u>. Personal Communication. June 2009.<br />
# Laible, Philip D. "Foreign Gene Expression in Photosynthetic Bacteria." <u>The Purple Phototropic Bacteria.</u> (2009): 839-860.<br />
# Lee, Jeong K. and Samuel Kaplan. "Transcriptional Regulation of ''puc'' Operon Expression in ''Rhodobacter sphaeroides.''" <u>Journal of Biological Chemistry.</u> Vol. 270.35 (1995): 20,453-20,458.<br />
# Levskaya, Anselmetal et al. "Engineering ''Escherichia coli'' to See Light." <u>Nature.</u> Vol. 438 (2005): 441-442.<br />
# "Life Sciences Catalog 2009-2010." <u>National Diagnostics.</u> (2009).<br />
# Melis, Anastasios. "Solar Energy Conversion Efficiencies in Photosynthesis: Minimizing the Chlorophyll Antennae to Maximize Efficiency." <u>Plant Science.</u> (2009).<br />
# Mizuno, Takeshi and Shoji Mizushima. "Characterization by Deletion and Localized Mutagenesis in Vitro of the Promoter Region of the ''Escherichia coli'' OmpC Gene and Importance of the Upstream DNA Domain in Positive Regulation by the OmpR Protein." <u>Journal of Bacteriology.</u> Vol. 168.1 (1986): 86-95.<br />
# Moskvin, Oleg V. et al. "Transcriptome Analysis of the ''Rhodobacter sphaeroides'' PspR Regulon: PspR as a Master Regulator of Photosystem Development." <u>Journal of Bacteriology.</u> Vol. 187.6 (2005): 2,148-2,156.<br />
# Mussgnug, Jan H. et al. "Engineering photosynthetic light capture: impacts on improved solar energy to biomass conversion." <u> Plant Biotechnology Journal</u> Vol. 5 Issue 6. (2007) pp. 802-814<br />
# "NEBCutter V2.0." <u>New England BioLabs.</u> 2009. 6 June 2009 <http://tools.neb.com/NEBcutter2/index.php>.<br />
# "OligoAnalyzer 3.1." <u>Integrated DNA Technologies.</u> 2009. 6 June 2009. <http://www.idtdna.com/analyzer/Applications/OligoAnalyzer/><br />
# Sambrook, Joseph and David W. Russell. <u>Molecular Cloning: A Laboratory Manual.</u> Vol. 1-3. New York: Cold Springs Harbor Laboratory Press, 2001.<br />
# Scheuring, Simon and Sturgis, James N. "Chromatic Adaptation of Photosynthetic Membranes" <u>Science</u> Vol. 309 (2005) pp.484-487<br />
# Sener, Melih K. et al. "Atomic-level structural and functional model of a bacterial photosynthetic membrane vesicle" <u>Proceedings of the National Academy of Sciences of the United States of America.</u> Vol. 104, Number 40,pp. 15723-15728<br />
# Shimizu-Sato, Sae et al. "A Light-Switchable Gene Promoter System." <u>Nature Publishing Group.</u> Vol. 20 (2002): 1,041-1044.<br />
# Smith, Harry and M. Geoffrey Holmes. <u>Techniquies in Photomorphogenesis.</u> London: Academic Press, Inc., 1984.<br />
# <u>System modeling in cellular biology from concepts to nuts and bolts.</u> Cambridge, MA: MIT P, 2006.<br />
# Terry, Matthew J. "Biosynthesis and Analysis of Bilins." <u>Heme, Chlorophyll, and Bilins: Methods and Protocols.</u> (2002) 273-291. <br />
# Wang, Wanneng et al. "Heterologous Synthesis and Assembly of Functional LHII Antenna Complexes from ''Rhodovulum sulfidophilum'' in ''Rhodobacter sphaeroides'' Mutant." <u>Molecular Biology Reports.</u> Vol. 12.3 (2008).<br />
# Walz, Thomas et al. "Projection Structures of Three Photosynthetic Complexes from Rhodobacter sphaeroides: LH2 at 6 A, LH1 and RC-LH1 at 25 A" <u>Journal of Molecular Biology</u> Vol. 282 pp. 833-845<br />
# Yakovlev, A. G. et al. "Light Control Over the Size of an Antenna Unit Building Block as an Efficient Strategy for Light Harvesting in Photosynthesis." <u>Federation of European Biochemical Societies.</u> (2002):129-132.<br />
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{{WashUbottom}}</div>Brendan1http://2009.igem.org/Team:Wash_U/ProjectTeam:Wash U/Project2009-10-22T02:39:56Z<p>Brendan1: /* Results */</p>
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__NOTOC__<br />
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== '''Introduction''' == <br />
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Our project goal is to maximize the photosynthetic productivity for a series of photobioreactors containing ''Rhodobacter sphaeroides'' under both high and low light intensities by synthetically regulating the size of the light-harvesting antenna complex LH2. We chose to undertake such a project in ''R. sphaeroides'' due to its well-characterized photosynthetic and genetic system. <br />
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The antenna system functions to expand the spectrum of light available for photosynthesis by absorbing different wavelengths than that of the reaction center. Essentially, it is like the dish around the main receiver of any antenna. Marine bacteria, such as ''R. sphaeroides'', evolved very large antenna complexes to absorb light in a natural environment where there is great competition for photons. As a result, the photosynthetic machinery is saturated at low light intensities in a synthetic non-competitive environment, such as a photobioreactor. This causes up to 95% of incidental photons to be dissipated as heat or fluorescence by bacteria through a process called Non-Photochemical Quenching (NPQ) (Mussgnug et al., 2007). In essence, these photons are being wasted as NPQ reduces light penetration into and across bioreactors and starves the shaded cells for for photons. One method that has been shown to improve photosynthetic productivity in photobioreactors is the reduction of light-harvesting antenna sizes (Polle et al., 2002, Mussgnug et al., 2007). Though, current approaches to this end are difficult to precisely control from the perspective of biological engineering and synthetic biology. <br />
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Our intention is to create a dynamic system to vary antenna size that is dependent on incident light intensity and that can be readily optimized using bioengineering principles. This synthetic regulation of the LH2 complex should result in the bacteria grown under high light intensities expressing fewer LH2 complexes than the cells that are more shaded, leading to a reduction of wasted incident photons while maintaining a high overall absorbance across subsequent bioreactors. Consequently, we expect to see an increase in the total photosynthetic productivity for our mutant ''Rhodobacter sphaeroides'' when compared to the wild type grown under similar conditions. <br />
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|<font size="2" style="color:black;">'''Synthetically Regulated Antenna Energy Production in Bioreactors'''<br />
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== '''Organism''' ==<br />
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''Rhodobacter sphaeroides'' is a purple Alphaproteobacteria. It is flexible metabolically and can grow heterotrophically via aerobic and anaerobic respiration, as well as phototrophically under anaerobic conditions with light. <br />
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''R. sphaeroides'' is one of the best understood photosynthetic organisms. The photosystem is located in intracytoplasmic membrane invaginations and the main components are the Light Haresting Complex 2 (LH2), Light Harvesting Complex 1 (LH1), and the Reaction Center (RC). These pigment-protein complexes non-covalently bind bacteriochlorophylls and carotenoids. LH1 and RC make up the core complex and are often found in the ratio 1:1. LH2 is more peripheral and naturally ranges in the ratio to LH1/RC of 3.0:1 to 6.7:1 under varying light conditions (Scheuring et al., 2005). LH2 absorbs photons maximally at the wavelengths of 850 and 800 nm and funnels its energy to LH1 and the reaction center for photochemistry. (Scheuring et al., 2005). <br />
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The two subunits of LH2 are coded for by the pucB/A genes and are naturally promoted by the puc promoter. <br />
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<br><div style="text-align: center;">'''ICM of ''R. sphaeroides'' '''[[Image:R sphaeroides photosystem.png| 500 px | center]]<center>source:Sener et al.2007<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:black;">'''Plate of ''R. sphaeroides'' 2.4.1'''<br />
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[[Image:R sphaeroides aborbance.png| 450px | left]][[Image:R sphaeroides 0986.png| 450px | right]]<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br>source: Walz et al. 1998<br><br />
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====Online Resources for ''R. sphaeroides''====<br />
[http://www.kazusa.or.jp/codon/cgi-bin/showcodon.cgi?species=272943 Codon Usage Table for ''R. sphaeroides'' 2.41]<br />
<br> [http://www.ncbi.nlm.nih.gov/sites/entrez?db=genome&cmd=Retrieve&dopt=Overview&list_uids=18843 ''R. sphaeroides'' 2.41 NCBI Genome Tools]<br />
<br> [http://archaea.ucsc.edu/cgi-bin/hgPcr?wp_target=&db=0&org=Rhodobacter+sphaeroides+2+4+1&wp_f=&wp_r=&wp_size=2000&wp_perfect=15&wp_good=15&wp_showPage=true&hgsid=159191 ''In silico'' PCR of ''R. sphaeroides'' 2.41 genome]<br />
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=='''Regulation'''==<br />
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[[Image:pucregulation-fullpanels.png|950px]]<br />
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'''The Natural System (Left)''' Under high oxygen tension, PpsR prevents transcription from the puc promoter of the pucBA genes, and thus prevents the expression of LH2(1st panel). Under low oxygen tension, this repression is removed by activated AppA, thus allowing expression of LH2 from pucBA(2nd panel).<br><br />
'''Synthetic Regulation System (Right)''' Our system takes advantage of constitutive protein expression from the puc promoter at low oxygen tension, and replaces pucBA with Cph8 at this site. Placing pucBA expression under the control of the ompC promoter allows for Cph8/OmpR control of LH2 expression under varying light conditions. Under high intensity of incident light, Cph8 will not be autophosphorylated, preventing expression of pucBA (3rd panel). When light intensity is lower, Cph8 will autophosphorylate, thus phosphorylating and activating OmpR and promoting expression of LH2 from the ompC promoter.<br><br />
This regulation system was designed such that light is able to penetrate deeper into and through photobioreactors where light is plentiful.<br />
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<br>[https://2009.igem.org/Team:Wash_U/Project Back To Top]<br />
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== '''Results''' ==<br />
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For methods, materials, and procedures regarding these results, please see [https://2009.igem.org/Team:Wash_U/Protocol#Tissue_Flask_Experiment Tissue Flask Experiment] or [https://2009.igem.org/Team:Wash_U/Biological_Parts#Modeling Modeling]<br />
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'''Figure 1'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:#black;">'''b''' Tissue Flask Position 2 Optical Density of Wild Type and DCBomega<br />
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[[Image:Tissue Flask 1b.png| 450 px| left]][[Image:Tissue Flask 2b.png| 450px | right]]<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:#black;">'''c''' Tissue Flask Position 3 Optical Density of Wild Type and DCBomega<br />
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|<font size="2" style="color:#black;">'''d''' Tissue Flask Position 4 Optical Density of Wild Type and DCBomega<br />
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[[Image:Tissue Flask 3b.png| 450px | left]][[Image:Tissue Flask 4b.png| 450px | right]]<br />
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'''e''' Tissue Flask Position 5 Optical Density of Wild Type and DBComega<br />
[[Image:Tissue Flask 5b.png| 450px | left]]<br />
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Figure 1a,b,c,d,e are the optical density of a tissue flask, as assessed at 600 nm, over the course of the 6 days of the experiment.<br />
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'''Figure 2'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:#black;">'''b''' The Change in the Optical Density of the Tissue Flasks for the WT<br />
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[[Image:3D 241.png| 450 px | left]][[Image:3D DBCOmega2.png| 450 px | right]]<br />
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Figure 2a and 2b display the growth of the tissue flasks as measured by OD assessed as absorbance at 600 nm. The graph is organized such that those closest to the "days" axis were closest to the light source (i.e. flask 1 is closer than flask 2).<br />
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'''Figure 3'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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[[Image:Bar 241 b.png| 450px | left]][[Image:Bar DBCOmega b.png| 450px | right]]<br />
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Figure 3a and 3b display the cumulative growth of the tissue flasks as measured by the sum of the optical density of the respective cell type's tissue flasks at a given day. Furthermore, the contribution of a given tissue flask to cumulative growth is displayed.<br><br />
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'''Figure 4'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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[[image:sphaeroides tissue flask.jpg|450 px|left|]][[image:dbc tissue flask.jpg|450px|right]]<br />
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Figure 4A and 4B display the appearance of the tissue flasks for both strains on day 6. They are arranged from left to right with left being closed to the light source (flask 1) and right furthest (flask 5)<br />
<div style="text-align: center;"> <br />
'''Figure 5'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:#black;">'''b''' R. sphaeroides Spectrum by Flask Distance from Source<br />
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[[Image: WT irradiance 3d.png| 450 px | left]][[Image: DBComega irradiance 3d.png| 450 px | right]]<br />
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Contour of Absolute Irradiance by Flash distance from Source<br />
{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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[[Image: r sphaeroides day 2 a.jpg| 450px | left]][[Image: dbc day 4b.jpg| 450px | right]]<br />
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'''e''' Wild Type Absolute Irradiance Behind Flask 1: Day 6<br />
[[Image: irradiance WT flask 1 day6.png| 450px | center]]<br><br><br />
Caption for 5e<br />
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'''Figure 6'''<br />
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Relative growth of DBComega to WT in Flask 2 at OD 600<br />
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[[Image:relative growth.png| 600px | center]]<br />
This data illustrates the contribution of LH1 to growth relative to that of LH2 <br><br />
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'''Figure 7'''<br><br />
Nonlinear least-squares estimation of WT LH2 saturation curve<br><br />
[[Image: saturation for WT as inferred.png| 450 px]]<br />
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Caption for figure 7<br />
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'''Figure 8'''<br><br />
Simulated Optical Density of Mutant and Wild Type Bioreactors<br><br />
Layers One and Two . Layers Three, Four and Five<br />
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[[Image: OD after flask 1 and 2.jpg| 200px ]][[Image: OD after 3,4, and 5.jpg| 200px ]]<br />
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[https://2009.igem.org/Team:Wash_U/Project Back To Top]<br />
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== '''Analysis''' ==<br />
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The tissue flask experiment was an interesting way to observe the effects of a full knockout of the Light Harvesting Antenna Complex 2 (LH2) vs the Wild Type on an array of conseuctive photobioreactors. A couple of conclusions can be drawn from this data that can be used in modeling our synthetic regulation system for LH2 and in the future design of systems for the optimization of Light Harvesting Antenna size for photobioreactors . <br><br><br />
The most striking difference between the Wild Type Rhodobacter Sphaeroides 2.41 and DBCOmega (LH2 knockout) growth patterns is that the first flask in the WT (closest to the light source) grew less than the second flask, while the converse was true in DBCOmega (figure 2a and 2b). This can be attributed to the photosystem saturation curves for the respective cultures. The Wild Type has an LH2 complex, meaning that their antenna size is inherently larger and that their photosystem will be saturated at lower light intensities than the LH2 deficient mutant. We observed that the incident light intensity used in this experiment led to oversaturation for the first WT tissue flask and resulted in growth-inhibiting photodamage, as is evidenced by its lesser growth relative to the second culture. In contrast, photodamage was not observed in DBCOmega as is evidenced by the first tissue flask that grew at the fastest respective rate. Furthermore, it appears that this photodamage slowed the growth of the first WT tissue flask to the point that the OD of this tissue flask was nearly equal to that of DBCOmega after 5 days of growth(.987 vs. .967) (figure 1a). As such, the contribution of LH2 to the growth of the WT in flask 1 can be assessed to be little after day 3 when the growth curve slows- though, this light (at 800 and 850 nm) was still greatly absorbed by flask 1 ('''see irradiance figure for wt flask 1 day 6'''). It is likely that much of this light was then wasted through Non-Photochemical Quenching (NPQ), depleting the flasks behind flask 1 of photons at the LH2 absorbtion peaks and decreasing the cumulative productivity of flasks 1-5. This effect of wasting photons though NPQ under high light intensities is one that we sought to minimize in the design of our synthetic regulation for the pucB/A genes. <br><br><br />
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A further a comparison of the WT and DBCOmega experiments reveals that the 3rd tissue flask in each had almost identical growth rates (figure 1c), while the DBCOmega 4th and 5th tissue flask outperformed that of the WT (figure 1d and 1e). This can simply be attributed to the higher density of cells in the 2nd flask of the WT that led to an overall lesser quantity of photons passing through to the 4th and 5th flasks in the WT experiment and likely resulting in heterotrophic growth (though this will need to be confirmed with a "dark control" experiment that should be completed in time for the Jamboree). This decrease in available photons for the WT can be seen by viewing the absolute irradiance data (incident light intensity at a given wavelength) after the third culture for the WT (figure 5a and 5c). This decrease in irradiance is the most pronounced for the LH2 absorption bands at 800 and 850 nm, as was expected. Our synthetic regulation system for pucB/A was designed with the intention of allowing greater penetration of light through the first couple of flasks at the LH2 wavelengths, as expression of pucB/A is inversely correlated to incident light intensity.<br><br><br />
'''Thomas, add in some further comments about the sprectroradiometer stuff. Specifically about how it changes in the WT vs. that of DBCOmega using some specific examples on the graphs'''<br><br><br />
Overall, the cumulative culture growth of the WT exceeded that of DBCOmega (figure 3) due to the performance of the second tissue flask of the WT relative to that of DBCOmega (figure 1b) despite the photodamage that occurred in tissue flask 1 for the WT. This cumulative culture growth can be considered photosynthetic productivty and is proportional to the amount of energy/desired product that can be obtained from these cultures. <br><br><br />
'''It is an interesting exercise to model how our mutant system would perform under these same conditions: Thomas add in some comments about referencing that diagram and what we took from this tissue flask experiment to construct that (and upload it when you are done with it).'''<br />
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'''add stuff here about the second flasks and approaching 20% contribution of LH1 to growth'''<br />
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=='''Conclusion and Future Work'''==<br />
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Based on model, should have had feedback mechanism- should be something that sets a minimum expression level for LH2, that is...<br><br />
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-tissue flask w prkcbc3 before jamboree and compare/interpret using characterization data<br><br />
-tissue flask w. full mutant in future- if this doesn't reveal cph8 to responding in an exponentially decaying fashion to light intensity, try w. a system that would<br />
-characterize puc promoter under light conditions and additional oxygen tensions<br />
-complete other characterization work<br />
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== '''References''' ==<br />
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# "2007-2008 Catalog & Technical Reference." <u>New England BioLabs Inc.</u> (2007).<br />
# Alon, Uri. <u>Introduction to systems biology and the design principles of biological networks</u>. Boca Raton, FL: Chapman & Hall, 2006.<br />
# Ausubel, Fred et al. <u>Short Protocols in Molecular Biology: Third Edition.</u> Canada: John Wiley and Sons, Inc., 1999.<br />
# "BioBrick Assembly Help." <u>Registry of Standard Biological Parts.</u> 2009. 6 July 2009. <http://partsregistry.org/Main_Page>.<br />
# Bower, James M. <u>Computational Modeling of Genetic and Biochemical Networks (Computational Molecular Biology).</u> New York: M.I.T. PRESS, 2001.<br />
# Braatsch, Stephan et al. "A single Flavoprotein AppA, Integrates Both Redox and Light Signals in ''Rhodobacter sphaeroides.''" <u>Molecular Microbiology.</u> Vol. 45.3 (2002): 827-836.<br />
# Brown, Stanley B. et al. "Bile Pigment Synthesis in Plants: Incorporation of Haem into Phycocyanobilin and Phycocyanobiliproteins in Cyanidium Caldarium." <u>Biochemistry.</u> Vol. 194 (1981): 137-147.<br />
# Dragnea, Vladimira et al. "Time-Resolved Spectroscopic Studies of the AppA Blue-Light Receptor BLUF Domain from ''Rhodobacter sphaeroides'' <u>Biochemistry.</u> Vol. 44 (2005): 15,978-15,985.<br />
# "Entrez Nucleotide Search." <u>National Center for Biotechnology Information.</u> 2009. 6 June 2009 <http://www.ncbi.nlm.nih.gov/nuccore/49175990?from=3533887&to=3534606&report=gbwithparts>.<br />
# Fowler, Gregory J. S. and C. Neil Hunter. "The Synthesis and Assembly of Functional High and Low Light LH2 Antenna Complexes from ''Rhodopseudomonas palustris'' in ''Rhodobacter sphaeroides.''" <u>The Journal of Biological Chemistry.</u> Vol. 271.23 (1996): 13,356-13,361.<br />
# Gabrielsen, Mads et al. "Peripheral Complexes of Purple Bacteria." <u>The Purple Phototropic Bacteria.</u> (2009): 135-153.<br />
# "GenElute HP Plasmid Miniprep Kit: User Guide." <u>Sigma-Aldrich</u>. (2008).<br />
# Harwood, Caroline S. "Nitrogenase-Catalyzed Hydrogen Production by Purple Nonsulfur Photosynthetic Bacteria." <u>Bioenergy.</u> (2008): 259-271.<br />
# Jager, Andreas et al. "The AppA and PpsR Proteins from ''Rhodobacter sphaeroides'' Can Establish a Redox-Dependent Signal Chain but Fail to Transmit Blue-Light Signals in Other Bacteria." <u>Journal of Bacteriology.</u> Vol. 189.6 (2007): 2,274-2,282.<br />
# Jones, M. R. et al. "Mutants of ''Rhodobacter sphaeroides'' Lacking One or More Pigment-Protein Complexes and Complementation with Reaction-Centre, LH1, and LH2 Genes." <u>Molecular Microbiology.</u> Vol. 6.9 (1992): 1,173-1,184.<br />
# Kondo, Toshihiko et al. "Enhancement of Hydrogen Production by a Photosynthetic Bacterium Mutant with Reduced Pigment." <u>Journal of Bioscience and Bioengineering.</u> Vol. 93.2 (2002): 145-150.<br />
# Lagarias, J. Clark. <u>PCB from Spirulina</u>. Personal Communication. June 2009.<br />
# Laible, Philip D. "Foreign Gene Expression in Photosynthetic Bacteria." <u>The Purple Phototropic Bacteria.</u> (2009): 839-860.<br />
# Lee, Jeong K. and Samuel Kaplan. "Transcriptional Regulation of ''puc'' Operon Expression in ''Rhodobacter sphaeroides.''" <u>Journal of Biological Chemistry.</u> Vol. 270.35 (1995): 20,453-20,458.<br />
# Levskaya, Anselmetal et al. "Engineering ''Escherichia coli'' to See Light." <u>Nature.</u> Vol. 438 (2005): 441-442.<br />
# "Life Sciences Catalog 2009-2010." <u>National Diagnostics.</u> (2009).<br />
# Melis, Anastasios. "Solar Energy Conversion Efficiencies in Photosynthesis: Minimizing the Chlorophyll Antennae to Maximize Efficiency." <u>Plant Science.</u> (2009).<br />
# Mizuno, Takeshi and Shoji Mizushima. "Characterization by Deletion and Localized Mutagenesis in Vitro of the Promoter Region of the ''Escherichia coli'' OmpC Gene and Importance of the Upstream DNA Domain in Positive Regulation by the OmpR Protein." <u>Journal of Bacteriology.</u> Vol. 168.1 (1986): 86-95.<br />
# Moskvin, Oleg V. et al. "Transcriptome Analysis of the ''Rhodobacter sphaeroides'' PspR Regulon: PspR as a Master Regulator of Photosystem Development." <u>Journal of Bacteriology.</u> Vol. 187.6 (2005): 2,148-2,156.<br />
# Mussgnug, Jan H. et al. "Engineering photosynthetic light capture: impacts on improved solar energy to biomass conversion." <u> Plant Biotechnology Journal</u> Vol. 5 Issue 6. (2007) pp. 802-814<br />
# "NEBCutter V2.0." <u>New England BioLabs.</u> 2009. 6 June 2009 <http://tools.neb.com/NEBcutter2/index.php>.<br />
# "OligoAnalyzer 3.1." <u>Integrated DNA Technologies.</u> 2009. 6 June 2009. <http://www.idtdna.com/analyzer/Applications/OligoAnalyzer/><br />
# Sambrook, Joseph and David W. Russell. <u>Molecular Cloning: A Laboratory Manual.</u> Vol. 1-3. New York: Cold Springs Harbor Laboratory Press, 2001.<br />
# Scheuring, Simon and Sturgis, James N. "Chromatic Adaptation of Photosynthetic Membranes" <u>Science</u> Vol. 309 (2005) pp.484-487<br />
# Sener, Melih K. et al. "Atomic-level structural and functional model of a bacterial photosynthetic membrane vesicle" <u>Proceedings of the National Academy of Sciences of the United States of America.</u> Vol. 104, Number 40,pp. 15723-15728<br />
# Shimizu-Sato, Sae et al. "A Light-Switchable Gene Promoter System." <u>Nature Publishing Group.</u> Vol. 20 (2002): 1,041-1044.<br />
# Smith, Harry and M. Geoffrey Holmes. <u>Techniquies in Photomorphogenesis.</u> London: Academic Press, Inc., 1984.<br />
# <u>System modeling in cellular biology from concepts to nuts and bolts.</u> Cambridge, MA: MIT P, 2006.<br />
# Terry, Matthew J. "Biosynthesis and Analysis of Bilins." <u>Heme, Chlorophyll, and Bilins: Methods and Protocols.</u> (2002) 273-291. <br />
# Wang, Wanneng et al. "Heterologous Synthesis and Assembly of Functional LHII Antenna Complexes from ''Rhodovulum sulfidophilum'' in ''Rhodobacter sphaeroides'' Mutant." <u>Molecular Biology Reports.</u> Vol. 12.3 (2008).<br />
# Walz, Thomas et al. "Projection Structures of Three Photosynthetic Complexes from Rhodobacter sphaeroides: LH2 at 6 A, LH1 and RC-LH1 at 25 A" <u>Journal of Molecular Biology</u> Vol. 282 pp. 833-845<br />
# Yakovlev, A. G. et al. "Light Control Over the Size of an Antenna Unit Building Block as an Efficient Strategy for Light Harvesting in Photosynthesis." <u>Federation of European Biochemical Societies.</u> (2002):129-132.<br />
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[https://2009.igem.org/Team:Wash_U/Project Back To Top]<br />
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{{WashUbottom}}</div>Brendan1http://2009.igem.org/Team:Wash_U/Biological_PartsTeam:Wash U/Biological Parts2009-10-22T02:37:32Z<p>Brendan1: /* Simulating a Bioreactor */</p>
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The Registry of Standard Biological Parts is a library of DNA sequences combined with online characterization resources. The Registry has created a standard protocol for making segments of DNA compatible with all other segments of DNA, regardless of order or size. BioBrick is the term given to such segments of DNA, a term alluding to the fact that any number of bricks may be combined in any order to produce complex, unique systems. This is accomplished by standardizing the restriction enzymes used to surround BioBricks, as well as the plasmids used to transform them. For a graphical representation of of the process, please click [http://ginkgobioworks.com/support/BioBrick_Assembly_Manual.jpg here]. A powerful online database provides information and characterization of all of the BioBricks in the Registry and uses the wiki format (the same one used in wikipedia) which encourages others to edit content directly on the page. Below are list of parts that were used/created for this project. <br />
<div style="text-align: left;"><font size="4" style="color:black"><br />
=='''Parts'''==<br />
<center><br />
<font size="2"><br />
Parts used to characterize and build our final project<br />
{|cellpadding="9" cellspacing="0" style="background-color:#FFEFD5;" border=1<br />
|'''Component'''<br />
|'''Part/Accession #'''<br />
|'''Base Pairs'''<br />
|'''Plasmid'''<br />
|'''Resistance'''<br />
|'''Well'''<br />
|-<br />
|RBS-34<br />
|[http://partsregistry.org/Part:BBa_B0034 BBa_B0034]<br />
|12<br />
|pSB1A2<br />
|Ampicillin<br />
|plate 1, 2M<br />
|-<br />
|Cph8<br />
|[http://partsregistry.org/Part:BBa_I15010 BBa_I15010]<br />
|2,238<br />
|pSB2K3<br />
|Kanamycin<br />
|N/A<br />
|-<br />
|RFP<br />
|[http://partsregistry.org/Part:BBa_J04051 BBa_J04051]<br />
|720<br />
|N/A<br />
|N/A<br />
|N/A<br />
|-<br />
|OmpR (E. coli)<br />
|[http://partsregistry.org/wiki/index.php?title=Part:BBa_K098011 BBa_K098011]<br />
|720<br />
|pSB1T3<br />
|Tetracycline<br />
|N/A<br />
|-<br />
|OmpR (R. sphaeroides)<br />
|[http://partsregistry.org/Part:BBa_K227010 BBa_K227010]<br />
|720<br />
|New<br />
|New<br />
|New<br />
|-<br />
|Terminator<br />
|[http://partsregistry.org/Part:BBa_B0015 BBa_B0015]<br />
|129<br />
|pSB1AK3<br />
|Ampicillin <br>and Kanamycin<br />
|plate 1, 23L<br />
|-<br />
|RBS +OmpR(sph) + Terminator<br />
includes prefix and suffix<br />
|[[media:OmpR_+_terminator.txt|sequence]]<br />
[http://partsregistry.org/Part:BBa_K227011 BBa_K227011]<br />
|875/916<br />
|pSB1k3<br />
|Kanamycin<br />
|synthesized<br />
|-<br />
|OmpC promoter<br />
|[http://partsregistry.org/Part:BBa_R0082 BBa_R0082]<br />
|108<br />
|pSB1A2<br />
|Ampicillin<br />
|plate 1, 16K<br />
|-<br />
|puc promoter<br />
|[http://partsregistry.org/Part:BBa_K227007 BBa_K227007]<br />
|651<br />
|pSB1k3<br />
|Kanamycin<br />
|New<br />
|-<br />
|puc BA<br />
|[http://partsregistry.org/Part:BBa_K227006 BBa_K227006]<br />
|336<br />
|pSB1k3<br />
|Kanamycin<br />
|New<br />
|-<br />
|puc B<br />
|[http://partsregistry.org/Part:BBa_K227005 BBa_K227005]<br />
|156<br />
|pSB1k3<br />
|Kanamycin<br />
|New<br />
|-<br />
|puc A<br />
|[http://partsregistry.org/Part:BBa_K227004 BBa_K227004]<br />
|165<br />
|pSB1k3<br />
|Kanamycin<br />
|New<br />
|-<br />
|OmpC promoter+pucBA<br />
|[[media:OmpC_promoter_+_puc_BA.txt|sequence]]<br />
[http://partsregistry.org/Part:BBa_K227008 BBa_K227008]<br />
|492/539<br />
|pSB1k3<br />
|Kanamycin<br />
|synthesized<br />
|-<br />
|Green Fluorescent Protein<br />
|[http://partsregistry.org/Part:BBa_E0240 BBa_E0240]<br />
|876<br />
|pSB1A2<br />
|Ampicillin<br />
|plate 1, 12M<br />
|}<br />
<br />
<br />
Plasmids used to create and characterize our project<br />
{|cellpadding="9" cellspacing="0" style="background-color:#FFEFD5;" border=1<br />
|'''Plasmid'''<br />
|'''Base Pairs'''<br />
|'''Resistance'''<br />
|'''Copy Number'''<br />
|-<br />
|[http://partsregistry.org/Part:pSB1A2 pSB1A2]<br />
|2,079<br />
|Ampicillin<br />
|high<br />
|-<br />
|[http://partsregistry.org/Part:pSB1K3 pSB1K3]<br />
|2,206<br />
|Kanamycin<br />
|high<br />
|-<br />
|[http://partsregistry.org/Part:pSB1A3 pSB1A3]<br />
|2,157<br />
|Ampicillin<br />
|high<br />
|-<br />
|[http://partsregistry.org/Part:pSB2K3 pSB2K3]<br />
|4,425<br />
|Kanamycin<br />
|variable<br />
|-<br />
|[http://partsregistry.org/Part:pSB1T3 pSB1T3]<br />
|2,463<br />
|Tetracycline<br />
|high<br />
|-<br />
|pRKCBC3<br />
|~11.5kb<br />
|Tetracycline<br />
|4<br />
|}<font size="2"><br />
<br />
<br />
<br />
Parts submitted to the Registry of Standard Biological Parts<br />
{|cellpadding="9" cellspacing="0" style="background-color:#FFEFD5;" border=1<br />
|'''Part/Accession #Component'''<br />
|'''Component'''<br />
|'''Type'''<br />
|'''Base Pairs'''<br />
|'''Plasmid'''<br />
|'''Resistance'''<br />
|-<br />
|[http://partsregistry.org/Part:BBa_I15010 BBa_I15010]<br />
|Cph8 (planned resubmission)<br />
|Coding<br />
|2,238<br />
|plasmid<br />
|Resistance<br />
|-<br />
|[http://partsregistry.org/Part:BBa_K227004 BBa_K227004]<br />
|puc A<br />
|Coding<br />
|165<br />
|pSB1K3<br />
|Kanamycin<br />
|-<br />
|[http://partsregistry.org/Part:BBa_K227005 BBa_K227005]<br />
|puc B<br />
|Coding<br />
|156<br />
|pSB1K3<br />
|Kanamycin<br />
|-<br />
|[http://partsregistry.org/Part:BBa_K227006 BBa_K227006]<br />
|puc BA<br />
|Coding<br />
|336<br />
|pSB1K3<br />
|Kanamycin<br />
|-<br />
|[http://partsregistry.org/Part:BBa_K227007 BBa_K227007]<br />
|puc promoter<br />
|Regulatory<br />
|651<br />
|pSB1K3<br />
|Kanamycin<br />
|-<br />
|[http://partsregistry.org/Part:BBa_K227008 BBa_K227008]<br />
|ompC+PucBA (synthesized)<br />
|Composite<br />
|492<br />
|pSB1AT3<br />
|Ampicilin,Tetracycline<br />
|-<br />
|[http://partsregistry.org/Part:BBa_K227009 BBa_K227009]<br />
|PucPromotor+GFP<br />
|Composite<br />
|1377<br />
|pSB1A2<br />
|Ampicilin<br />
|-<br />
|[http://partsregistry.org/Part:BBa_K227011 BBa_K227011]<br />
|RBS34+OmpR(sph)+Term (synthesized)<br />
|Composite<br />
|875<br />
|pSB1K3<br />
|Kanamycin<br />
|-<br />
|[http://partsregistry.org/Part:BBa_K227012 BBa_K227012]<br />
|RBS34+OmpR(sph)+Term+OmpC+PucB/A<br />
|Composite<br />
|1375<br />
|pSB1K3<br />
|Kanamycin<br />
|-<br />
|[http://partsregistry.org/Part:BBa_K227013 BBa_K227013]<br />
|ompCpro + GFP<br />
|Composite<br />
|992<br />
|pSB1A2<br />
|Ampicilin<br />
|-<br />
|[http://partsregistry.org/Part:BBa_K227014 BBa_K227014]<br />
|pucpro+pucBA<br />
|Composite<br />
|1035<br />
|pSB1K3<br />
|Kanamycin<br />
|-<br />
|[http://partsregistry.org/Part:BBa_K227015 BBa_K227015]<br />
|RBS34+OmpR(sph)+Term+OmpC+GFP<br />
|Composite<br />
|1875<br />
|pSB1A2<br />
|Ampicilin<br />
|-<br />
<br />
|}<br />
<div style="text-align: left;"><br />
[https://2009.igem.org/Team:Wash_U/Biological_Parts Back To Top]<font size="4"><br />
<br />
=='''Characterization'''==<br />
<font size="2"> <br><br />
'''pucB/A'''<br><br />
pucB/A are the genes that code for the beta and alpha subunits of the light harvesting complex LH2. Here, we measured the expression level of pucB/A from the puc promoter in the genome vs. on a low copy plasmid, pRKCBC3. This data allows us to utilize pucB/A as a reporter gene for expression from promoters, in complement to its role in cellular growth. <br> <br />
'''Method'''<br><br />
Relative LH2 expression in R. sphaeroides 2.4.1, R. sphaeroides DBCΩ and R. sphaeroides DBCΩ pRKCBC3 grown anaerobically in the dark<br><br />
<br />
Growth conditions:<br />
-Cultures grown in the dark at 34° C, shaking at 160 rpm<br />
- R. sphaeroides 2.4.1<br />
-15 ml culture in polystyrene tube<br />
-15ml M22 was inoculated with 1ml inoculant with OD600nm = 0.270<br />
- OD600nm (Volume) = 0.270<br />
-Culture was allowed to grow 19hrs<br />
- R. sphaeroides DBCΩ<br />
-15 ml culture in polystyrene tube<br />
-15ml M22 was inoculated with 865ul inoculant with OD600nm = 0.312<br />
- OD600nm (Volume) = 0.270<br />
-Culture was allowed to grow 19hrs<br />
- R. sphaeroides DBCΩ pRKCBC3<br />
-10 ml culture in polystyrene tube<br />
-10 ml M22 tet 5ug/ml was inoculated with loop of R. sphaeroides DBCΩ pRKCBC3 and capped with rubber stopper<br />
-Culture was allowed to grow for 3 days so that density was high enough to get a good reading<br />
<br />
Analysis:<br />
Cultures were removed from the incubtor and placed on ice to slow changes in cellular composition. 1 ml was extracted from each culture and a UV-vis absorption spectra of the culture was taken from 300-950 nm. <br />
The optical density of the cultures at 600nm was used to normalize the absorption spectrum by division by this value. Background subtraction of spectrophotometer data was performed in Origin 6.1 Software. A ten-point baseline was created by a "positive peak" algorithm then modified to approximate the scattering curve that falls as the inverse fourth power of wavelength. <br><br />
'''Results'''<br><br />
[[Image:puc.jpg|420px|]]<br><br />
'''Conclusion'''<br><br />
This data indicates that pucB/A can be utilized as a reporter gene as the LH2 absorption bands at 800 and 850 nm are not present in the LH2 deficient mutant DBComega. Furthermore, changes in the expression conditions of pucB/A are reflected on the absorption spectrum. In this case, expression is higher when the genes are expressed from the puc promoter within genomic DNA than on plasmid pRKCBC3, despite the fact that pRKCBC3 is maintained at 4-5 copies in a cell. <br><br />
<br />
<br />
<br />
<br />
<br />
'''Puc Promoter'''<br><br />
[[Image:Slide1.jpg|480px|left]] The puc promoterpromotes transcription of the LH2 pucB/A genes naturally in ''Rhodobacter sphaeroides''. It is important that we are able to compare the transcription rate of the puc promoter in the natural system vs. our mutant system so that we can determine exactly how much efficiency is gained by adding a red light sensor. The absorption spectra of a DBCOmega mutant (LH2 deficient) transformed with pRKCBC3 containing the puc promoter and pucB/A genes will allow us to characterize the puc promoter under high and low oxygen conditions. <br />
More absorption of light at the LH2 spectra peaks normalized to culture OD corresponds with more transcription and vis versa.<br />
<br><br />
'''Method'''<br><br />
Cultures were grown in the dark at 34° C, shaking at 160 rpm. The anaerobic test condition was established by inoculating a 10 ml culture tube with 10 ml M22 tet 5ug/ml with a loop of R. sphaeroides DBCΩ pRKCBC3 and capped with a rubber stopper. The aerobic test condition was established by innoculating a 10 ml culture tube with 5ml M22 tet 5ug/ml with a loop of R. sphaeroides DBCΩ pRKCBC3 and covered with vented cap- the vented cap and relatively low culture volume left significant headroom in the culture for oxygen exchange. Oxygen tension was not able to be quantitatively measured due to the nature of the experiment<br><br><br />
For measurement of pucB/A expression from the puc promoter: <br><br />
Cultures were removed from the incubtor and placed on ice to slow changes in cellular composition. 1 ml was extracted from each culture and a UV-vis absorption spectra of the culture was taken from 300-950 nm. <br />
The optical density of the cultures at 600nm was used to normalize the absorption spectrum by division by this value. Background subtraction of spectrophotometer data was performed in Origin 6.1 Software. A ten-point baseline was created by a "positive peak" algorithm then modified to approximate the scattering curve that falls as the inverse fourth power of wavelength. <br><br />
'''Results'''<br><br />
[[Image:02 puc pro characterization graph a.png|700px|]]<br><br />
'''Conclusion'''<br><br />
This data matches the literature for expression from the puc promoter at different oxygen tensions and as such confirms the assumptions that we have made in modeling our system. <br><br />
See: Braatsch et al. 2002<br />
<br />
=='''Future Characterization'''==<br />
<br>[[Image:Slidex.jpg|480px|left]] The next step in our characterization of our synthetic red light response system is to analyze changes in phosphorylation of ompR in ''Rhodobacter sphaeroides''. In our final system, we only want puc genes to be transcribed and expressed via halting the autophosphorylation of ompR, not simply the puc promoter as it naturally occurs. By placing the ompR coding region downstream of the red light sensor and upstream of a terminator our modified system controls expression of the puc genes by the red light sensor. It should be impossible for the puc promoter to directly cause the transcription of puc genes due to the terminator, but instead, transcription of the puc genes must be activated via decreasing the presence of phosphorylated ompR. The end target of ompR transcription is the ompC promoter, located directly upstream of the puc genes. Placing GFP on the ompC transcript will show how often the promoter is transcribed and how often ompR is phoshorylated.<br />
<br><br><br><br><br><br><br><br><br>[[Image:Slidey.jpg|480px|left]] Part three of our characterization measures the effectiveness of the red light sensor in downregulating the phosphorylation of ompR. This setup is identical to that of part two except we have introduced the red light sensor. Now, the rate of ompR autophosphorylation will be halted by binding to a domain on the light-activated EnvZ kinase analogue. GFP is still attached to the end product, the ompC promoter. By comparing the fluorescence of GFP in this scenario compared with the second scenario the decrease in rate of phosphorylation should be apparent due to the activity of the red light sensor.<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br>[[Image:Slidez.jpg|480px|left]] This is the final construct that will be our actual functioning model in ''Rhodobacter sphaeroides''. This finished product will be compared to the wild type over various intensities of light and cell culture densities can be compared to see which strain, the wild type or the modified strain with the red light sensor was more efficient in harvesting light with varying intensities.<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><font size="2"><br />
[https://2009.igem.org/Team:Wash_U/Biological_Parts Back To Top]<br />
<br />
<br />
<font size="4"><br />
<br />
=='''Modeling'''==<br />
<br />
[[Image:pucBAModelDiagram.jpg|thumb|200px|pucBA Expression Model Diagram]]<br />
[[Image:pucBAModelEq.jpg|thumb|200px|pucBA Model Equations]]<br />
[[Image:pucBAModelRxn.jpg|thumb|200px|pucBA Model Reactions]]<br />
[[Image:pucBAModelTestSim.jpg|thumb|200px|Test Simulation Output]]<br />
'''Modeling the Gene Regulatory Network'''<br />
<font size="2"><br />
:Our group seeks to assess the optimality of the synthetic system that modulates pucB/A gene expression and LH2 complex assembly in ''Rhodobacter sphaeroides''. Here we employ a mathematical model of this system to generate predictions about the behavior of the active system in response to light input. Features of the system that the model may help investigate include the time scale of response to light signals, the robustness of the system in response to fluctuations in light intensity, and the translation between changes in gene expression and the absorbance spectrum of the engineered cells.<br />
<br><br><br><br />
<br />
:Though the context of the model can extend back to the transcription of PrrA/B genes involved in integration oxygen and light signals, a preliminary testing model was developed using assumptions of certain initial conditions to isolate the light signal's effect. Since Cph8 and OmpR are located on the same transcript downstream of the puc promoter region, it was assumed that their associated protein and mRNA had already reached steady state concentrations, and the phosphorylation reaction had already reached steady state. Moreover, the concentrations of the factors were assumed to be equal at this state. The model whose diagram was constructed in the Simbiology Toolbox distributed by MathWorks details key reactions leading to the translation of the pucB/A genes. The reaction rate equation used for the lack of phosphorylation of OmpR when the light signal reaches Cph8 bound to OmpR is captured in a modified form of Michaelis-Menten kinetics. A logic function that corresponds to light ON/OFF (1/0) multiplies the maximum reaction rate in the numerator of the phosphorylation equation. Thus, the model assumes that no phosphorylation occurs by this mechanism in the presence of light. The OmpC promoter binding equation was based on the Hill Equation for an Activator(1). <br />
<br><br><br><br />
<br />
:Component characterization steps and literature searches are underway in order to obtain quantitative parameters for the reaction rates. In order to simulate behavior of the system, putative values were included that exaggerate true concentrations and time scales. OmpR was given an initial concentration normalized to one, and all other components were assumed insignificant initially to this value. An ideal light pulse was introduced at an instant and removed thirty simulation seconds later. From this rudimentary simulation it can be drawn that the nonlinearities of the phosphorylation and transcription factor binding kinetics effectively smooth the sharp light input. By design, the light switch ON yields no phosphorylation of OmpR and repression of the pucB/A genes which would give rise to LH2. Conversely, when left OFF, the concentration of pucB/A recovers and increases until the steady state determined by its translation and degradation rates.<br />
<br><br><br><br />
<br />
'''References'''<br />
<br />
1. Alon, Uri. <u>Introduction to systems biology and the design principles of biological networks</u>. Boca Raton, FL: Chapman & Hall, 2006.<br />
<br><br />
2. Bower, James M. <u>Computational Modeling of Genetic and Biochemical Networks (Computational Molecular Biology).</u> New York: M.I.T. PRESS, 2001.<br />
<br><br />
3. <u>System modeling in cellular biology from concepts to nuts and bolts.</u> Cambridge, MA: MIT P, 2006.<br />
<br />
<br><br />
<br><br><br><br />
=='''Simulating a Bioreactor'''==<br />
Simulation Equations <br><br />
[[image:simulationequations.png|200px|left]]<br><br><br />
<br />
Nonlinear least-squares estimation of LH2 saturation curve <br><br><br><br><br><br><br><br />
[[Image: saturation for WT as inferred.png|450px|left]] <br><br />
<br />
Exponential response curve for mutant LH2 saturation coefficients <br><br />
[[image:lh2adaption.jpg|450px|left|]] <br><br />
<br />
<font size="2"><br />
'''Problem:''' In a typical reactor, cells at the surface absorb more than enough light to saturate their photosynthetic apparatus, transmitting less energy to deeper layers. For wild type cells, the “saturation curve” is approximately the same for all cells, regardless of their incident light intensity.<br><br />
'''Simulating our Mutants advantage in a Bioreactor'''<br />
For our mutant cells, this curve scales as a function of light intensity, due to negative regulation of LH2 complex production<br />
<br />
<div style="text-align: left;"><br />
Saturation curve: Absorbance as a function of incident light intensity. <br>The coefficient changes with intensity in the mutant only.<br><br><br />
Assumptions:<br />
- Light intensity at next layer is given by transmittance from previous layer (assume no backscattering).<br><br />
- Total energy funneled to photosynthetic pathways is estimated as the sum of light absorbed by each layer.<br />
<br />
<br><br />
<br><br />
<font size="2"><br />
[[Image:wtvsmutsim1.jpg|450px|left]][[Image:wtvsmutsim2.jpg|450px|right]]<br />
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type here1<br />
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[[Image:wtvsmutsim3.jpg|450px|left]][[Image:wtvsmutsim4.jpg|450px|right]]<br />
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type here2<br />
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[[Image:wtvsmutsim5.jpg|600px|center]]<br />
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type here3<br />
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'''References'''<br />
<br />
#Steven, Spilatro R. "Photosynthesis Investigation Study." Photosynthesis Investigation Study. Department of Biology, Marietta College, Marietta, Ohio, 1998. Web. 21 Oct. 2009. <http://www.marietta.edu/~spilatrs/biol103/photolab/photosyn.html>.<br />
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<div style="text-align: left;"><br />
[https://2009.igem.org/Team:Wash_U/Biological_Parts Back To Top]<br />
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{{WashUbottom}}</div>Brendan1http://2009.igem.org/Team:Wash_U/Biological_PartsTeam:Wash U/Biological Parts2009-10-22T02:37:01Z<p>Brendan1: /* Simulating a Bioreactor */</p>
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<div>{{WashUback}}<br />
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The Registry of Standard Biological Parts is a library of DNA sequences combined with online characterization resources. The Registry has created a standard protocol for making segments of DNA compatible with all other segments of DNA, regardless of order or size. BioBrick is the term given to such segments of DNA, a term alluding to the fact that any number of bricks may be combined in any order to produce complex, unique systems. This is accomplished by standardizing the restriction enzymes used to surround BioBricks, as well as the plasmids used to transform them. For a graphical representation of of the process, please click [http://ginkgobioworks.com/support/BioBrick_Assembly_Manual.jpg here]. A powerful online database provides information and characterization of all of the BioBricks in the Registry and uses the wiki format (the same one used in wikipedia) which encourages others to edit content directly on the page. Below are list of parts that were used/created for this project. <br />
<div style="text-align: left;"><font size="4" style="color:black"><br />
=='''Parts'''==<br />
<center><br />
<font size="2"><br />
Parts used to characterize and build our final project<br />
{|cellpadding="9" cellspacing="0" style="background-color:#FFEFD5;" border=1<br />
|'''Component'''<br />
|'''Part/Accession #'''<br />
|'''Base Pairs'''<br />
|'''Plasmid'''<br />
|'''Resistance'''<br />
|'''Well'''<br />
|-<br />
|RBS-34<br />
|[http://partsregistry.org/Part:BBa_B0034 BBa_B0034]<br />
|12<br />
|pSB1A2<br />
|Ampicillin<br />
|plate 1, 2M<br />
|-<br />
|Cph8<br />
|[http://partsregistry.org/Part:BBa_I15010 BBa_I15010]<br />
|2,238<br />
|pSB2K3<br />
|Kanamycin<br />
|N/A<br />
|-<br />
|RFP<br />
|[http://partsregistry.org/Part:BBa_J04051 BBa_J04051]<br />
|720<br />
|N/A<br />
|N/A<br />
|N/A<br />
|-<br />
|OmpR (E. coli)<br />
|[http://partsregistry.org/wiki/index.php?title=Part:BBa_K098011 BBa_K098011]<br />
|720<br />
|pSB1T3<br />
|Tetracycline<br />
|N/A<br />
|-<br />
|OmpR (R. sphaeroides)<br />
|[http://partsregistry.org/Part:BBa_K227010 BBa_K227010]<br />
|720<br />
|New<br />
|New<br />
|New<br />
|-<br />
|Terminator<br />
|[http://partsregistry.org/Part:BBa_B0015 BBa_B0015]<br />
|129<br />
|pSB1AK3<br />
|Ampicillin <br>and Kanamycin<br />
|plate 1, 23L<br />
|-<br />
|RBS +OmpR(sph) + Terminator<br />
includes prefix and suffix<br />
|[[media:OmpR_+_terminator.txt|sequence]]<br />
[http://partsregistry.org/Part:BBa_K227011 BBa_K227011]<br />
|875/916<br />
|pSB1k3<br />
|Kanamycin<br />
|synthesized<br />
|-<br />
|OmpC promoter<br />
|[http://partsregistry.org/Part:BBa_R0082 BBa_R0082]<br />
|108<br />
|pSB1A2<br />
|Ampicillin<br />
|plate 1, 16K<br />
|-<br />
|puc promoter<br />
|[http://partsregistry.org/Part:BBa_K227007 BBa_K227007]<br />
|651<br />
|pSB1k3<br />
|Kanamycin<br />
|New<br />
|-<br />
|puc BA<br />
|[http://partsregistry.org/Part:BBa_K227006 BBa_K227006]<br />
|336<br />
|pSB1k3<br />
|Kanamycin<br />
|New<br />
|-<br />
|puc B<br />
|[http://partsregistry.org/Part:BBa_K227005 BBa_K227005]<br />
|156<br />
|pSB1k3<br />
|Kanamycin<br />
|New<br />
|-<br />
|puc A<br />
|[http://partsregistry.org/Part:BBa_K227004 BBa_K227004]<br />
|165<br />
|pSB1k3<br />
|Kanamycin<br />
|New<br />
|-<br />
|OmpC promoter+pucBA<br />
|[[media:OmpC_promoter_+_puc_BA.txt|sequence]]<br />
[http://partsregistry.org/Part:BBa_K227008 BBa_K227008]<br />
|492/539<br />
|pSB1k3<br />
|Kanamycin<br />
|synthesized<br />
|-<br />
|Green Fluorescent Protein<br />
|[http://partsregistry.org/Part:BBa_E0240 BBa_E0240]<br />
|876<br />
|pSB1A2<br />
|Ampicillin<br />
|plate 1, 12M<br />
|}<br />
<br />
<br />
Plasmids used to create and characterize our project<br />
{|cellpadding="9" cellspacing="0" style="background-color:#FFEFD5;" border=1<br />
|'''Plasmid'''<br />
|'''Base Pairs'''<br />
|'''Resistance'''<br />
|'''Copy Number'''<br />
|-<br />
|[http://partsregistry.org/Part:pSB1A2 pSB1A2]<br />
|2,079<br />
|Ampicillin<br />
|high<br />
|-<br />
|[http://partsregistry.org/Part:pSB1K3 pSB1K3]<br />
|2,206<br />
|Kanamycin<br />
|high<br />
|-<br />
|[http://partsregistry.org/Part:pSB1A3 pSB1A3]<br />
|2,157<br />
|Ampicillin<br />
|high<br />
|-<br />
|[http://partsregistry.org/Part:pSB2K3 pSB2K3]<br />
|4,425<br />
|Kanamycin<br />
|variable<br />
|-<br />
|[http://partsregistry.org/Part:pSB1T3 pSB1T3]<br />
|2,463<br />
|Tetracycline<br />
|high<br />
|-<br />
|pRKCBC3<br />
|~11.5kb<br />
|Tetracycline<br />
|4<br />
|}<font size="2"><br />
<br />
<br />
<br />
Parts submitted to the Registry of Standard Biological Parts<br />
{|cellpadding="9" cellspacing="0" style="background-color:#FFEFD5;" border=1<br />
|'''Part/Accession #Component'''<br />
|'''Component'''<br />
|'''Type'''<br />
|'''Base Pairs'''<br />
|'''Plasmid'''<br />
|'''Resistance'''<br />
|-<br />
|[http://partsregistry.org/Part:BBa_I15010 BBa_I15010]<br />
|Cph8 (planned resubmission)<br />
|Coding<br />
|2,238<br />
|plasmid<br />
|Resistance<br />
|-<br />
|[http://partsregistry.org/Part:BBa_K227004 BBa_K227004]<br />
|puc A<br />
|Coding<br />
|165<br />
|pSB1K3<br />
|Kanamycin<br />
|-<br />
|[http://partsregistry.org/Part:BBa_K227005 BBa_K227005]<br />
|puc B<br />
|Coding<br />
|156<br />
|pSB1K3<br />
|Kanamycin<br />
|-<br />
|[http://partsregistry.org/Part:BBa_K227006 BBa_K227006]<br />
|puc BA<br />
|Coding<br />
|336<br />
|pSB1K3<br />
|Kanamycin<br />
|-<br />
|[http://partsregistry.org/Part:BBa_K227007 BBa_K227007]<br />
|puc promoter<br />
|Regulatory<br />
|651<br />
|pSB1K3<br />
|Kanamycin<br />
|-<br />
|[http://partsregistry.org/Part:BBa_K227008 BBa_K227008]<br />
|ompC+PucBA (synthesized)<br />
|Composite<br />
|492<br />
|pSB1AT3<br />
|Ampicilin,Tetracycline<br />
|-<br />
|[http://partsregistry.org/Part:BBa_K227009 BBa_K227009]<br />
|PucPromotor+GFP<br />
|Composite<br />
|1377<br />
|pSB1A2<br />
|Ampicilin<br />
|-<br />
|[http://partsregistry.org/Part:BBa_K227011 BBa_K227011]<br />
|RBS34+OmpR(sph)+Term (synthesized)<br />
|Composite<br />
|875<br />
|pSB1K3<br />
|Kanamycin<br />
|-<br />
|[http://partsregistry.org/Part:BBa_K227012 BBa_K227012]<br />
|RBS34+OmpR(sph)+Term+OmpC+PucB/A<br />
|Composite<br />
|1375<br />
|pSB1K3<br />
|Kanamycin<br />
|-<br />
|[http://partsregistry.org/Part:BBa_K227013 BBa_K227013]<br />
|ompCpro + GFP<br />
|Composite<br />
|992<br />
|pSB1A2<br />
|Ampicilin<br />
|-<br />
|[http://partsregistry.org/Part:BBa_K227014 BBa_K227014]<br />
|pucpro+pucBA<br />
|Composite<br />
|1035<br />
|pSB1K3<br />
|Kanamycin<br />
|-<br />
|[http://partsregistry.org/Part:BBa_K227015 BBa_K227015]<br />
|RBS34+OmpR(sph)+Term+OmpC+GFP<br />
|Composite<br />
|1875<br />
|pSB1A2<br />
|Ampicilin<br />
|-<br />
<br />
|}<br />
<div style="text-align: left;"><br />
[https://2009.igem.org/Team:Wash_U/Biological_Parts Back To Top]<font size="4"><br />
<br />
=='''Characterization'''==<br />
<font size="2"> <br><br />
'''pucB/A'''<br><br />
pucB/A are the genes that code for the beta and alpha subunits of the light harvesting complex LH2. Here, we measured the expression level of pucB/A from the puc promoter in the genome vs. on a low copy plasmid, pRKCBC3. This data allows us to utilize pucB/A as a reporter gene for expression from promoters, in complement to its role in cellular growth. <br> <br />
'''Method'''<br><br />
Relative LH2 expression in R. sphaeroides 2.4.1, R. sphaeroides DBCΩ and R. sphaeroides DBCΩ pRKCBC3 grown anaerobically in the dark<br><br />
<br />
Growth conditions:<br />
-Cultures grown in the dark at 34° C, shaking at 160 rpm<br />
- R. sphaeroides 2.4.1<br />
-15 ml culture in polystyrene tube<br />
-15ml M22 was inoculated with 1ml inoculant with OD600nm = 0.270<br />
- OD600nm (Volume) = 0.270<br />
-Culture was allowed to grow 19hrs<br />
- R. sphaeroides DBCΩ<br />
-15 ml culture in polystyrene tube<br />
-15ml M22 was inoculated with 865ul inoculant with OD600nm = 0.312<br />
- OD600nm (Volume) = 0.270<br />
-Culture was allowed to grow 19hrs<br />
- R. sphaeroides DBCΩ pRKCBC3<br />
-10 ml culture in polystyrene tube<br />
-10 ml M22 tet 5ug/ml was inoculated with loop of R. sphaeroides DBCΩ pRKCBC3 and capped with rubber stopper<br />
-Culture was allowed to grow for 3 days so that density was high enough to get a good reading<br />
<br />
Analysis:<br />
Cultures were removed from the incubtor and placed on ice to slow changes in cellular composition. 1 ml was extracted from each culture and a UV-vis absorption spectra of the culture was taken from 300-950 nm. <br />
The optical density of the cultures at 600nm was used to normalize the absorption spectrum by division by this value. Background subtraction of spectrophotometer data was performed in Origin 6.1 Software. A ten-point baseline was created by a "positive peak" algorithm then modified to approximate the scattering curve that falls as the inverse fourth power of wavelength. <br><br />
'''Results'''<br><br />
[[Image:puc.jpg|420px|]]<br><br />
'''Conclusion'''<br><br />
This data indicates that pucB/A can be utilized as a reporter gene as the LH2 absorption bands at 800 and 850 nm are not present in the LH2 deficient mutant DBComega. Furthermore, changes in the expression conditions of pucB/A are reflected on the absorption spectrum. In this case, expression is higher when the genes are expressed from the puc promoter within genomic DNA than on plasmid pRKCBC3, despite the fact that pRKCBC3 is maintained at 4-5 copies in a cell. <br><br />
<br />
<br />
<br />
<br />
<br />
'''Puc Promoter'''<br><br />
[[Image:Slide1.jpg|480px|left]] The puc promoterpromotes transcription of the LH2 pucB/A genes naturally in ''Rhodobacter sphaeroides''. It is important that we are able to compare the transcription rate of the puc promoter in the natural system vs. our mutant system so that we can determine exactly how much efficiency is gained by adding a red light sensor. The absorption spectra of a DBCOmega mutant (LH2 deficient) transformed with pRKCBC3 containing the puc promoter and pucB/A genes will allow us to characterize the puc promoter under high and low oxygen conditions. <br />
More absorption of light at the LH2 spectra peaks normalized to culture OD corresponds with more transcription and vis versa.<br />
<br><br />
'''Method'''<br><br />
Cultures were grown in the dark at 34° C, shaking at 160 rpm. The anaerobic test condition was established by inoculating a 10 ml culture tube with 10 ml M22 tet 5ug/ml with a loop of R. sphaeroides DBCΩ pRKCBC3 and capped with a rubber stopper. The aerobic test condition was established by innoculating a 10 ml culture tube with 5ml M22 tet 5ug/ml with a loop of R. sphaeroides DBCΩ pRKCBC3 and covered with vented cap- the vented cap and relatively low culture volume left significant headroom in the culture for oxygen exchange. Oxygen tension was not able to be quantitatively measured due to the nature of the experiment<br><br><br />
For measurement of pucB/A expression from the puc promoter: <br><br />
Cultures were removed from the incubtor and placed on ice to slow changes in cellular composition. 1 ml was extracted from each culture and a UV-vis absorption spectra of the culture was taken from 300-950 nm. <br />
The optical density of the cultures at 600nm was used to normalize the absorption spectrum by division by this value. Background subtraction of spectrophotometer data was performed in Origin 6.1 Software. A ten-point baseline was created by a "positive peak" algorithm then modified to approximate the scattering curve that falls as the inverse fourth power of wavelength. <br><br />
'''Results'''<br><br />
[[Image:02 puc pro characterization graph a.png|700px|]]<br><br />
'''Conclusion'''<br><br />
This data matches the literature for expression from the puc promoter at different oxygen tensions and as such confirms the assumptions that we have made in modeling our system. <br><br />
See: Braatsch et al. 2002<br />
<br />
=='''Future Characterization'''==<br />
<br>[[Image:Slidex.jpg|480px|left]] The next step in our characterization of our synthetic red light response system is to analyze changes in phosphorylation of ompR in ''Rhodobacter sphaeroides''. In our final system, we only want puc genes to be transcribed and expressed via halting the autophosphorylation of ompR, not simply the puc promoter as it naturally occurs. By placing the ompR coding region downstream of the red light sensor and upstream of a terminator our modified system controls expression of the puc genes by the red light sensor. It should be impossible for the puc promoter to directly cause the transcription of puc genes due to the terminator, but instead, transcription of the puc genes must be activated via decreasing the presence of phosphorylated ompR. The end target of ompR transcription is the ompC promoter, located directly upstream of the puc genes. Placing GFP on the ompC transcript will show how often the promoter is transcribed and how often ompR is phoshorylated.<br />
<br><br><br><br><br><br><br><br><br>[[Image:Slidey.jpg|480px|left]] Part three of our characterization measures the effectiveness of the red light sensor in downregulating the phosphorylation of ompR. This setup is identical to that of part two except we have introduced the red light sensor. Now, the rate of ompR autophosphorylation will be halted by binding to a domain on the light-activated EnvZ kinase analogue. GFP is still attached to the end product, the ompC promoter. By comparing the fluorescence of GFP in this scenario compared with the second scenario the decrease in rate of phosphorylation should be apparent due to the activity of the red light sensor.<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br>[[Image:Slidez.jpg|480px|left]] This is the final construct that will be our actual functioning model in ''Rhodobacter sphaeroides''. This finished product will be compared to the wild type over various intensities of light and cell culture densities can be compared to see which strain, the wild type or the modified strain with the red light sensor was more efficient in harvesting light with varying intensities.<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><font size="2"><br />
[https://2009.igem.org/Team:Wash_U/Biological_Parts Back To Top]<br />
<br />
<br />
<font size="4"><br />
<br />
=='''Modeling'''==<br />
<br />
[[Image:pucBAModelDiagram.jpg|thumb|200px|pucBA Expression Model Diagram]]<br />
[[Image:pucBAModelEq.jpg|thumb|200px|pucBA Model Equations]]<br />
[[Image:pucBAModelRxn.jpg|thumb|200px|pucBA Model Reactions]]<br />
[[Image:pucBAModelTestSim.jpg|thumb|200px|Test Simulation Output]]<br />
'''Modeling the Gene Regulatory Network'''<br />
<font size="2"><br />
:Our group seeks to assess the optimality of the synthetic system that modulates pucB/A gene expression and LH2 complex assembly in ''Rhodobacter sphaeroides''. Here we employ a mathematical model of this system to generate predictions about the behavior of the active system in response to light input. Features of the system that the model may help investigate include the time scale of response to light signals, the robustness of the system in response to fluctuations in light intensity, and the translation between changes in gene expression and the absorbance spectrum of the engineered cells.<br />
<br><br><br><br />
<br />
:Though the context of the model can extend back to the transcription of PrrA/B genes involved in integration oxygen and light signals, a preliminary testing model was developed using assumptions of certain initial conditions to isolate the light signal's effect. Since Cph8 and OmpR are located on the same transcript downstream of the puc promoter region, it was assumed that their associated protein and mRNA had already reached steady state concentrations, and the phosphorylation reaction had already reached steady state. Moreover, the concentrations of the factors were assumed to be equal at this state. The model whose diagram was constructed in the Simbiology Toolbox distributed by MathWorks details key reactions leading to the translation of the pucB/A genes. The reaction rate equation used for the lack of phosphorylation of OmpR when the light signal reaches Cph8 bound to OmpR is captured in a modified form of Michaelis-Menten kinetics. A logic function that corresponds to light ON/OFF (1/0) multiplies the maximum reaction rate in the numerator of the phosphorylation equation. Thus, the model assumes that no phosphorylation occurs by this mechanism in the presence of light. The OmpC promoter binding equation was based on the Hill Equation for an Activator(1). <br />
<br><br><br><br />
<br />
:Component characterization steps and literature searches are underway in order to obtain quantitative parameters for the reaction rates. In order to simulate behavior of the system, putative values were included that exaggerate true concentrations and time scales. OmpR was given an initial concentration normalized to one, and all other components were assumed insignificant initially to this value. An ideal light pulse was introduced at an instant and removed thirty simulation seconds later. From this rudimentary simulation it can be drawn that the nonlinearities of the phosphorylation and transcription factor binding kinetics effectively smooth the sharp light input. By design, the light switch ON yields no phosphorylation of OmpR and repression of the pucB/A genes which would give rise to LH2. Conversely, when left OFF, the concentration of pucB/A recovers and increases until the steady state determined by its translation and degradation rates.<br />
<br><br><br><br />
<br />
'''References'''<br />
<br />
1. Alon, Uri. <u>Introduction to systems biology and the design principles of biological networks</u>. Boca Raton, FL: Chapman & Hall, 2006.<br />
<br><br />
2. Bower, James M. <u>Computational Modeling of Genetic and Biochemical Networks (Computational Molecular Biology).</u> New York: M.I.T. PRESS, 2001.<br />
<br><br />
3. <u>System modeling in cellular biology from concepts to nuts and bolts.</u> Cambridge, MA: MIT P, 2006.<br />
<br />
<br><br />
<br><br><br><br />
=='''Simulating a Bioreactor'''==<br />
Simulation Equations <br><br />
[[image:simulationequations.png|200px|left]] <br> <br> <br> <br><br />
<br />
Nonlinear least-squares estimation of LH2 saturation curve <br><br><br><br><br><br><br><br />
[[Image: saturation for WT as inferred.png|450px|left]] <br><br />
<br />
Exponential response curve for mutant LH2 saturation coefficients <br><br />
[[image:lh2adaption.jpg|450px|left|]] <br><br />
<br />
<font size="2"><br />
'''Problem:''' In a typical reactor, cells at the surface absorb more than enough light to saturate their photosynthetic apparatus, transmitting less energy to deeper layers. For wild type cells, the “saturation curve” is approximately the same for all cells, regardless of their incident light intensity.<br><br />
'''Simulating our Mutants advantage in a Bioreactor'''<br />
For our mutant cells, this curve scales as a function of light intensity, due to negative regulation of LH2 complex production<br />
<br />
<div style="text-align: left;"><br />
Saturation curve: Absorbance as a function of incident light intensity. <br>The coefficient changes with intensity in the mutant only.<br><br><br />
Assumptions:<br />
- Light intensity at next layer is given by transmittance from previous layer (assume no backscattering).<br><br />
- Total energy funneled to photosynthetic pathways is estimated as the sum of light absorbed by each layer.<br />
<br />
<br><br />
<br><br />
<font size="2"><br />
[[Image:wtvsmutsim1.jpg|450px|left]][[Image:wtvsmutsim2.jpg|450px|right]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
type here1<br />
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<br />
[[Image:wtvsmutsim3.jpg|450px|left]][[Image:wtvsmutsim4.jpg|450px|right]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
type here2<br />
<br />
[[Image:wtvsmutsim5.jpg|600px|center]]<br />
<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
type here3<br />
<br />
<div style="text-align: center;"><br />
text<br />
<br><br><br />
<br />
<div style="text-align: left;"><br />
[https://2009.igem.org/Team:Wash_U/Biological_Parts Back To Top]<br />
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<br />
{{WashUbottom}}</div>Brendan1http://2009.igem.org/Team:Wash_U/ProjectTeam:Wash U/Project2009-10-22T02:30:59Z<p>Brendan1: /* Results */</p>
<hr />
<div>{{WashUback}}<br />
__NOTOC__<br />
<br />
<br />
<div style="text-align: left;"> <font size="4" style="color:black"><br />
== '''Introduction''' == <br />
<font size="2"><br />
Our project goal is to maximize the photosynthetic productivity for a series of photobioreactors containing ''Rhodobacter sphaeroides'' under both high and low light intensities by synthetically regulating the size of the light-harvesting antenna complex LH2. We chose to undertake such a project in ''R. sphaeroides'' due to its well-characterized photosynthetic and genetic system. <br />
<br />
The antenna system functions to expand the spectrum of light available for photosynthesis by absorbing different wavelengths than that of the reaction center. Essentially, it is like the dish around the main receiver of any antenna. Marine bacteria, such as ''R. sphaeroides'', evolved very large antenna complexes to absorb light in a natural environment where there is great competition for photons. As a result, the photosynthetic machinery is saturated at low light intensities in a synthetic non-competitive environment, such as a photobioreactor. This causes up to 95% of incidental photons to be dissipated as heat or fluorescence by bacteria through a process called Non-Photochemical Quenching (NPQ) (Mussgnug et al., 2007). In essence, these photons are being wasted as NPQ reduces light penetration into and across bioreactors and starves the shaded cells for for photons. One method that has been shown to improve photosynthetic productivity in photobioreactors is the reduction of light-harvesting antenna sizes (Polle et al., 2002, Mussgnug et al., 2007). Though, current approaches to this end are difficult to precisely control from the perspective of biological engineering and synthetic biology. <br />
<br />
Our intention is to create a dynamic system to vary antenna size that is dependent on incident light intensity and that can be readily optimized using bioengineering principles. This synthetic regulation of the LH2 complex should result in the bacteria grown under high light intensities expressing fewer LH2 complexes than the cells that are more shaded, leading to a reduction of wasted incident photons while maintaining a high overall absorbance across subsequent bioreactors. Consequently, we expect to see an increase in the total photosynthetic productivity for our mutant ''Rhodobacter sphaeroides'' when compared to the wild type grown under similar conditions. <br />
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<div style="text-align: center;"><br />
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|<font size="2" style="color:black;">'''Wild Type Energy Production in a series of Bioreactors'''<br />
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|<font size="2" style="color:black;">'''Synthetically Regulated Antenna Energy Production in Bioreactors'''<br />
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[[Image:Slide09b.jpg| 450px | left]][[Image:Slide10 a.jpg| 450px | right]]<br />
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[https://2009.igem.org/Team:Wash_U/Project Back To Top]<br />
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== '''Organism''' ==<br />
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''Rhodobacter sphaeroides'' is a purple Alphaproteobacteria. It is flexible metabolically and can grow heterotrophically via aerobic and anaerobic respiration, as well as phototrophically under anaerobic conditions with light. <br />
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''R. sphaeroides'' is one of the best understood photosynthetic organisms. The photosystem is located in intracytoplasmic membrane invaginations and the main components are the Light Haresting Complex 2 (LH2), Light Harvesting Complex 1 (LH1), and the Reaction Center (RC). These pigment-protein complexes non-covalently bind bacteriochlorophylls and carotenoids. LH1 and RC make up the core complex and are often found in the ratio 1:1. LH2 is more peripheral and naturally ranges in the ratio to LH1/RC of 3.0:1 to 6.7:1 under varying light conditions (Scheuring et al., 2005). LH2 absorbs photons maximally at the wavelengths of 850 and 800 nm and funnels its energy to LH1 and the reaction center for photochemistry. (Scheuring et al., 2005). <br />
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The two subunits of LH2 are coded for by the pucB/A genes and are naturally promoted by the puc promoter. <br />
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<br><div style="text-align: center;">'''ICM of ''R. sphaeroides'' '''[[Image:R sphaeroides photosystem.png| 500 px | center]]<center>source:Sener et al.2007<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:black;">'''Plate of ''R. sphaeroides'' 2.4.1'''<br />
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[[Image:R sphaeroides aborbance.png| 450px | left]][[Image:R sphaeroides 0986.png| 450px | right]]<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br>source: Walz et al. 1998<br><br />
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====Online Resources for ''R. sphaeroides''====<br />
[http://www.kazusa.or.jp/codon/cgi-bin/showcodon.cgi?species=272943 Codon Usage Table for ''R. sphaeroides'' 2.41]<br />
<br> [http://www.ncbi.nlm.nih.gov/sites/entrez?db=genome&cmd=Retrieve&dopt=Overview&list_uids=18843 ''R. sphaeroides'' 2.41 NCBI Genome Tools]<br />
<br> [http://archaea.ucsc.edu/cgi-bin/hgPcr?wp_target=&db=0&org=Rhodobacter+sphaeroides+2+4+1&wp_f=&wp_r=&wp_size=2000&wp_perfect=15&wp_good=15&wp_showPage=true&hgsid=159191 ''In silico'' PCR of ''R. sphaeroides'' 2.41 genome]<br />
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[https://2009.igem.org/Team:Wash_U/Project Back To Top]<br />
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=='''Regulation'''==<br />
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[[Image:pucregulation-fullpanels.png|950px]]<br />
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'''The Natural System (Left)''' Under high oxygen tension, PpsR prevents transcription from the puc promoter of the pucBA genes, and thus prevents the expression of LH2(1st panel). Under low oxygen tension, this repression is removed by activated AppA, thus allowing expression of LH2 from pucBA(2nd panel).<br><br />
'''Synthetic Regulation System (Right)''' Our system takes advantage of constitutive protein expression from the puc promoter at low oxygen tension, and replaces pucBA with Cph8 at this site. Placing pucBA expression under the control of the ompC promoter allows for Cph8/OmpR control of LH2 expression under varying light conditions. Under high intensity of incident light, Cph8 will not be autophosphorylated, preventing expression of pucBA (3rd panel). When light intensity is lower, Cph8 will autophosphorylate, thus phosphorylating and activating OmpR and promoting expression of LH2 from the ompC promoter.<br><br />
This regulation system was designed such that light is able to penetrate deeper into and through photobioreactors where light is plentiful.<br />
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<br>[https://2009.igem.org/Team:Wash_U/Project Back To Top]<br />
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== '''Results''' ==<br />
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For methods, materials, and procedures regarding these results, please see [https://2009.igem.org/Team:Wash_U/Protocol#Tissue_Flask_Experiment Tissue Flask Experiment] or [https://2009.igem.org/Team:Wash_U/Biological_Parts#Modeling Modeling]<br />
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'''Figure 1'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:#black;">'''a''' Tissue Flask Position 1 Optical Density of Wild Type and DCBomega<br />
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|<font size="2" style="color:#black;">'''b''' Tissue Flask Position 2 Optical Density of Wild Type and DCBomega<br />
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[[Image:Tissue Flask 1b.png| 450 px| left]][[Image:Tissue Flask 2b.png| 450px | right]]<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:#black;">'''c''' Tissue Flask Position 3 Optical Density of Wild Type and DCBomega<br />
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|<font size="2" style="color:#black;">'''d''' Tissue Flask Position 4 Optical Density of Wild Type and DCBomega<br />
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[[Image:Tissue Flask 3b.png| 450px | left]][[Image:Tissue Flask 4b.png| 450px | right]]<br />
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'''e''' Tissue Flask Position 5 Optical Density of Wild Type and DBComega<br />
[[Image:Tissue Flask 5b.png| 450px | left]]<br />
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Figure 1a,b,c,d,e are the optical density of a tissue flask, as assessed at 600 nm, over the course of the 6 days of the experiment.<br />
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'''Figure 2'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:#black;">'''a''' The Change in the Optical Density of the Tissue Flasks for the WT<br />
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|<font size="2" style="color:#black;">'''b''' The Change in the Optical Density of the Tissue Flasks for the WT<br />
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[[Image:3D 241.png| 450 px | left]][[Image:3D DBCOmega2.png| 450 px | right]]<br />
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Figure 2a and 2b display the growth of the tissue flasks as measured by OD assessed as absorbance at 600 nm. The graph is organized such that those closest to the "days" axis were closest to the light source (i.e. flask 1 is closer than flask 2).<br />
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'''Figure 3'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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[[Image:Bar 241 b.png| 450px | left]][[Image:Bar DBCOmega b.png| 450px | right]]<br />
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Figure 3a and 3b display the cumulative growth of the tissue flasks as measured by the sum of the optical density of the respective cell type's tissue flasks at a given day. Furthermore, the contribution of a given tissue flask to cumulative growth is displayed.<br><br />
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'''Figure 4'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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[[image:sphaeroides tissue flask.jpg|450 px|left|]][[image:dbc tissue flask.jpg|450px|right]]<br />
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Figure 4A and 4B display the appearance of the tissue flasks for both strains on day 6. They are arranged from left to right with left being closed to the light source (flask 1) and right furthest (flask 5)<br />
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'''Figure 5'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:#black;">'''a''' DBComega Spectrum by Flask Distance from Source<br />
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|<font size="2" style="color:#black;">'''b''' R. sphaeroides Spectrum by Flask Distance from Source<br />
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[[Image: WT irradiance 3d.png| 450 px | left]][[Image: DBComega irradiance 3d.png| 450 px | right]]<br />
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Contour of Absolute Irradiance by Flash distance from Source<br />
{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:#black;">'''c''' R. Sphaeroides 2.4.1<br />
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|<font size="2" style="color:#black;">'''d''' DBComega<br />
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[[Image: r sphaeroides day 2 a.jpg| 450px | left]][[Image: dbc day 4b.jpg| 450px | right]]<br />
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'''c''' Wild Type Absolute Irradiance Behind Flask 1: Day 6<br />
[[Image: irradiance WT flask 1 day6.png| 450px | center]]<br />
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'''Figure 6'''<br />
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Relative growth of DBComega to WT in Flask 2 at OD 600<br />
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[[Image:relative growth.png| 600px | center]]<br />
This data illustrates the contribution of LH1 to growth relative to that of LH2 <br><br />
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'''Figure 7'''<br><br />
Nonlinear least-squares estimation of WT LH2 saturation curve<br><br />
[[Image: saturation for WT as inferred.png| 450 px]]<br />
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'''Figure 8'''<br><br />
Simulated Optical Density of Mutant and Wild Type Bioreactors<br><br />
Layers One and Two . Layers Three, Four and Five<br />
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[[Image: OD after flask 1 and 2.jpg| 200px ]][[Image: OD after 3,4, and 5.jpg| 200px ]]<br />
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[https://2009.igem.org/Team:Wash_U/Project Back To Top]<br />
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== '''Analysis''' ==<br />
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The tissue flask experiment was an interesting way to observe the effects of a full knockout of the Light Harvesting Antenna Complex 2 (LH2) vs the Wild Type on an array of conseuctive photobioreactors. A couple of conclusions can be drawn from this data that can be used in modeling our synthetic regulation system for LH2 and in the future design of systems for the optimization of Light Harvesting Antenna size for photobioreactors . <br><br><br />
The most striking difference between the Wild Type Rhodobacter Sphaeroides 2.41 and DBCOmega (LH2 knockout) growth patterns is that the first flask in the WT (closest to the light source) grew less than the second flask, while the converse was true in DBCOmega (figure 2a and 2b). This can be attributed to the photosystem saturation curves for the respective cultures. The Wild Type has an LH2 complex, meaning that their antenna size is inherently larger and that their photosystem will be saturated at lower light intensities than the LH2 deficient mutant. We observed that the incident light intensity used in this experiment led to oversaturation for the first WT tissue flask and resulted in growth-inhibiting photodamage, as is evidenced by its lesser growth relative to the second culture. In contrast, photodamage was not observed in DBCOmega as is evidenced by the first tissue flask that grew at the fastest respective rate. Furthermore, it appears that this photodamage slowed the growth of the first WT tissue flask to the point that the OD of this tissue flask was nearly equal to that of DBCOmega after 5 days of growth(.987 vs. .967) (figure 1a). As such, the contribution of LH2 to the growth of the WT in flask 1 can be assessed to be little after day 3 when the growth curve slows- though, this light (at 800 and 850 nm) was still greatly absorbed by flask 1 ('''see irradiance figure for wt flask 1 day 6'''). It is likely that much of this light was then wasted through Non-Photochemical Quenching (NPQ), depleting the flasks behind flask 1 of photons at the LH2 absorbtion peaks and decreasing the cumulative productivity of flasks 1-5. This effect of wasting photons though NPQ under high light intensities is one that we sought to minimize in the design of our synthetic regulation for the pucB/A genes. <br><br><br />
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A further a comparison of the WT and DBCOmega experiments reveals that the 3rd tissue flask in each had almost identical growth rates (figure 1c), while the DBCOmega 4th and 5th tissue flask outperformed that of the WT (figure 1d and 1e). This can simply be attributed to the higher density of cells in the 2nd flask of the WT that led to an overall lesser quantity of photons passing through to the 4th and 5th flasks in the WT experiment and likely resulting in heterotrophic growth (though this will need to be confirmed with a "dark control" experiment that should be completed in time for the Jamboree). This decrease in available photons for the WT can be seen by viewing the absolute irradiance data (incident light intensity at a given wavelength) after the third culture for the WT (figure 5a and 5c). This decrease in irradiance is the most pronounced for the LH2 absorption bands at 800 and 850 nm, as was expected. Our synthetic regulation system for pucB/A was designed with the intention of allowing greater penetration of light through the first couple of flasks at the LH2 wavelengths, as expression of pucB/A is inversely correlated to incident light intensity.<br><br><br />
'''Thomas, add in some further comments about the sprectroradiometer stuff. Specifically about how it changes in the WT vs. that of DBCOmega using some specific examples on the graphs'''<br><br><br />
Overall, the cumulative culture growth of the WT exceeded that of DBCOmega (figure 3) due to the performance of the second tissue flask of the WT relative to that of DBCOmega (figure 1b) despite the photodamage that occurred in tissue flask 1 for the WT. This cumulative culture growth can be considered photosynthetic productivty and is proportional to the amount of energy/desired product that can be obtained from these cultures. <br><br><br />
'''It is an interesting exercise to model how our mutant system would perform under these same conditions: Thomas add in some comments about referencing that diagram and what we took from this tissue flask experiment to construct that (and upload it when you are done with it).'''<br />
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'''add stuff here about the second flasks and approaching 20% contribution of LH1 to growth'''<br />
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=='''Conclusion and Future Work'''==<br />
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Based on model, should have had feedback mechanism- should be something that sets a minimum expression level for LH2, that is...<br><br />
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-tissue flask w prkcbc3 before jamboree and compare/interpret using characterization data<br><br />
-tissue flask w. full mutant in future- if this doesn't reveal cph8 to responding in an exponentially decaying fashion to light intensity, try w. a system that would<br />
-characterize puc promoter under light conditions and additional oxygen tensions<br />
-complete other characterization work<br />
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[https://2009.igem.org/Team:Wash_U/Project Back To Top]<br />
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== '''References''' ==<br />
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# "2007-2008 Catalog & Technical Reference." <u>New England BioLabs Inc.</u> (2007).<br />
# Alon, Uri. <u>Introduction to systems biology and the design principles of biological networks</u>. Boca Raton, FL: Chapman & Hall, 2006.<br />
# Ausubel, Fred et al. <u>Short Protocols in Molecular Biology: Third Edition.</u> Canada: John Wiley and Sons, Inc., 1999.<br />
# "BioBrick Assembly Help." <u>Registry of Standard Biological Parts.</u> 2009. 6 July 2009. <http://partsregistry.org/Main_Page>.<br />
# Bower, James M. <u>Computational Modeling of Genetic and Biochemical Networks (Computational Molecular Biology).</u> New York: M.I.T. PRESS, 2001.<br />
# Braatsch, Stephan et al. "A single Flavoprotein AppA, Integrates Both Redox and Light Signals in ''Rhodobacter sphaeroides.''" <u>Molecular Microbiology.</u> Vol. 45.3 (2002): 827-836.<br />
# Brown, Stanley B. et al. "Bile Pigment Synthesis in Plants: Incorporation of Haem into Phycocyanobilin and Phycocyanobiliproteins in Cyanidium Caldarium." <u>Biochemistry.</u> Vol. 194 (1981): 137-147.<br />
# Dragnea, Vladimira et al. "Time-Resolved Spectroscopic Studies of the AppA Blue-Light Receptor BLUF Domain from ''Rhodobacter sphaeroides'' <u>Biochemistry.</u> Vol. 44 (2005): 15,978-15,985.<br />
# "Entrez Nucleotide Search." <u>National Center for Biotechnology Information.</u> 2009. 6 June 2009 <http://www.ncbi.nlm.nih.gov/nuccore/49175990?from=3533887&to=3534606&report=gbwithparts>.<br />
# Fowler, Gregory J. S. and C. Neil Hunter. "The Synthesis and Assembly of Functional High and Low Light LH2 Antenna Complexes from ''Rhodopseudomonas palustris'' in ''Rhodobacter sphaeroides.''" <u>The Journal of Biological Chemistry.</u> Vol. 271.23 (1996): 13,356-13,361.<br />
# Gabrielsen, Mads et al. "Peripheral Complexes of Purple Bacteria." <u>The Purple Phototropic Bacteria.</u> (2009): 135-153.<br />
# "GenElute HP Plasmid Miniprep Kit: User Guide." <u>Sigma-Aldrich</u>. (2008).<br />
# Harwood, Caroline S. "Nitrogenase-Catalyzed Hydrogen Production by Purple Nonsulfur Photosynthetic Bacteria." <u>Bioenergy.</u> (2008): 259-271.<br />
# Jager, Andreas et al. "The AppA and PpsR Proteins from ''Rhodobacter sphaeroides'' Can Establish a Redox-Dependent Signal Chain but Fail to Transmit Blue-Light Signals in Other Bacteria." <u>Journal of Bacteriology.</u> Vol. 189.6 (2007): 2,274-2,282.<br />
# Jones, M. R. et al. "Mutants of ''Rhodobacter sphaeroides'' Lacking One or More Pigment-Protein Complexes and Complementation with Reaction-Centre, LH1, and LH2 Genes." <u>Molecular Microbiology.</u> Vol. 6.9 (1992): 1,173-1,184.<br />
# Kondo, Toshihiko et al. "Enhancement of Hydrogen Production by a Photosynthetic Bacterium Mutant with Reduced Pigment." <u>Journal of Bioscience and Bioengineering.</u> Vol. 93.2 (2002): 145-150.<br />
# Lagarias, J. Clark. <u>PCB from Spirulina</u>. Personal Communication. June 2009.<br />
# Laible, Philip D. "Foreign Gene Expression in Photosynthetic Bacteria." <u>The Purple Phototropic Bacteria.</u> (2009): 839-860.<br />
# Lee, Jeong K. and Samuel Kaplan. "Transcriptional Regulation of ''puc'' Operon Expression in ''Rhodobacter sphaeroides.''" <u>Journal of Biological Chemistry.</u> Vol. 270.35 (1995): 20,453-20,458.<br />
# Levskaya, Anselmetal et al. "Engineering ''Escherichia coli'' to See Light." <u>Nature.</u> Vol. 438 (2005): 441-442.<br />
# "Life Sciences Catalog 2009-2010." <u>National Diagnostics.</u> (2009).<br />
# Melis, Anastasios. "Solar Energy Conversion Efficiencies in Photosynthesis: Minimizing the Chlorophyll Antennae to Maximize Efficiency." <u>Plant Science.</u> (2009).<br />
# Mizuno, Takeshi and Shoji Mizushima. "Characterization by Deletion and Localized Mutagenesis in Vitro of the Promoter Region of the ''Escherichia coli'' OmpC Gene and Importance of the Upstream DNA Domain in Positive Regulation by the OmpR Protein." <u>Journal of Bacteriology.</u> Vol. 168.1 (1986): 86-95.<br />
# Moskvin, Oleg V. et al. "Transcriptome Analysis of the ''Rhodobacter sphaeroides'' PspR Regulon: PspR as a Master Regulator of Photosystem Development." <u>Journal of Bacteriology.</u> Vol. 187.6 (2005): 2,148-2,156.<br />
# Mussgnug, Jan H. et al. "Engineering photosynthetic light capture: impacts on improved solar energy to biomass conversion." <u> Plant Biotechnology Journal</u> Vol. 5 Issue 6. (2007) pp. 802-814<br />
# "NEBCutter V2.0." <u>New England BioLabs.</u> 2009. 6 June 2009 <http://tools.neb.com/NEBcutter2/index.php>.<br />
# "OligoAnalyzer 3.1." <u>Integrated DNA Technologies.</u> 2009. 6 June 2009. <http://www.idtdna.com/analyzer/Applications/OligoAnalyzer/><br />
# Sambrook, Joseph and David W. Russell. <u>Molecular Cloning: A Laboratory Manual.</u> Vol. 1-3. New York: Cold Springs Harbor Laboratory Press, 2001.<br />
# Scheuring, Simon and Sturgis, James N. "Chromatic Adaptation of Photosynthetic Membranes" <u>Science</u> Vol. 309 (2005) pp.484-487<br />
# Sener, Melih K. et al. "Atomic-level structural and functional model of a bacterial photosynthetic membrane vesicle" <u>Proceedings of the National Academy of Sciences of the United States of America.</u> Vol. 104, Number 40,pp. 15723-15728<br />
# Shimizu-Sato, Sae et al. "A Light-Switchable Gene Promoter System." <u>Nature Publishing Group.</u> Vol. 20 (2002): 1,041-1044.<br />
# Smith, Harry and M. Geoffrey Holmes. <u>Techniquies in Photomorphogenesis.</u> London: Academic Press, Inc., 1984.<br />
# <u>System modeling in cellular biology from concepts to nuts and bolts.</u> Cambridge, MA: MIT P, 2006.<br />
# Terry, Matthew J. "Biosynthesis and Analysis of Bilins." <u>Heme, Chlorophyll, and Bilins: Methods and Protocols.</u> (2002) 273-291. <br />
# Wang, Wanneng et al. "Heterologous Synthesis and Assembly of Functional LHII Antenna Complexes from ''Rhodovulum sulfidophilum'' in ''Rhodobacter sphaeroides'' Mutant." <u>Molecular Biology Reports.</u> Vol. 12.3 (2008).<br />
# Walz, Thomas et al. "Projection Structures of Three Photosynthetic Complexes from Rhodobacter sphaeroides: LH2 at 6 A, LH1 and RC-LH1 at 25 A" <u>Journal of Molecular Biology</u> Vol. 282 pp. 833-845<br />
# Yakovlev, A. G. et al. "Light Control Over the Size of an Antenna Unit Building Block as an Efficient Strategy for Light Harvesting in Photosynthesis." <u>Federation of European Biochemical Societies.</u> (2002):129-132.<br />
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{{WashUbottom}}</div>Brendan1http://2009.igem.org/Team:Wash_U/ProjectTeam:Wash U/Project2009-10-22T02:29:58Z<p>Brendan1: /* Results */</p>
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__NOTOC__<br />
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== '''Introduction''' == <br />
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Our project goal is to maximize the photosynthetic productivity for a series of photobioreactors containing ''Rhodobacter sphaeroides'' under both high and low light intensities by synthetically regulating the size of the light-harvesting antenna complex LH2. We chose to undertake such a project in ''R. sphaeroides'' due to its well-characterized photosynthetic and genetic system. <br />
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The antenna system functions to expand the spectrum of light available for photosynthesis by absorbing different wavelengths than that of the reaction center. Essentially, it is like the dish around the main receiver of any antenna. Marine bacteria, such as ''R. sphaeroides'', evolved very large antenna complexes to absorb light in a natural environment where there is great competition for photons. As a result, the photosynthetic machinery is saturated at low light intensities in a synthetic non-competitive environment, such as a photobioreactor. This causes up to 95% of incidental photons to be dissipated as heat or fluorescence by bacteria through a process called Non-Photochemical Quenching (NPQ) (Mussgnug et al., 2007). In essence, these photons are being wasted as NPQ reduces light penetration into and across bioreactors and starves the shaded cells for for photons. One method that has been shown to improve photosynthetic productivity in photobioreactors is the reduction of light-harvesting antenna sizes (Polle et al., 2002, Mussgnug et al., 2007). Though, current approaches to this end are difficult to precisely control from the perspective of biological engineering and synthetic biology. <br />
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Our intention is to create a dynamic system to vary antenna size that is dependent on incident light intensity and that can be readily optimized using bioengineering principles. This synthetic regulation of the LH2 complex should result in the bacteria grown under high light intensities expressing fewer LH2 complexes than the cells that are more shaded, leading to a reduction of wasted incident photons while maintaining a high overall absorbance across subsequent bioreactors. Consequently, we expect to see an increase in the total photosynthetic productivity for our mutant ''Rhodobacter sphaeroides'' when compared to the wild type grown under similar conditions. <br />
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|<font size="2" style="color:black;">'''Wild Type Energy Production in a series of Bioreactors'''<br />
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|<font size="2" style="color:black;">'''Synthetically Regulated Antenna Energy Production in Bioreactors'''<br />
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[[Image:Slide09b.jpg| 450px | left]][[Image:Slide10 a.jpg| 450px | right]]<br />
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[https://2009.igem.org/Team:Wash_U/Project Back To Top]<br />
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== '''Organism''' ==<br />
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''Rhodobacter sphaeroides'' is a purple Alphaproteobacteria. It is flexible metabolically and can grow heterotrophically via aerobic and anaerobic respiration, as well as phototrophically under anaerobic conditions with light. <br />
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''R. sphaeroides'' is one of the best understood photosynthetic organisms. The photosystem is located in intracytoplasmic membrane invaginations and the main components are the Light Haresting Complex 2 (LH2), Light Harvesting Complex 1 (LH1), and the Reaction Center (RC). These pigment-protein complexes non-covalently bind bacteriochlorophylls and carotenoids. LH1 and RC make up the core complex and are often found in the ratio 1:1. LH2 is more peripheral and naturally ranges in the ratio to LH1/RC of 3.0:1 to 6.7:1 under varying light conditions (Scheuring et al., 2005). LH2 absorbs photons maximally at the wavelengths of 850 and 800 nm and funnels its energy to LH1 and the reaction center for photochemistry. (Scheuring et al., 2005). <br />
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The two subunits of LH2 are coded for by the pucB/A genes and are naturally promoted by the puc promoter. <br />
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<br><div style="text-align: center;">'''ICM of ''R. sphaeroides'' '''[[Image:R sphaeroides photosystem.png| 500 px | center]]<center>source:Sener et al.2007<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:black;">'''Plate of ''R. sphaeroides'' 2.4.1'''<br />
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[[Image:R sphaeroides aborbance.png| 450px | left]][[Image:R sphaeroides 0986.png| 450px | right]]<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br>source: Walz et al. 1998<br><br />
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====Online Resources for ''R. sphaeroides''====<br />
[http://www.kazusa.or.jp/codon/cgi-bin/showcodon.cgi?species=272943 Codon Usage Table for ''R. sphaeroides'' 2.41]<br />
<br> [http://www.ncbi.nlm.nih.gov/sites/entrez?db=genome&cmd=Retrieve&dopt=Overview&list_uids=18843 ''R. sphaeroides'' 2.41 NCBI Genome Tools]<br />
<br> [http://archaea.ucsc.edu/cgi-bin/hgPcr?wp_target=&db=0&org=Rhodobacter+sphaeroides+2+4+1&wp_f=&wp_r=&wp_size=2000&wp_perfect=15&wp_good=15&wp_showPage=true&hgsid=159191 ''In silico'' PCR of ''R. sphaeroides'' 2.41 genome]<br />
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[https://2009.igem.org/Team:Wash_U/Project Back To Top]<br />
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=='''Regulation'''==<br />
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[[Image:pucregulation-fullpanels.png|950px]]<br />
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'''The Natural System (Left)''' Under high oxygen tension, PpsR prevents transcription from the puc promoter of the pucBA genes, and thus prevents the expression of LH2(1st panel). Under low oxygen tension, this repression is removed by activated AppA, thus allowing expression of LH2 from pucBA(2nd panel).<br><br />
'''Synthetic Regulation System (Right)''' Our system takes advantage of constitutive protein expression from the puc promoter at low oxygen tension, and replaces pucBA with Cph8 at this site. Placing pucBA expression under the control of the ompC promoter allows for Cph8/OmpR control of LH2 expression under varying light conditions. Under high intensity of incident light, Cph8 will not be autophosphorylated, preventing expression of pucBA (3rd panel). When light intensity is lower, Cph8 will autophosphorylate, thus phosphorylating and activating OmpR and promoting expression of LH2 from the ompC promoter.<br><br />
This regulation system was designed such that light is able to penetrate deeper into and through photobioreactors where light is plentiful.<br />
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<br>[https://2009.igem.org/Team:Wash_U/Project Back To Top]<br />
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== '''Results''' ==<br />
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For methods, materials, and procedures regarding these results, please see [https://2009.igem.org/Team:Wash_U/Protocol#Tissue_Flask_Experiment Tissue Flask Experiment] or [https://2009.igem.org/Team:Wash_U/Biological_Parts#Modeling Modeling]<br />
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'''Figure 1'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:#black;">'''a''' Tissue Flask Position 1 Optical Density of Wild Type and DCBomega<br />
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|<font size="2" style="color:#black;">'''b''' Tissue Flask Position 2 Optical Density of Wild Type and DCBomega<br />
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[[Image:Tissue Flask 1b.png| 450 px| left]][[Image:Tissue Flask 2b.png| 450px | right]]<br><br><br><br><br><br><br><br><br><br><br><br><br><br><br><br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:#black;">'''c''' Tissue Flask Position 3 Optical Density of Wild Type and DCBomega<br />
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|<font size="2" style="color:#black;">'''d''' Tissue Flask Position 4 Optical Density of Wild Type and DCBomega<br />
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[[Image:Tissue Flask 3b.png| 450px | left]][[Image:Tissue Flask 4b.png| 450px | right]]<br />
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'''e''' Tissue Flask Position 5 Optical Density of Wild Type and DBComega<br />
[[Image:Tissue Flask 5b.png| 450px | left]]<br />
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Figure 1a,b,c,d,e are the optical density of a tissue flask, as assessed at 600 nm, over the course of the 6 days of the experiment.<br />
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'''Figure 2'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:#black;">'''b''' The Change in the Optical Density of the Tissue Flasks for the WT<br />
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[[Image:3D 241.png| 450 px | left]][[Image:3D DBCOmega2.png| 450 px | right]]<br />
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Figure 2a and 2b display the growth of the tissue flasks as measured by OD assessed as absorbance at 600 nm. The graph is organized such that those closest to the "days" axis were closest to the light source (i.e. flask 1 is closer than flask 2).<br />
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'''Figure 3'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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[[Image:Bar 241 b.png| 450px | left]][[Image:Bar DBCOmega b.png| 450px | right]]<br />
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Figure 3a and 3b display the cumulative growth of the tissue flasks as measured by the sum of the optical density of the respective cell type's tissue flasks at a given day. Furthermore, the contribution of a given tissue flask to cumulative growth is displayed.<br><br />
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'''Figure 4'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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[[image:sphaeroides tissue flask.jpg|450 px|left|]][[image:dbc tissue flask.jpg|450px|right]]<br />
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Figure 4A and 4B display the appearance of the tissue flasks for both strains on day 6. They are arranged from left to right with left being closed to the light source (flask 1) and right furthest (flask 5)<br />
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'''Figure 5'''<br />
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{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:#black;">'''b''' R. sphaeroides Spectrum by Flask Distance from Source<br />
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[[Image: WT irradiance 3d.png| 450 px | left]][[Image: DBComega irradiance 3d.png| 450 px | right]]<br />
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Contour of Absolute Irradiance by Flash distance from Source<br />
{|style="background-color:#ffefd5; width:100%" <font size="2"><br />
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|<font size="2" style="color:#black;">'''c''' R. Sphaeroides 2.4.1<br />
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|<font size="2" style="color:#black;">'''d''' DBComega<br />
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[[Image: r sphaeroides day 2 a.jpg| 450px | left]][[Image: dbc day 4b.jpg| 450px | right]]<br />
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'''c''' Wild Type Absolute Irradiance Behind Flask 1: Day 6<br />
[[Image: irradiance WT flask 1 day6.png| 450px | center]]<br />
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'''Figure 6'''<br />
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Relative growth of DBComega to WT in Flask 2 at OD 600<br />
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[[Image:relative growth.png| 600px | center]]<br />
This data illustrates the contribution of LH1 to growth relative to that of LH2 <br><br />
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'''Figure 7'''<br><br />
Nonlinear least-squares estimation of WT LH2 saturation curve<br><br />
[[Image: saturation for WT as inferred.png| 450 px]]<br />
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'''Figure 8'''<br><br />
Simulated Optical Density of Mutant and Wild Type Bioreactors<br><br />
Layers One and Two . Layers Three, Four and Five<br />
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[[Image: OD after flask 1 and 2.jpg| 200px ]][[Image: OD after 3,4, and 5.jpg| 200px ]]<br />
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[https://2009.igem.org/Team:Wash_U/Project Back To Top]<br />
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== '''Analysis''' ==<br />
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The tissue flask experiment was an interesting way to observe the effects of a full knockout of the Light Harvesting Antenna Complex 2 (LH2) vs the Wild Type on an array of conseuctive photobioreactors. A couple of conclusions can be drawn from this data that can be used in modeling our synthetic regulation system for LH2 and in the future design of systems for the optimization of Light Harvesting Antenna size for photobioreactors . <br><br><br />
The most striking difference between the Wild Type Rhodobacter Sphaeroides 2.41 and DBCOmega (LH2 knockout) growth patterns is that the first flask in the WT (closest to the light source) grew less than the second flask, while the converse was true in DBCOmega (figure 2a and 2b). This can be attributed to the photosystem saturation curves for the respective cultures. The Wild Type has an LH2 complex, meaning that their antenna size is inherently larger and that their photosystem will be saturated at lower light intensities than the LH2 deficient mutant. We observed that the incident light intensity used in this experiment led to oversaturation for the first WT tissue flask and resulted in growth-inhibiting photodamage, as is evidenced by its lesser growth relative to the second culture. In contrast, photodamage was not observed in DBCOmega as is evidenced by the first tissue flask that grew at the fastest respective rate. Furthermore, it appears that this photodamage slowed the growth of the first WT tissue flask to the point that the OD of this tissue flask was nearly equal to that of DBCOmega after 5 days of growth(.987 vs. .967) (figure 1a). As such, the contribution of LH2 to the growth of the WT in flask 1 can be assessed to be little after day 3 when the growth curve slows- though, this light (at 800 and 850 nm) was still greatly absorbed by flask 1 ('''see irradiance figure for wt flask 1 day 6'''). It is likely that much of this light was then wasted through Non-Photochemical Quenching (NPQ), depleting the flasks behind flask 1 of photons at the LH2 absorbtion peaks and decreasing the cumulative productivity of flasks 1-5. This effect of wasting photons though NPQ under high light intensities is one that we sought to minimize in the design of our synthetic regulation for the pucB/A genes. <br><br><br />
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A further a comparison of the WT and DBCOmega experiments reveals that the 3rd tissue flask in each had almost identical growth rates (figure 1c), while the DBCOmega 4th and 5th tissue flask outperformed that of the WT (figure 1d and 1e). This can simply be attributed to the higher density of cells in the 2nd flask of the WT that led to an overall lesser quantity of photons passing through to the 4th and 5th flasks in the WT experiment and likely resulting in heterotrophic growth (though this will need to be confirmed with a "dark control" experiment that should be completed in time for the Jamboree). This decrease in available photons for the WT can be seen by viewing the absolute irradiance data (incident light intensity at a given wavelength) after the third culture for the WT (figure 5a and 5c). This decrease in irradiance is the most pronounced for the LH2 absorption bands at 800 and 850 nm, as was expected. Our synthetic regulation system for pucB/A was designed with the intention of allowing greater penetration of light through the first couple of flasks at the LH2 wavelengths, as expression of pucB/A is inversely correlated to incident light intensity.<br><br><br />
'''Thomas, add in some further comments about the sprectroradiometer stuff. Specifically about how it changes in the WT vs. that of DBCOmega using some specific examples on the graphs'''<br><br><br />
Overall, the cumulative culture growth of the WT exceeded that of DBCOmega (figure 3) due to the performance of the second tissue flask of the WT relative to that of DBCOmega (figure 1b) despite the photodamage that occurred in tissue flask 1 for the WT. This cumulative culture growth can be considered photosynthetic productivty and is proportional to the amount of energy/desired product that can be obtained from these cultures. <br><br><br />
'''It is an interesting exercise to model how our mutant system would perform under these same conditions: Thomas add in some comments about referencing that diagram and what we took from this tissue flask experiment to construct that (and upload it when you are done with it).'''<br />
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'''add stuff here about the second flasks and approaching 20% contribution of LH1 to growth'''<br />
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=='''Conclusion and Future Work'''==<br />
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Based on model, should have had feedback mechanism- should be something that sets a minimum expression level for LH2, that is...<br><br />
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-tissue flask w prkcbc3 before jamboree and compare/interpret using characterization data<br><br />
-tissue flask w. full mutant in future- if this doesn't reveal cph8 to responding in an exponentially decaying fashion to light intensity, try w. a system that would<br />
-characterize puc promoter under light conditions and additional oxygen tensions<br />
-complete other characterization work<br />
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[https://2009.igem.org/Team:Wash_U/Project Back To Top]<br />
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== '''References''' ==<br />
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# "2007-2008 Catalog & Technical Reference." <u>New England BioLabs Inc.</u> (2007).<br />
# Alon, Uri. <u>Introduction to systems biology and the design principles of biological networks</u>. Boca Raton, FL: Chapman & Hall, 2006.<br />
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# Bower, James M. <u>Computational Modeling of Genetic and Biochemical Networks (Computational Molecular Biology).</u> New York: M.I.T. PRESS, 2001.<br />
# Braatsch, Stephan et al. "A single Flavoprotein AppA, Integrates Both Redox and Light Signals in ''Rhodobacter sphaeroides.''" <u>Molecular Microbiology.</u> Vol. 45.3 (2002): 827-836.<br />
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# Moskvin, Oleg V. et al. "Transcriptome Analysis of the ''Rhodobacter sphaeroides'' PspR Regulon: PspR as a Master Regulator of Photosystem Development." <u>Journal of Bacteriology.</u> Vol. 187.6 (2005): 2,148-2,156.<br />
# Mussgnug, Jan H. et al. "Engineering photosynthetic light capture: impacts on improved solar energy to biomass conversion." <u> Plant Biotechnology Journal</u> Vol. 5 Issue 6. (2007) pp. 802-814<br />
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# Scheuring, Simon and Sturgis, James N. "Chromatic Adaptation of Photosynthetic Membranes" <u>Science</u> Vol. 309 (2005) pp.484-487<br />
# Sener, Melih K. et al. "Atomic-level structural and functional model of a bacterial photosynthetic membrane vesicle" <u>Proceedings of the National Academy of Sciences of the United States of America.</u> Vol. 104, Number 40,pp. 15723-15728<br />
# Shimizu-Sato, Sae et al. "A Light-Switchable Gene Promoter System." <u>Nature Publishing Group.</u> Vol. 20 (2002): 1,041-1044.<br />
# Smith, Harry and M. Geoffrey Holmes. <u>Techniquies in Photomorphogenesis.</u> London: Academic Press, Inc., 1984.<br />
# <u>System modeling in cellular biology from concepts to nuts and bolts.</u> Cambridge, MA: MIT P, 2006.<br />
# Terry, Matthew J. "Biosynthesis and Analysis of Bilins." <u>Heme, Chlorophyll, and Bilins: Methods and Protocols.</u> (2002) 273-291. <br />
# Wang, Wanneng et al. "Heterologous Synthesis and Assembly of Functional LHII Antenna Complexes from ''Rhodovulum sulfidophilum'' in ''Rhodobacter sphaeroides'' Mutant." <u>Molecular Biology Reports.</u> Vol. 12.3 (2008).<br />
# Walz, Thomas et al. "Projection Structures of Three Photosynthetic Complexes from Rhodobacter sphaeroides: LH2 at 6 A, LH1 and RC-LH1 at 25 A" <u>Journal of Molecular Biology</u> Vol. 282 pp. 833-845<br />
# Yakovlev, A. G. et al. "Light Control Over the Size of an Antenna Unit Building Block as an Efficient Strategy for Light Harvesting in Photosynthesis." <u>Federation of European Biochemical Societies.</u> (2002):129-132.<br />
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