Team:Wash U/Biological Parts

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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.

Parts

Parts used to characterize and build our final project

Component	Part/Accession #	Base Pairs	Plasmid	Resistance	Well
RBS-34	[http://partsregistry.org/Part:BBa_B0034 BBa_B0034]	12	pSB1A2	Ampicillin	plate 1, 2M
Cph8	[http://partsregistry.org/Part:BBa_I15010 BBa_I15010]	2,238	pSB2K3	Kanamycin	N/A
RFP	[http://partsregistry.org/Part:BBa_J04051 BBa_J04051]	720	N/A	N/A	N/A
OmpR (E. coli)	[http://partsregistry.org/wiki/index.php?title=Part:BBa_K098011 BBa_K098011]	720	pSB1T3	Tetracycline	N/A
OmpR (R. sphaeroides)	[http://partsregistry.org/Part:BBa_K227010 BBa_K227010]	720	New	New	New
Terminator	[http://partsregistry.org/Part:BBa_B0015 BBa_B0015]	129	pSB1AK3	Ampicillin and Kanamycin	plate 1, 23L
RBS +OmpR(sph) + Terminator includes prefix and suffix	sequence [http://partsregistry.org/Part:BBa_K227011 BBa_K227011]	875/916	pSB1k3	Kanamycin	synthesized
OmpC promoter	[http://partsregistry.org/Part:BBa_R0082 BBa_R0082]	108	pSB1A2	Ampicillin	plate 1, 16K
puc promoter	[http://partsregistry.org/Part:BBa_K227007 BBa_K227007]	651	pSB1k3	Kanamycin	New
puc BA	[http://partsregistry.org/Part:BBa_K227006 BBa_K227006]	336	pSB1k3	Kanamycin	New
puc B	[http://partsregistry.org/Part:BBa_K227005 BBa_K227005]	156	pSB1k3	Kanamycin	New
puc A	[http://partsregistry.org/Part:BBa_K227004 BBa_K227004]	165	pSB1k3	Kanamycin	New
OmpC promoter+pucBA	sequence [http://partsregistry.org/Part:BBa_K227008 BBa_K227008]	492/539	pSB1k3	Kanamycin	synthesized
Green Fluorescent Protein	[http://partsregistry.org/Part:BBa_E0240 BBa_E0240]	876	pSB1A2	Ampicillin	plate 1, 12M

Plasmids used to create and characterize our project

Plasmid	Base Pairs	Resistance	Copy Number
[http://partsregistry.org/Part:pSB1A2 pSB1A2]	2,079	Ampicillin	high
[http://partsregistry.org/Part:pSB1K3 pSB1K3]	2,206	Kanamycin	high
[http://partsregistry.org/Part:pSB1A3 pSB1A3]	2,157	Ampicillin	high
[http://partsregistry.org/Part:pSB2K3 pSB2K3]	4,425	Kanamycin	variable
[http://partsregistry.org/Part:pSB1T3 pSB1T3]	2,463	Tetracycline	high
pRKCBC3	~11.5kb	Tetracycline	4

Parts submitted to the Registry of Standard Biological Parts

Part/Accession #Component	Component	Type	Base Pairs	Plasmid	Resistance
[http://partsregistry.org/Part:BBa_I15010 BBa_I15010]	Cph8 (planned resubmission)	Coding	2,238	plasmid	Resistance
[http://partsregistry.org/Part:BBa_K227004 BBa_K227004]	puc A	Coding	165	pSB1K3	Kanamycin
[http://partsregistry.org/Part:BBa_K227005 BBa_K227005]	puc B	Coding	156	pSB1K3	Kanamycin
[http://partsregistry.org/Part:BBa_K227006 BBa_K227006]	puc BA	Coding	336	pSB1K3	Kanamycin
[http://partsregistry.org/Part:BBa_K227007 BBa_K227007]	puc promoter	Regulatory	651	pSB1K3	Kanamycin
[http://partsregistry.org/Part:BBa_K227008 BBa_K227008]	ompC+PucBA (synthesized)	Composite	492	pSB1AT3	Ampicilin,Tetracycline
[http://partsregistry.org/Part:BBa_K227009 BBa_K227009]	PucPromotor+GFP	Composite	1377	pSB1A2	Ampicilin
[http://partsregistry.org/Part:BBa_K227011 BBa_K227011]	RBS34+OmpR(sph)+Term (synthesized)	Composite	875	pSB1K3	Kanamycin
[http://partsregistry.org/Part:BBa_K227012 BBa_K227012]	RBS34+OmpR(sph)+Term+OmpC+PucB/A	Composite	1375	pSB1K3	Kanamycin
[http://partsregistry.org/Part:BBa_K227013 BBa_K227013]	ompCpro + GFP	Composite	992	pSB1A2	Ampicilin
[http://partsregistry.org/Part:BBa_K227014 BBa_K227014]	pucpro+pucBA	Composite	1035	pSB1K3	Kanamycin
[http://partsregistry.org/Part:BBa_K227015 BBa_K227015]	RBS34+OmpR(sph)+Term+OmpC+GFP	Composite	1875	pSB1A2	Ampicilin

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Characterization

pucB/A
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.
Method
Relative LH2 expression in R. sphaeroides 2.4.1, R. sphaeroides DBCΩ and R. sphaeroides DBCΩ pRKCBC3 grown anaerobically in the dark

Growth conditions: -Cultures grown in the dark at 34° C, shaking at 160 rpm - R. sphaeroides 2.4.1 -15 ml culture in polystyrene tube -15ml M22 was inoculated with 1ml inoculant with OD600nm = 0.270 - OD600nm (Volume) = 0.270 -Culture was allowed to grow 19hrs - R. sphaeroides DBCΩ -15 ml culture in polystyrene tube -15ml M22 was inoculated with 865ul inoculant with OD600nm = 0.312 - OD600nm (Volume) = 0.270 -Culture was allowed to grow 19hrs - R. sphaeroides DBCΩ pRKCBC3 -10 ml culture in polystyrene tube -10 ml M22 tet 5ug/ml was inoculated with loop of R. sphaeroides DBCΩ pRKCBC3 and capped with rubber stopper -Culture was allowed to grow for 3 days so that density was high enough to get a good reading

Analysis: 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. 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.
Results

Conclusion
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.

Puc Promoter

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.

More absorption of light at the LH2 spectra peaks normalized to culture OD corresponds with more transcription and vis versa.
Method
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

For measurement of pucB/A expression from the puc promoter:
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. 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.
Results

Conclusion
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.
See: Braatsch et al. 2002

Future Characterization

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.

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.

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.

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Modeling

pucBA Expression Model Diagram

pucBA Model Equations

pucBA Model Reactions

Test Simulation Output

Modeling the Gene Regulatory Network

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.

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).

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.

References

1. Alon, Uri. Introduction to systems biology and the design principles of biological networks. Boca Raton, FL: Chapman & Hall, 2006.
2. Bower, James M. Computational Modeling of Genetic and Biochemical Networks (Computational Molecular Biology). New York: M.I.T. PRESS, 2001.
3. System modeling in cellular biology from concepts to nuts and bolts. Cambridge, MA: MIT P, 2006.

Simulating A Bioreactor

Simulation Equations

Nonlinear least-squares estimation of LH2 saturation curve

Exponential response curve for mutant LH2 saturation coefficients

Figure ?

Relative growth of DBComega to WT in Flask 2 at OD 600

This data illustrates the contribution of LH1 to growth relative to that of LH2

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. Cells operating past the saturation point

waste incident photons by non-photochemical quenching and possibly undergo photodamage. For wild type cells, the

“saturation curve” is assumed to be approximately the same for all cells in all layers, regardless of their

incident light intensity. This means that layers of cells on the exterior of the reactor nearest a light source

receive an overabundance of photons and in turn block the interior layers from receiving enough light. In an

optimal reactor, all layers would operate near their respective saturation points to maximize the photosynthetic

channeling of incident light energy. The total saturation curve for wild type R. Sphaeroides was fit with a nonlinear least squares regression of the

form in Equation A. Data points were generated from calculating absorbance as the negative logarithm of the ratio

of the absolute irradiance detected on the next layer to incident absolute irradiance on a layer of cells. A

logistic form was chosen to account for the diminishing returns to absorption of further photons past a threshold

operating capacity of the photosynthetic apparatus. (1)
Simulating our Mutant's advantage in a Bioreactor For our mutant cells, the LH2 saturation curve for each layer scales as a function of light intensity. This

predicted behavior in the mutant is due to negative regulation of LH2 complex production as incident light

intensity increases. The scalar of the magnitude of the saturation curve was altered according to a predicted

exponential curve of LH2 production in repsone to changes in incident light. It was assumed that the system could

vary expression levels such that at high light intensities, the saturation curve is scaled to 25% of that of the

wild type. At low light intensities, LH2 production was assumed to have the potential to be up-regulated to 150%

of wild type expression levels. The advantage this mutant would confer stems from the adaptive nature of the saturation curve heights. Cells

receiving the most light on the outside of the bioreactor saturate at low absorption levels. This allows more

light to transmit to further layers, which have elevated saturation curves due to lower incident light.

Assumptions: - Light intensity at next layer is given by transmittance from previous layer (assumes no backscattering).
- Total energy funneled to photosynthetic pathways is estimated as the sum of light absorbed by each layer. This

generalizes to the optical density measurement of cell culture density. - The constant wild type saturation curve inherently includes both LH2 and LH1 contributions to absorbance. The

mutant's variable saturation curve only accounts for LH2 absorbance, since this is the only complex under the

light-sensing regulation. To account for the component of absorbance provided by LH1, the proportion of total

optical density due strictly to LH1 was investigated by comparing the growth of wild type and LH2-knockout

(dbcOmega) cultures. It is evident that by day three the proportion of Optical Density accounted for by LH1

absorption converges to a value near 0.2. In other words, at the phase the layers of cells have grown in the model,

20% of total optical density can be attributed to LH1. To account for this, the absorption in the mutant was

divided by the factor (1-0.2) = 0.8. Then, the total optical density of the mutant cultures reflects total

absorption by both LH1 and LH2.

Model Assumptions
- Light intensity at next layer is given by transmittance from previous layer (assumes no backscattering).
- Total energy funneled to photosynthetic pathways is estimated as the sum of light absorbed by each layer. This

generalizes to the optical density measurement of cell culture density.
- The constant wild type saturation curve inherently includes both LH2 and LH1 contributions to absorbance. The

mutant's variable saturation curve only accounts for LH2 absorbance, since this is the only complex under the

light-sensing regulation. To account for the component of absorbance provided by LH1, the proportion of total

optical density due strictly to LH1 was investigated by comparing the growth of wild type and LH2-knockout

(dbcOmega) cultures. It is evident that by day three the proportion of Optical Density accounted for by LH1

absorption converges to a value near 0.2. In other words, at the phase the layers of cells have grown in the model,

20% of total optical density can be attributed to LH1. To account for this, the absorption in the mutant was

divided by the factor (1-0.2) = 0.8. Then, the total optical density of the mutant cultures reflects total

absorption by both LH1 and LH2.
- The model was revised upon gathering optical density data from the five layers of the bioreactor setup. (See

Results Figure 2a.) In the wild type, the optical density of the first flask of cells was much lower than

predicted, a phenomenon that was attributed to photodamage of the cells due to exposure to a large quantity of

light past the saturation point of the LH2 complexes. In the optical density data for the flasks, both the

dbcOmega knockout and the wild type logistically grew to an absorbance value of 1. This gave reason to put a hard

limit of 1 on the first flask's potential optical density. Any light left over from this cutoff was transmitted to

the next layer, as evidenced by in the increased growth of the second wild type flask in the Optical Density data. - The response curve for the coefficient of saturation for the mutant due to changes in light intensity was modeled

as an inverse exponential form. In other words, the system reacts to increasing light intensity by exponentially

tapering the coefficient of saturation.

Saturation curve: Absorbance as a function of incident light intensity.
The coefficient changes with intensity in the mutant only.

Assumptions: - Light intensity at next layer is given by transmittance from previous layer (assume no backscattering).
- Total energy funneled to photosynthetic pathways is estimated as the sum of light absorbed by each layer.

type here1

type here2

type here3

References

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>.

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