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| a Tissue Flask Position 1 Optical Density of WT and DCBomega
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| b Tissue Flask Position 2 Optical Density of WT and DCBomega
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| c Tissue Flask Position 3 Optical Density of WT and DCBomega
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| d Tissue Flask Position 4 Optical Density of WT and DCBomega
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e Tissue Flask Position 5 Optical Density of WT and DBComega
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.
Figure 2
a The Change in the Optical Density of the Tissue Flasks for the WT b The Change in the Optical Density of Tissue Flasks for DBComega
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).
Figure 3
a Cumulative Growth of the WT Tissue Flasks b Cumulative Growth of the DBComega Tissue Flasks
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.
Figure 4
A The WT tissue flasks B The DBComega tissue flasks
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)
Figure 5
A Title B title
C title D Title
450 px
description of figure 5
Figure 6 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
Figure 7 WT LH2 saturation curve as inferred from data
Figure 8Modeling Results
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Analysis
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 .
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.
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 (after flask 2 WT Irradiance day 3). 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.
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
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.
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).
add stuff here about the second flasks and approaching 20% contribution of LH1 to growth
Conclusion and Future Work
Based on model, should have had feedback mechanism- should be something that sets a minimum expression level for LH2, that is...
-tissue flask w prkcbc3 before jamboree and compare/interpret using characterization data
-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
-characterize puc promoter under light conditions and additional oxygen tensions
-complete other characterization work
- What actually happened?
- Ways to improve, redo parts of our experiment differently
- What further should be done to our end product to further its development, related back to what we initially tried to do?
- What are possible biofuel applications and how could our system be used to improve existing biofuels?
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