Team:ECUST/Demonstrate

Key Achivements

1. We successfully constructed the Knock out(BBa_K2308001) and Knock in(BBa_K2308002) plasmids with pDM4 backbone by Gibson assembly.


2. We successfully knocked in the sYFP2(BBa_K2308003) in Rhodobacter sphaeroides 2.4.1’s C terminus of H submit of reaction center. Then we did some experiments with strain RC-sYFP2.


3. We successfully constructed an inducible plasmid pIND4 with IacIq gene from pMCS-eq(BBa_K2308016) which is suitable for Rhodobacter sphaeroides 2.4.1 and characterized it both with optimized sYFP2(BBa_K2308003) and unoptimized sYFP2 (BBa_K864100).


4. We successfully manufacture a new type of photobioreactor and characterized it by some cold mold experiments.




Results


Figure 2. Room temperature absorption spectra of membranes from WT and WT RC-sYFP2 normalised to 590 nm



The RC-sYFP2 fusion was created in a wild type background. Absorption spectra of membranes from this strain recorded at room temperature show no isolated sYFP2 peak due to overlap with absorption of carotenoid at 514 nm.


Figure 3. Fluorescence image of whole cells of RC-sYFP2 cells when excited at 495nm



Fluorescence image shew sYFP2 which is fused with H submit of Rhodobacter sphaeroides 2.4.1 ’s reaction center folded correctly and was able to emit fluorescence.


Figure 4. Each data point is an average from three replicates. Light was provided using Xian Jin white LED bulbs at an intensity of 2000lx.



The energy absorbed by sYFP2 and then transferred to reaction center had only a little impact on the entire photosynthesis efficiency, so the mass of cells didn't increase evidently.

Figure 5. Each data point is an average from three replicates.(a) Growth curves of sYFP2(optimized), sYFP2(original) strains.(b) Fluorescence intensity of sYFP2(optimized), sYFP2(original) strains after induced for 6h.



Discussion: The strain with the expression of sYFP2 have a tendency to slow down on the growth curve. Comparing optimized sYFP2 with the original sYFP2 and their cell mass, we can observe that the expression level of optimized sYFP2 is much higher.

Figure 6. Each data point is an average from three replicates.
（a）H2 production of RC-sYFP2, sYFP2, Rhodobacter sphaeroides 2.4.1 with empty plasmid pIND4, WT after seven days.
（b）Growth curve of RC-sYFP2, sYFP2, Rhodobacter sphaeroides 2.4.1 with empty plasmid pIND4, WT




All gas is collected by drainage method, the gas of RC-sYFP2 drained out first after 45 hours and sYFP2 drained out after 68 hours, WT after 50 hours and Control after 80 hours. The volume of the gas is nearly the same, which means an additional light harvester didn’t bring up the amount of hydrogen production. But it should be noticed that the growth rate of four strains are different, and strains with sYFP2 can grow better and faster in fermentation culture media, and is able to reach the hydrogen production stage faster than others.The amount of hydrogen is far less than what we expected, which might be the result of the unsealed reactor, and the mixed gas of argon and hydrogen.



Figure 8. Individual velocity flow field distribution of light-emitting agitator



The flow field of the whole device is simulated by the principle of fluid mechanics, and the flow field distribution in the unidirectional flow model is good and has good stirring performance. Comparing the addition of the built-in light source into the reactor with the original reactor, we found during that the same amount of time the volume of light cover greatly increased, which helps the improvement of the efficiency of the Rhodobacter sphaeroides 2.4.1.


Figure 9. Light distribution of photobioreactor of which the light surrounds both outside and inside.



Comparing the light distribution effect in fermentor with both light-emitting agitator and light jacket with which only has light jacket, we can find that the light intensity has significantly increased. This characterizes that our new bioreactor meet the light need of photobioreactor.



Problems we face

1. Rhodobacter sphaeroides 2.4.1 is a strain that is difficult to do genetic manipulation. So we have had a lot of trouble when screening engineering bacteria through homologous recombination.
2. For the engineering strain RC-sYFP2 we constructed, because of the overlap of carotenoids absorbed in the range of 450-550 nm, it did not show an increase in the absorption peak or the growth rate compared to the wild type. But after knockout crtB gene, that circumstance will be improved.
3. We initially tried to use photosynthetic fermentation to produce hydrogen, but did not get the expected hydrogen, and we would continue to explore the medium conditions.
4. We used light-emitting agitator of plexiglass materials, which can not bare the high temperature sterilization. Therefore, we have to explore the heat-resistant material in practical applications.
5. On the top of the agitator, the use of the brush has not yet reached the waterproof requirements. So there is a need to improve the waterproof measures.
6. The shape of the blades needs to be improved. Taking into account that theRhodobacter sphaeroides 2.4.1 do not need oxygen to produce hydrogen, our design only using radial mixing paddles. In other situation, the effect of mass transfer needs to be improved.

Future works

Our project focused on improve the efficiency photoelectric conversion through construction of engineering bacteria and the new photobioreactor.



1、First of all, we will improve our homologous recombination skills, and try to use Red/ET recombination technique in Rhodobacter sphaeroides 2.4.1, which has higher successful rate, to make the genome manipulation more easy.
2、We will continue to screen for crtB knockout strains to eliminate the overlap effects of carotenoids on sYFP2 uptake.
3、With the discovery of brighter red fluorescent protein, we are able to expand the absorption spectrum through infusion.
4、Go on deep study of the conditions of the photosynthetic fermentation of hydrogen. Based on the production of hydrogen, we will further research wastewater fermentation in the food factory.
5、We will keep on looking for more suitable materials for the production of agitator, and improve waterproofing measures for the brush further.
6、For the improvement of the oxygen mass transfer coefficient, we may change the upper blades to axial mixing type.
7、We will optimize the design of the lamp strip’s electric lines, and compress the space.