Part1: 1.Photosynthetic microorganism system
Figure 5. Lactate production of engineered Synechocystis sp. PCC6803. Comparison of WT, ldhD-lldP, ldhDc-lldP, ldhDnARSdR-lldP, ldhDARSdR-lldP. Synechocystis grew for 7 days, then L-Rhamose(1g/L) was added. After induced 72 hours, lactate concentration has shown above.
The figure indicates that after transforming our circuit in bacteria, the production and releasing of lactate increase evidently, and ldhDARSdR-lldP is the most efficient one. In summary, our engineered bacteria, Synechocystis sp. PCC 6803(synechocystis), do achieve our goal to provide lactate to Shewanella.
1.3 Rhodopseudomonas palustris
We try to use mleS (convert pyruvate to D-lactate), ldhA (convert pyruvate to D-lactate) and lldP (L-lactate permease) to make sure their strains could produce lactate. We construct genes into the shuttle plasmid pMG105 in E.coli BL21, and then detect it .
For example of pMG105-PpckaA-RBS -lldP-RBS-ldhA-TT (Figure 3).
Figure 6. shows gene circuits are successfully constructed into E.coli BL21.
Real-Time Quantitative PCR
we do Real-Time Quantitative PCR to verify the expression of gene circuits by E.coli BL21.
Figure 7.Relative expression level of targeted genes in E.coli BL21. We choose mleS(malate dehydrogenase, the conversion of malic acid to L-lactate), lldP(L-lactate permease, the lactate is transported out of the cell) and ldhA(fermentative D-lactate dehydrogenase, NAD-dependent, convert pyruvate to D-lactate) as the reference genes and pMG105 as the standard quantity. (A). The expression level of mleS. (B). The expression level of lldP. (C). The expression level of ldhA. We could find that mleS, lldP and ldhA have a higher expression compared with pMG105. It means every genes can express efficiently.
It shows the verification of gene expression is correct. Gene mleS, lldP and ldhA have a good expression on mRNA level.
We detect lactate production of gene circuit in E.coli BL21 (Figure8)
Figure 8.LB is a kind of medium to cultivate bacterial. We detect lactate content of LB and others. PMG105 has the lowest lactate content because pMG105 doesn’t have any gene to help produce lactate. It proves that all of our gene circuits have ability to produce lactate. Besides, compared with ldhA and lldP, ldhA-lldP has a better production efficiency; compared with ldhA-lldP, lldP-ldhA has a better production efficiency; compared with ldhA-lldP, mleS-lldP has a better production efficiency; compared with other gene circuits, mleS-lldP-ldhA has a better production efficiency. The result proves our modeling is correct.
Part2: Electrogenic microorganism system
Figure 10. Relative expression level of targeted genes in engineered Shewanella Oneidensis MR-1.We chose gyrB(encodes DNA gyrase B) as the reference gene and 1 as the standard quantity. (A). The expression level of pYYDT-dld. (B). The expression level of pYYDT-gapA-mdh. (C). The expression level of pYYDT-pflB-fdh.
As shown by figure 10, it is demonstrated that these genes could be expressed in engineered Shewanella Oneidensis MR-1.
Improvement about the efficiency of electricity production is our final goal. We compared the Shewanella contained targeted genes with the Shewanella with no-load vector pYYDT. If the former one could produce more electricity, our construction could be proved to receive a success.
Part3: Whole design
There may be more potential interactions in the co-culture of Rhodopseudomonas palustris and Shewanella, which can greatly improve the coulombic efficiency of our MFC system.
Figure 11. The interaction between Rhodopseudomonas palustris and Shewanella.
Figure 12.The comparison of electricity production between wild type Rhodopseudomonas palustris and type Synechocystis PCC6803.
Figure 13.The comparison between Shewanella with no-load vector and Shewanella with targeted genes.
We could learn from this figure that constructed bacteria could produce more electricity, which means that the targeted genes have been expressed successfully in the cell and helped to produce more electricity.