Team:HUST-China/Results
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Team:HUST-China/Results
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Team:HUST-China/Results
Results
Part1: Photosynthetic microorganism system
1.1 Synechocystis sp
1.1.1 Verification of transformation
pCK306 plasmid contains with yellow fluorescent protein. Therefore, to show pCK306 expresses successfully in Synechocystis sp. PCC 6803(cyanobacteria), we use fluorescence microscope to test whether it was transformed into bacteria or not. After cultivating for one week, we use 10μcyanobacteria cultivate dripping on the slides.
Figure 1. Results of yellow fluorescent protein of pCK306 plasmid in Synechocystis PCC6803. L-Rhamose(1g/L) was added and after 36 hours, engineered bacteria showed it expressed successfully.
As we can see from figure 1, yellow fluorescent protein achieves expression, which indicates our transformation method works.
1.1.2 Verification of gene expression
Affinity Chromatography
In addition to knowing transcription of the gene, we want to exhibit the expression of the protein, we insert 6Xhis-tag into lldP and did affinity chromatography to show we finally made it. The result is shown below:
Figure 2a. There were nearly 0.2g bacteria used to testify the expression of lldP.
After affinity chromatography, the protein was electrophoresed through SDS-PAGE. As the red box shows, lldP can be seen . Comparing to wild type, lldP is expressed in Synechocystis
Because of the slow growth of Synechocystis sp. PCC 6803 and time limitation, we did not have too much bacteria, so we only use nearly 0.2g of them to show whether lldP expressed. The color is pale but still can be seen. lldP expression succeeds.
Real-Time Quantitative PCR
To illustrate our project does work. We must prove that our circuit express successfully. Therefore, we did real-time quantitative PCR to demostrate the transcription of the circuit ldhD-lldP, ldhDc-lldP, ldhDnARSdR-lldP and ldhDARSdR-lldP. The result shows as the figure below:
Figure 2b. The figure shows that ldhD-lldP, ldhDc-lldP, ldhDnARSdR-lldP, ldhDARSdR-lldP express successfully.
1.1.3 Detection of Lactate
Finally, we should testify that the whole circuit in Synechocystis sp. PCC 6803(cyanobacteria) works. So we did lactate detection experiment. After adding L-Rhamose(1g/L) for 72h, we use Lactic Acid assay kit, provided by Nanjing Jiancheng Bioengineering, to quantify lactate concentration. The result is shown below, all the cyanobacteria cultivates are converting to OD750=1.
Figure 3. 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(cyanobacteria), do achieve our goal to provide lactate to Shewanella.
1.2 Rhodopseudomonas palustris (Rps)
1.2.1 Verification of gene expression
We transformed pMG105-PpckA-RBS-mleS-TT, pMG105-PpckA-RBS-lldP-TT, pMG105-PpckA-RBS-ldhA-TT, pMG105-PpckA-RBS-mleS-lldP-TT, pMG105-PpckA-RBS-lldP-RBS-ldhA-TT, pMG105-PpckA-RBS-ldhA-RBS-lldP-TT, pMG105-PpckA-RBS-mleS-RBS-lldP-RBS-ldhA-TT into the E.coli BL21. And then, we do Real-Time Quantitative PCR to verificate targeted gene (Figure 4).
(A)
(B)
(C)
Figure 4. 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.
Figure 4 shows that our genes express successfully on mRNA level.
1.2.2 Detection of Lactate
We detect lactate production of every gene circuits in E.coli BL21 after incubating for 4h (Figure 5).
Figure 5. LB is a kind of medium to incubate bacterial. We detect lactate content of LB and the 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.
The figure 5 shows that our modification is effective. Every gene circuits can help strains produce lactate, and mleS-lldP-ldhA is the most efficient one. Therefore, our construction of gene circuits achieve the goal to help strains produce lactate.
Part2: Electrogenic microorganism system
Shewanella Oneidensis
2.1 Lactate utilization
Since lactate is the carbon source of Shewanella Oneidensis MR-1, overexpression of lactate dehydrogenase might help the bacteria to utilize lactate more efficiently and produce more electricity. We chose dld(encodes D-lactate dehydrogenase) and lldEFG(encodes L-lactate dehydrogenase) to overexpress in Shewanella Oneidensis MR-1.
2.1.1 Gene Expression
To demonstrate that the targeted genes are expressed by engineered Shewanella, we did Real-Time Quantitative PCR.
Figure 6.(A). The expression level of mleS. (B). The epression level of lldP. (C). The expression of ldhA.
As we can see from figure 6, dld could be overexpressed by engineered Shewanella. For further verification, we used the engineered bacteria to produce electricity.
2.1.2 Electrogenesis
By comparing the ability of producing electricity, we might find out whether pYYDT-dld and pYYDT-lldEFG could effectively help Shewanella to produce more electricity.
Figure 7. The comparison of electricity production between engineered Shewanella. (A). Voltage output of WT and recombinant S. oneidensis.
It could be demonstrated that targeted genes could be expressed in the engineered cells. More lactate has been utilized by engineered bacteria, which helps to produce more electricty.
2.2.2 NADH production
NADH is an important part for electrogenesis as it is one of the most important electron carriers in the cell. We hypothesized that more NADH produced by the cell would cause more electricity to be generated. Thus, we chose gapA(encodes glyceraldehyde-3-phosphate dehydrogenase), mdh(encodes NAD-dependent malate dehydrogenase), pflB(encodes pyruvate formate-lyase) and fdh(encodes formate dehydrogenase) to overexpress in Shewanella Oneidensis MR-1.
(1). Gene Expression
To demonstrate that the targeted genes are expressed by engineered Shewanella, we did Real-Time Quantitative PCR.
(A)
(B)
(C)
(D)
(E)
Figure 8. Relative expression level of targeted genes in engineered Shewanella Oneidensis MR-1. We chose gyrB(encodes DNA gyrase B) as the reference genes and 1 as the standard quantity. (A). The expression level of pYYDT-gapA, there was no signal in bacteria which contained pYYDT. (B). The expression level of pYYDT-mdh. (C). The expression level of pYYDT-gapA-mdh. (D). The expression level of pYYDT-pflB. (E). The expression level of pYYDT-pflB-fdh.
As figure 8 shows, gapA, mdh, pflB and fdh could be overexpressed by engineered Shewanella. For further verification, we used the engineered bacteria to produce electricity.
(2). Electrogenesis
By comparing the ability of producing electricity, we might find out whether pYYDT-gapA-mdh and pYYDT-pflB-fdh could effectively help Shewanella to produce more electricity.
Figure 9. The comparison of electricity production between engineered Shewanella. (A). Voltage output of WT and recombinant S. oneidensis.
It could be demonstrated that targeted genes could be expressed in the engineered cells. More NADH has been produced by engineered bacteria, which helps to produce more electricty.
Part3: Whole design
3.1 Pre-experiment of electrogenesis
In the formal experiment. We compared the ability of electricity production between blank medium, Shewanella Oneidensis MR-1 and E.coli to make sure that Shewanella could produce electricity in our device.
Figure 10. The comparison of ability of electricity production between wild type Shewanella Oneidensis MR-1 and E.coli.
We can find that Shewanella could produce electricity and the amount of electricity produced by Shewanella is much more than E.coli.
3.2 Lactate contrast
Then we designed two groups in order to explore the ability of electricity production when we added lactate in Shewanella Oneidensis MR-1.
Figure 11. The influence when adding lactate in wild type Shewanella Oneidensis MR-1.
According to our experiments, we can make a conclusion: compared with medium that doesn’t add lactate, Shewanella oneidensis generates electricity more efficiently in lactate-added medium.
3.3 The difference between carbon rods and carbon cloth
We used carbon rods as electrodes at first. However, we were told by our adviser that the carbon cloth might have better effect for electrogenesis since more bacteria could attach on it.
Figure 12. The comparison of electricity production between using carbon rod and carbon cloth as the electrode.
It is easy to draw the conclusion that the carbon cloth has better effect than carbon rods as electrode.
3.4 The influence of oxygen on electrogenesis
During searching, we found that Shewanella Oneidensis MR-1 had commonly been used in electrogenesis at anaerobic condition. Since we chose Synechocystis PCC6803 as the lactate producer, the oxygen produced by Synechocystis PCC6803 could not be avoided. So we did this group of electrogenesis experiment to find out whether the oxygen would have a negative influence on producing electricity.
Figure 13. The comparison of electricity production between anaerobic circumstance and aerobic circumstance.
Shewanella could produce more electricity at anaerobic circumstance, which means that Synechocystis PCC6803 is not an ideal lactate producer in this electrogenesis system.
3.5 Contrast the symbiotic effect of wild-type strains`
We tried to compare the effects of Synechocystis PCC6803 and Rhodopseudomonas palustris by constructing a MFC system.
Figure 14. The comparison of electricity production between Synechocystis PCC6803 and Rhodopseudomonas palustris
The figure shows that the effect of Rhodopseudomonas palustris is better than Synechocystis PCC6803. We believe that the oxygen produced by Synechocystis PCC6803 mainly reduces the electricity production effect.
3.6 Functional verification of engineered Synechocystis PCC6803
Figure 15. The comparison of electricity production between wild type and engineered Synechocystis PCC6803
In this figure we can find that the engineered Synechocystis PCC6803 shows its function in MFC system.
