Difference between revisions of "Team:HUST-China/Results"

As we can see from figure 1, yellow fluorescent protein achieves expression, which indicates our transformation method works.

1.1.2 Verification of transformation

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:

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.

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.

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.

(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).

2.1.1 Gene Expression

To demonstrate that the targeted genes are expressed by engineered Shewanella, we did Real-Time Quantitative PCR.

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.

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.

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.

We can find that Shewanella could produce electricity and the amount of electricity produced by Shewanella is much more than E.coli.

3.2 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.

It is easy to draw the conclusion that the carbon cloth has better effect than carbon rods as electrode.

3.3 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.

Shewanella could produce more electricity at anaerobic circumstance, which means that Synechocystis PCC6803 is not an ideal lactate producer in this electrogenesis system.

3.4 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.

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.

3.5 Functional verification of engineered Synechocystis PCC6803

In this figure we can find that the engineered Synechocystis PCC6803 shows its function in MFC system.