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

Synechocystis sp. PCC6803

Since synechocystis itself lacks a pathway for producing lactate, and as a photoautotrophic microorganism, synechocystis lacks a lactate transporter to transport lactate out of the cell[1]. Therefore, we combined the lactate dehydrogenase gene with the lactate transporter gene in one circuit to achieve lactate production and transportation. For lactate dehydrogenase gene, we chose ldhD, and ldhDc is a codon-optimized version of ldhD, ldhDnARSdR is ldhD with D176A/I177R/F178S/N180R, and ldhDARSdR is the codon-optimized version of ldhDnARSdR. These codon optimizations are aim at increasing the production of lactate. The lldP ​​protein gene is used to transport the lactate out of the cell. We successfully construct genes ldhD-lldp, ldhDC-lldp, ldhDnARSdR-lldp and ldhDARSdR-lldp.

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 .

1.2. Verification of gene expression

Synechocystis sp. PCC6803

Detection of yellow fluorescent protein

And all the genes were constructed into the plasmid pck306 and transformed into synechocystis. Since pck306 plasmid contain the gene of yellow fluorescent protein, so we put engineered cynobacteria under the fluorescence microscope to verify the transformation in cynobacteria./p>

We could draw the conclusion that these genes were successfully constructed into engineered Synechocystis sp. PCC6803.

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.

Rhodopseudomonas palustris

It shows the verification of gene expression is correct. Gene mleS, lldP and ldhA have a good expression on mRNA level.

Synechocystis sp. PCC6803

Finally, to testify how the whole circuit in Synechocystis sp. PCC 6803(synechocystis) works, 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 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.

Rhodopseudomonas palustris

We detect lactate production of gene circuit in E.coli BL21 (Figure3)