Construction of genes

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.

1.2 Synechocystis sp. PCC6803

Verification of gene expression

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.

Figure 3. Results of yellow fluorescent protein of pck306 plasmid in Synechocystis sp. PCC6803. L-Rhamose(1g/L) was added and after 36 hours, engineered bacteria showed it expressed successfully.

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:

Figure 4a. 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 sp. PCC 6803.

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:

Lactate Detection

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.