Part:BBa_K2533038

RBS-pflB-RBS-fdh

It encodes pyruvate formate-lyase and formate dehydrogenase cytochrome b.

Usage and biology

pflB helps to transform pyruvate into Acetyl-CoA and fdh helps to transform formate into CO₂. With the overexpression of pflB-fdh, Shewanella could produce NADH more efficiently, which brings more electricity to be produced.

Characterization

This is one section contained two genes for NADH production part.

Figure1. RBS-pflB-RBS-fdh

DNA Gel Electrophoretic

To make sure that we get the target gene, we did the DNA gel electrophoretic to separate different gene. And here is the result.

Figure2. Verification of successful transformation of pYYDT-pflB-fdh

Our target length is 9314bp, and as the marker is marker4, we could be sure that the bright band in this picture is our target gene.

Real-Time Quantitative PCR

To demonstrate that pflB-fdh could be overexpressed by engineered Shewanella, we did Real-Time Quantitative PCR.

Figure3. Relative expression level of pflB-fdh in engineered Shewanella Oneidensis MR-1.

There was no signal in bacteria which contained pYYDT so we chose pYYDT-pflB-fdh as standard quantity.

Electrogenesis

By comparing the ability of producing electricity, we might find out whether pflB-fdh could effectively help Shewanella to produce more electricity.

Figure4. The comparison of electricity production between Shewanella contained pYYDT and pYYDT-pflB-fdh.

It could be demonstrated that targeted gene could be expressed in the engineered cells. More NADH has been produced by engineered bacteria, which helps to produce more electricty.