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 bands in this picture are our target genes.

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.