Part:BBa_K2533035

RBS-gapA-RBS-mdh-TT

It encodes glyceraldehyde-3-phosphate dehydrogenase and NAD dependent malate dehydrogenase.

Usage and biology

gapA could transform 3- phosphoglyceraldehyde into 1,3- diphosphoglycerate and mdh could transform malate into pyruvate. With the overexpression of gapA-mdh, Shewanella could produce NADH more efficiently, which brings more electricity being produced.

Characterization

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

Figure1:RBS-gapA-RBS-mdh

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-gapA-mdh

Our target genes are 2666bp, and as the marker is DS5000, we could be sure that the bright bands in this picture are our target genes.

Real-Time Quantitative PCR

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

Figure3:Relative expression level of gapA-mdh in engineered Shewanella Oneidensis MR-1.

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

Electrogenesis

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

Figure4:The comparison of electricity production between Shewanella contained pYYDT and pYYDT-gapA-mdh.

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.