Team:MichiganState/Results/Biosensor
Results
Biosensor Results
We incorporated a plasmid (pfhlA) into E. coli and S. oneidensis that was designed to express GFP in response to formate concentrations. We compared fluorescence with different formate concentrations and also compared to results from a plasmid without the formate-sensitive promoter (pRL814).
Contrary to hypothesized results, levels of green fluorescent protein (GFP) expression in both E. coli WM3064 and S. oneidensis MR-1 containing the engineered pRL814-fdhF-fhlA plasmid were not substantially higher than those of control strains containing the unmodified pRL814 plasmid. Levels of GFP expression in the control strains were observed to increase in response to formate at a similar rate as the modified strain. Data from experiments performed in LB medium are shown in Figure 1.
The unmodified pRL814 plasmid originally contained the T7A promoter, which was fused to the coding sequence for GFP, which is inducible by both lactose and IPTG. Lactose is a common metabolic pathway intermediate in bacteria. Therefore, it is possible that, during rapid growth with ample nutrients, the bacteria could have produced enough lactose to weakly activate the T7A promoter, initiating higher levels of GFP expression. This is a possible explanation for why the ‘background’ of GFP expression in the control strain is notably high.
Modeling of the FhlA primary amino acid sequence, as well as analysis of this model, was performed to determine how to proceed in future studies; refer to the Modeling tab. Upon this analysis, further site-directed mutagenesis experiments were suggested to tune the responsiveness of the protein to formate induction.