Part:BBa_K3206000

Glycine biosensor with gcvBp promoter region and RFP reporter

This composite part acts as a glycine biosensor as it becomes active in the presence of glycine, thus acting as a glycine biosensor. The gcvBp promoter has been coupled with a fluorescent reporter, which will trigger transcription of the reporter in the presence of glycine. The biosensor has been found to be more active when glycine levels are elevated, resulted in higher relative fluorescing units.

Glycine biosensor characterisation

Aim: Newcastle's 2019 team aimed to characterise a glycine biosensor designed using a gcvBp promoter which becomes active in the presence of glycine and a BBa_E1010 RFP gene as a reporter. The aim was to characterise the part by determining if higher concentrations of glycine results in higher levels of RFP output and whether this response is specific to this amino acid.

Figure 1. The optical density (OD600), RFP fluorescence and RFP fluorescence/OD600 (+/- S.E) for E. coli DH5-alpha transformed with the glycine biosensor when grown in LB at different concentrations of glycine and cysteine (mM). Measurements were taken after 48-hour incubation at 37 degrees Celsius rotating at 200 rpm. Excitation wavelength= 582 nm. Emission wavelength= 607 nm. Measurements were taken in a Thermo Fisher Varioskan™ LUX multimode microplate reader (data not normalised for background fluorescence).

Results:Figure 1 shows that when E. coli DH5-alpha transformed with this glycine biosensor is exposed to high concentrations of glycine (mM) results in higher levels of fluorescence. Additionally, this response is specific to this amino acid. This is because the level of fluorescence increases when glycine concentration increases, and fluorescence remains constant when cysteine levels are increased. The levels of fluorescence for the bacteria grown with elevated levels of cysteine can be attributed to the glycine already present to the growth media. Furthermore, when looking at the fluorescence levels relative to OD600, it is clear that fluorescence increases exponentially when glycine concentration increases.

Conclusion: We have shown that increased levels of glycine results in higher levels of fluorescence. Furthermore, this response is specific to glycine as similar levels of cysteine was not able to elicit a similar response.

More information can be found here: https://2019.igem.org/Team:Newcastle/Results/Glutathione

Sequence and Features BBa_K3206000 SequenceAndFeatures