Part:BBa_K4644022:Design
PZE-glnAP2-mcherry
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
BUCT constructed the pZe-glnAP2-mCherry plasmid to overexpress the promoter under different external nitrogen source concentrations, observing the expression level of the fluorescent protein to determine the responsiveness of the promoter.
In the characterization of the native promoter, we aimed to investigate whether this component could respond to different nitrogen source concentrations. In this induction phase, we used its direct precursor, glutamine, for induction.
After electroporating the overexpression plasmid and performing selection, individual colonies were picked and cultured in a 96-well plate with shaking in LB medium for 12 hours. Then, the cultures were transferred to an enzyme-linked immunosorbent assay (ELISA) plate. In nitrogen-depleted medium, different concentrations of glutamine were added for induction, and continuous induction was carried out for 24 hours.
References
Fan J Y, Cui Z Q, Wei H P, et al. Split mCherry as a new red bimolecular fluorescence complementation system for visualizing protein–protein interactions in living cells[J]. Biochemical and biophysical research communications, 2008, 367(1): 47-53.