Team:HBUT-China/Design
Design
There are two parts used in this project, ncrB and pncrA. These two parts come from the nickel resistance determinant ncrABCY. Within this operon, ncrA and ncrC encode two membrane proteins that form an efflux system, and ncrB encodes NcrB, which belongs to an uncharacterized family (DUF156) of proteins.
There is data which indicates that the expression of the nickel resistance determinant is induced by nickel ions, because ncrB encodes the transcriptional protein which binds with, and inhibits, the pncrA promoter, but nickel ions bind with this protein, thus activating the pncrA promoter.
So we replaced the ncrA gene with the RFP (Red Fluorescent Protein) gene, so that the presence of nickel ion would now promote RFP which can be easily detected. We used a spectrophotometer to detect this fluorescence.
As for our experiments and tests, we consider the concentration of nickle ions, cultivation time and the initial fluorescence to design the whole process. At first, we transferred the plasmids with the part mentioned above into the E. coli. Then we cultivate the E. coli in LB culture mediums with different concentrations of nickel ion. Ultimately, we measure the fluorescence intensity over set time periods and analyze the results. In this way our team was able to identify the best time of cultivation and other factors.
