Within our first iteration of the project the main objective was to generate a single cell biosensor for phosphate detection. At low phosphate levels the utilized phoA promoter is active which leads to the expression of the lacI repressor. By binding of lacI to the lacI/lacO binding site (operator) the expression of GFP is prohibited. At high phosphate concentrations the phoA promoter gets deactivated which prohibits the expression of lacI. The repressor dissociates from the operator, and since a constitutive promoter is used GFP expression can happen. A visible GFP signal should therefore indicate high phosphate concentration in the media.
At first we theoretical constructed the plasmid in benchling with severals parts of the iGEM catalog. The vector backbone (pSB1C3) was obtained from the iGEM distribution kit 2021, while our construct was ordered as two individual sequences since both parts together would have been to long to synthesize. The first task in the lab was to add restriction sites (EcoRI, BamHI, pstI) to our ordered constructs (part1, part2), via an overlap extension PCR. To prepare the vector backbone and the inserts for ligation, a restriction digest was performed, followed by a purification step via gel electrophoresis. The purified products were used for ligation of our construct and transformed into E.coli TOP10 cells via heat shock transformation. Currently we are waiting for the results of the transformation.