We conducted SDS-PAGE on whole-cell lysates of uninduced and induced cell. Gag-pol polyprotein is expressed as a whole with its stop codon mutated into its readthrough product, glutamine. The polyprotein has three functional parts, capsid protein, protease and reverse transcriptase. We made Y586F mutation on reverse transcriptase to increase its mutation rate. From the PAGE gel, we can see that all three bands could be seen when induced (Fig. 3).

Placing 2 wild-type loxP on both ends of the target sequence (mCherry) in the same direction, and expressing it under a stable promoter (J23101). By co-transforming the target plasmid with another plasmid carrying Cre recombinase, we verified that our Cre protein functions accordingly by excising the mCherry sequence from the promoter (Fig. 4). Through PCR amplification with the primers annealing to sequences outside the target, and subsequent electrophoresis, we found that the band from bacteria co-transforming Cre corresponds to the excision of mCherry.

When we were carrying out integrated human practice, we were warned by Prof. Wang that two homologous loxP would be excised at a much higher efficiency than performing recombination as we wished, so we searched the literature and selected 3 mutants that are said to be incompatible with wt-loxP but are compatible with themselves, they are lox511, lox2272 and lox5171. We tested their incompatibility with wt-loxP by replacing one of wt-loxP into the mutant at the ends of mCherry, and co-transformed the plasmid with Cre (Fig. 5 & 7). The result we obtained showed that lox5171-mCherry-wt_loxP performs best, and used it in further analysis (Fig. 7).

When our modelling demonstrated to us that the expression level of Cre needs to be much lower than that of RT, we introduced degradation tags. By attaching them to the C terminal of Cre recombinase, the protein would be rapidly recognized and degraded by the E. coli’s native SsrA-SmpB degradation system. This construct could also solve the problem of basal leakage and continued existence after inducer removal. Apart from the native AANDENYALAA tag, we also modified its last three or five amino acids into YALAV, YALVA, YALVV and WVLAA. We tested the stable expression level, as well as the degradation dynamic of each tag by attaching them to the C terminal of EGFP protein and measuring the change in fluorescence level (Fig. 7). The stable state expression increases as the number of mutated amino acids increase, or the mutated site nears the N’ of the tag. Supported by our modelling result, we deemed that the WVLAA tag performs best and chose to use it in further experiments.