The primary additions to the directed evolution expression cassette are the Kanamycin resistance marker (KanR) and the sacB gene, which autolysis the cell when the cell is exposed to sucrose. The KanR was placed downstream of the sacB gene purposefully; we can easily test if the KanR gene is getting expressed by growing the cultures on Kanamycin plates, and if they grow, then the sacB must be getting transcribed as well.
The KanR serves as our selection marker. The cells are grown on Kanamycin (Kan) plates in the presence of the proper inducer (EtOH or n-alkanes) so that the promoter is turned on, which then begins transcription of the downstream genes, nameably our RFP, KanR, and sacB. If the system isn’t working properly, the transcription of the KanR will not occur, and these cells will die, due to the Kan in the plates. This selection step should help raise the upper end on our fluorescence/OD vs. analyte concentration correlation curves.
Our counter-selection step utilizes the sacB gene. The cells will be grown on plates with sucrose, and without the necessary inducer to promote transcription of the genes downstream of the promoter. If transcription occurs without the presence of our analyte, the promoter is considered “leaky,” which gives us a false positive reading/baseline. In this counter-selection step, the cells that have leaky promoters will be eliminated, as the sacB will be transcribed, and since the cells are grown on media with sucrose, the successfully expressed sacB will lyse the cell. This counter-selection procedure should help us lower the bottom end of the fluorescence/OD vs. analyte concentration correlation curve.
Additionally, we are using Mutazyme® (Stratagene) to generate mutations in the gene we want to improve (eventually!) via error-prone PCR. Each gene in our directed evolution construct has been given unique flanking cut sites for restriction enzymes. Digesting the sensor and mutant gene with the correct enzyme will give each piece compatiple "sticky ends" that can be ligated together for quick and easy mutant libraries! The aforementioned screening process will be used to determine favorable mutants.
To test our proof of concept, we are doing error-prone PCR on the araC promoter. This promoter should only turn on transcription in the presence of arabinose, however, it has been found to turn on transcription in the presence of a common arabinose analogue, IPTG. Cells will be grown on arabinose + KanR plates to select the mutants that correctly respond to arabinose. They will then be grown on IPTG + sucrose plates to counter-select for the mutants that only respond to arabinose, not IPTG.
Initial testing of the efficacy of the sacB gene shows that it is especially prone to mutation in the cell proliferative cycle, since loss of function allows the mutant to survive the counter selection. For more information see parts K634007 and K322921. We have attempted to better understand the nature of these mutations, in hopes of improving stability. To verify that the change in phenotype was due to a mutant, and not simply a result of a loss of sucrose in the media, we recultured cells, as well as just the plasmids (in new DH10B cells) of the final cell populations into identical starting conditions. We found that growth of this second round did not exhibit the same lag in growth seen in the initial population, and rather immediately entered exponential growth. This data is shown below, and indicates that after "counter-selection" the plasmid alone bears a mutation which removes sucrose sensitivity.
Image Source: http://www.protein.ethz.ch/people/kast/PKast_DEcycles_big.png