Team:Wisconsin-Madison/directedevolution

We are undertaking a directed evolution experiment to help find a better correlation between the amount of RFP produced by our biosensor in response to different concentrations of analyte. We are doing this in several ways with the single plasmid construct, using a series of selection and counter-selection markers. First, we are using a constitutive promoter (J23100) that has a similar activity to the fully induced pBAD promoter for the transcription of either exaDE or alkS. With this, these proteins are constantly being produced, and consequently are always available to interact with the second half of the plasmid construct, which we are looking to improve.

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.

These two steps will be repeated alternatingly, until we achieve a better response for our biosensors. This is the part I’m not well informed on: Additionally, we are using Mutazyme® to generate mutations in our construct, and by using the aforementioned procedure, we will screen for the most effective biosensor mutant.

Learn more about plate reader experiments.

Image Source: http://www.protein.ethz.ch/people/kast/PKast_DEcycles_big.png