In vivo TF selection system

As the first step in our pipeline we propose a new in vivo method for the automated selection of mutant transcription factors or transcription factor binding sites from large and diverse libraries containing millions of variants. The novelty of our approach lies in the fact that we use negative selection, or “survival of the weakest” as the selection strategy. In our method, a functional variant activates lysis of the host, leading to release of the plasmid DNA coding for the functional variant. The plasmid DNA can then be amplified, transformed, or directly sequenced to determine which variants were functional. We believe that our negative selection scheme is a potentially powerful approach when coupled to next-generation sequencing. Using a proof-of-concept version of the system, consisting of a T7 driven lysis cassette we were able to show that:

1. Cells can be specifically lysed upon induction of the lysis cassette.
2. DNA can be recovered from the lysed cells.
3. We get an enrichment of plasmids originating from lysed cells in a mock selection experiment.

In vivo TF characterization

To be able to determine the in vivo activity and specificity of novel transcription factor variants we generated a suite of reporter plasmid systems. We successfully characterized a number of our reporter systems for functionality. We created a number of reporter plasmids to measure TetR activity, which lead to improved characterization data for the Partsregistry.