Kill Genes: An active approach

Background

When designing our actual kill genes, we needed to consider again the challenges of our environment. Many of the restriction enzymes found in the registry such as BamHI and BglII are active only at temperatures around 37° C. Although our bioreactor may be at this temperature, the surrounding environment would be much cooler. Since the kill genes should be active in the surrounding environment, we needed to pick enzymes that would be active at lower temperatures. In addition to that we also wanted enzymes that would cut very frequently in the E. coli genome to limit the chance of horizontal gene transfer. Finally, as we chose to use an inducible system, which can easily be mutated, we wanted to introduce some redundancy by using two different kill genes.

We ended up choosing genes for two novel kill enzymes: S7 micrococcal nuclease and CviAII. Both of these enzymes are active at much lower temperatures than restriction enzymes. Through sequence analysis of the E. coli genome, we also determined that they cut extremely frequently in the genome, much more so than BglII or BamHI even combined (figure 1). [File:Bamandbgl uclagary.png