Rational Design of Evolutionarily Stable Microbial Kill Switches

Abstract

The evolutionary stability of synthetic genetic circuits is key to both the understanding and application of genetic control elements. One particularly useful but challenging situation is a switch between life and death depending on environment. Here are presented \"essentializer\" and \"cryodeath\" circuits, which act as kill switches in Escherichia coli. The essentializer element induces cell death upon the loss of a bi-stable cI/Cro memory switch. Cryodeath makes use of a cold-inducible promoter to express a toxin. We employ rational design and a novel toxin/antitoxin titering approach to produce and screen a small library of potential constructs, in order to select for constructs that are evolutionarily stable. Both kill switches were shown to maintain functionality in vitro for at least 140 generations. In addition, cryodeath was shown to control the growth environment of a bacterial population, with an escape rate of less than 1 in 105 after ten days in vivo.