Rational Design of Evolutionarily Stable Microbial Kill Switches

Abstract

Summary The evolutionary stability of synthetic genetic circuits is key to both the understanding and application of genetic control elements. One 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 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. Additionally, cryodeath was shown to control the growth environment of a population, with an escape frequency of less than 1 in 105 after ten days of growth in the mammalian gut.

Synthetic cassettes for pH-mediated sensing, counting and containment

“…To achieve these attributes, we constructed and screened a rationally designed toxin/antitoxin library with varying levels of expression dictated by degenerate bases in the promoter RNAP -10 and ribosome binding sites of both the toxin and the antitoxin. This method was previously used to build the temperature sensitive kill switch cryodeath (Stirling et al, 2017), and the successful construction of acidTRP demonstrates the broader application of the method.…”

“…Phd is expressed under a modified constitutive LacUV5 promoter (Malan & McClure, 1984), with Doc expression controlled by Pasr. In a similar technique to the creation of the temperature sensitive kill switch cryodeath (Stirling et al, 2017), degenerate bases were introduced at three locations in the antitoxin RBS and two locations in the antitoxin -10 RNA polymerase binding site, as well as at two locations in the toxin RBS and three locations in the toxin -10 RNA polymerase binding site ( Figure 1B). Each of these ten modifications can result in one of two possible nucleotides, giving a library size of 2 10 = 1024 possible combinations.…”

“…Induction began at pH 6, with over 6000-fold increase in GFP production between pH 6 and pH 4.4 ( Figure S1). AT09 was combined with cryodeath (Stirling et al, 2017) resulting in a multiplicative effect on survival ratio in non-permissive conditions. Cryodeath is a temperature sensitive kill switch that uses the regulatory system from cold shock protein A (cspA) and the toxin antitoxin system CcdB/CcdA.…”

“…If cell death is initiated by the up-regulation of a toxic protein, instability of a kill switch can be attributed to the basal expression of the toxic gene even in permissive conditions. Previously we and others have shown that the inclusion of an antitoxin, and careful balancing of toxin and antitoxin expression levels across relevant conditions, can mitigate this effect, resulting in a kill switch that is evolutionarily stable over biologically relevant time periods (Gallagher et al, 2015;Stirling et al, 2017).…”

“…A containment system must also have a survival ratio (defined as the fraction of the population able to survive in the non-permissive environment) low enough that the probability of an escape event is essentially zero. Previously published kill switches that respond to intrinsic characteristics of their environment have had survival ratios no lower than 10 -6 (Piraner et al, 2016;Stirling et al, 2017), corresponding roughly to the mutation rate of a single gene (Lee et al, 2012). In practical environmental applications, population sizes are much greater than 10 6 cells, so even lower survival ratios are desirable.…”