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 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 10 days of growth in the mammalian gut.

Keywords: CspA; antitoxin; cold shock; containment; kill switch; lambda; library; promoter; synthetic biology; toxin.

Figure 1. Overview of kill switch design concept

A) In permissive conditions the toxin is repressed. The antitoxin is expressed at a constitutive low level to accommodate for any leaky expression of the toxin. B) Upon a change of environment to non-permissive conditions the repression is lifted and toxin expression increases. The low level of antitoxin expression is no longer capable of preventing a lethal level of free toxic

Figure 2. Design and construction of the essentializer element kill switch

A) Expression of cI represses expression of cro and lacZ in the memory element (top cassette), whilst simultaneously repressing expression of ccdB in the essentializer element (bottom cassette). ccdA is expressed at a constitutive low level. B) Exposure to tetracycline leads to a pulse of expression of cro from the trigger element (boxed off). Expression of cro allows for the expression of lacZ, whilst simultaneously repressing cI and ccdB. C) Memory element is absent. Without repression from cI or cro, ccdB is expressed at lethal levels. D) The engineered region encompassing the regulatory DNA for ccdB and ccdA. Highlighted are loci that have a strong impact on expression level for both the toxin (red) and antitoxin (green), as well as operator binding sites for cI and Cro (grey). Key bases that have been varied are emphasized. N = A, C, T or G; Y = C or T, W = A or T; M = A or C; D = A, T or G. E) Overview of the screening process for identifying lethal essentializer element candidates. Displayed are the relevant genotype after each step has been completed and the fraction of candidates that passed a given screen. See also Figure S1.

Figure 3. Analysis of essentializer element candidates

A) Transduction Assay. A donor strain with ampicillin resistance ~3000 bp from the memory element was used to remove the memory element. Due to the spacing between the loci of the cassettes a small subset of transductants would have both cassettes. B) Percentage of colonies that retained the memory element after the transduction assay (Figure 3A), both before (red) and after (blue) passaging for 140 generations. TM = toxin mutant and NL = EE non-lethal. C) Candidate essentializer strains were streaked on a plate spanning sub and super induction levels of ATc. Super induction of Cro near the center represses lacZ expression from the memory element. An intermediate expression level of Cro allows stable switching to the cro state and lacZ expression. Sub induction levels results in remaining in the cI state. D) Six biological repeats of the transduction assay (Figure 3A) for candidates EE10 and EE11. E) Six biological repeats of a competitive growth assay conducted over 70 generations to compare the fitness of parental (MG1655) and engineered bacterial strains. See also Tables S1-3.

Figure 4. Structure and modularity of P_cspA

A) The regulatory region of cold shock protein A (CspA). B) Expression of GFP under P_cspA at 37°C, 30°C, 22°C, and 15° C for 10 days. GFP expression level is shown with and without a linker, under P_rpsL, with no promoter and with no plasmid present. Images are from the same picture. See also Figure S2.

Figure 5. Design of the temperature sensitive kill switch cryodeath

A) At 37 °C translation of CcdB is limited allowing cell survival. B) At colder temperatures, expression of CcdB increases, resulting in cell death C) The engineered region encompassing the regulatory DNA for CcdB and CcdA. Highlighted are segments that have a strong impact on expression level for both the toxin (red) and antitoxin (green). Varied bases are emphasized. Y = C or T; M = A or C; W = A or T; R = A or G. See also Figure S3.

Figure 6. Analysis of cryodeath cadidates

A) Survival assay used to test the extent of population termination at non-permissive temperatures. B) Survival ratio of the ten temperature sensitive candidates in DH10β (CD1-CD10) before and after 140 generations of growth at a permissive temperature. Data represents average of two technical repeats, with error bars showing range. Two biological repeats after a period of growth are shown. Non-Lethal = CD non-lethal. C) Survival ratio of candidates CD01 and CD10 in MG1655 at 30 °C, 22 °C, and 15 °C. Three technical repeats of each condition are shown. D) Six biological repeats of a competitive growth assay of 70 generations to compare the fitness of parental (MG1655) and engineered bacterial strains. E) Six biological replicates of survival ratio of candidates CD01 and CD10 in MG1655 after 140 generations, each biological replicate data point is the average of 3 technical replicates plotted with 3 technical repeats. Initial survival ratios are from the data collected for Figure 6C. See also Figure S4 and Table S5.

Figure 7. Testing cryodeath in vivo

A) Containment of transgenic bacteria to mammalian gut by temperature sensitive induction of cryodeath. B) Survival assay of cultures grown from feces of 3 mice gavaged with toxin mutant and three mice gavaged with CD10 in MG1655, both 1 day and 10 days after gavaging. Each data point is a separate biological repeat, made from the average of three technical repeats.