'Deadman' and 'Passcode' microbial kill switches for bacterial containment

Abstract

Biocontainment systems that couple environmental sensing with circuit-based control of cell viability could be used to prevent escape of genetically modified microbes into the environment. Here we present two engineered safeguard systems known as the 'Deadman' and 'Passcode' kill switches. The Deadman kill switch uses unbalanced reciprocal transcriptional repression to couple a specific input signal with cell survival. The Passcode kill switch uses a similar two-layered transcription design and incorporates hybrid LacI-GalR family transcription factors to provide diverse and complex environmental inputs to control circuit function. These synthetic gene circuits efficiently kill Escherichia coli and can be readily reprogrammed to change their environmental inputs, regulatory architecture and killing mechanism.

Figure 1. Deadman kill switch

(a) Deadman circuit control of toxin gene expression. Cell viability was measured by CFU count following removal of the survival signal (ATc) and is displayed as a ratio of cells without ATc to cells with ATc at each time point. (b) Deadman circuit control of targeted essential protein degradation. Inclusion of the mf-lon specific pdt#1 tag on the specified essential gene causes mf-Lon-mediated degradation of the essential protein upon Deadman circuit activation. (c) Combined control of toxin expression and targeted essential protein degradation increases Deadman-induced cell death. In particular, targeted MurC degradation and EcoRI expression reduced cell viability to below the limit of detection (< 1 × 10⁻⁷) after 6 hours (indicated by a “0”). All data points represent mean ± S.D. of three biological replicates.

Figure 2. The fail-safe mechanism for Deadman circuit activation

To demonstrate active control over host cell viability, cells grown under survival conditions (with ATc) were exposed to 1 mM IPTG to directly induce EcoRI and mf-Lon expression. Cell viability was measured by CFU count and is displayed as a ratio of cell survival with and without IPTG at each time point. Data points represent the mean ± S.D. of three biological replicates.

Figure 3. Passcode kill switch

(a) Passcode circuit schematic and logic gate behavior. Cell survival requires the continued presence of inputs a and b and the absence of input c. Loss of input a or b or the addition of input c cause the passcode circuit to activate toxin expression, leading to cell death. (b) Three versions of the Passcode kill switch were used to control expression of ecoRI, mf-lon-mediated MurC degradation (mf-lon), or both ecoRI and mf-lon. Cells containing each circuit were placed in each of eight possible combinations of the three input molecules, and cell viability was measured by CFU count after 8 hours. In each condition, cell survival is displayed as a ratio of cells in that condition to cells in the “survival” condition highlighted in green. Cell survival below the limit of detection (< 1 × 10⁻⁷) is indicated by a “0”. All data points represent mean ± S.D. of three biological replicates.

Figure 4. Long-term circuit stability

(a) and (b) Cells with Deadman or Passcode circuits containing one toxin (EcoRI) or two toxins (EcoRI and mf-Lon) were passaged under survival conditions for 4 days, and sub-populations of cells were periodically switched to nonpermissive media (Deadman: no ATc, Passcode: no inducer) for eight hours. The survival ratio is the ratio of cells that survive in the death state to those in the survival state. Data points represent the mean ± S.D. of six biological replicates. The passcode circuit was also passaged in E. coli MDS42pdu ΔrecA (MDS strain), which lacks recombinogenic and mobile genomic elements. Deadman and Passcode circuits that do not contain toxin modules displayed increased stability throughout the 4 day experiment (see Supplementary Figs. 20–21). (c) Cells containing Deadman and Passcode circuits that survived exposure to their respective death states were isolated, and the entire circuit and toxin(s) were sequenced to identify the inactivating mutations. Toxin gene disruption by genome-encoded insertion-sequence (IS) elements and large deletions were the predominant cause of circuit inactivation. In the two-toxin Deadman circuit, inactivating TetR mutations allowed continued LacI expression and repression of toxin genes in non-biocontainment conditions (see Fig. 1 for a visual aid).