"Passcode" Kill Switch: Modular Hybrid Transcription Factors for Programmable Biocontainment

With the widespread use of genetically engineered microbes in medical and industrial applications, it is increasingly important to develop security measures that prevent the release of these microorganisms into the natural environment. Biological containment systems use an environmental signal sensing strategy in which survival of target cells requires the presence of specific small molecules in their assigned environments. Alteration of survival requirements may be necessary when the engineered microbes are used for different applications, which is difficult for many pioneering biocontainment systems. Here we develop a safeguard genetic circuit with programmable biocontainment conditions in E. coli. To create modular environmental sensors for building this genetic circuit, we first used a biochemistry approach to identify a conserved boundary between environmental sensing modules (ESMs) and DNA recognition modules (DRMs) from transcription factors (TFs) in the LacI/GalR family, in which these discrete ESMs and DRMs can be mixed and matched to create hybrid TFs that link different environmental inputs to the control of a specific promoter for gene expression. Then, we harnessed these hybrid TFs to build a transcriptional repression-based genetic circuit that blocks toxin gene expression under only one of the eight potential combinations generated by three environmental inputs—the “passcode” for cell survival. Exposure of the engineered cells to any of the other seven combinations induces toxin gene expression and cell death. Importantly, we can reprogram the survival “passcode” by reconnecting input detection to promoter activity regulation with different hybrid TFs. Our development of a genetic circuit-based kill switch demonstrates a promising approach to generate biocontainment systems with complex and customizable environmental requirements for cell survival and proliferation.