Engineered riboregulation of essential genes in genetically stabilized E coli for biocontainment

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Ryan Gallagher, Farren Isaacs

Yale University, United States

Since the Asilomar Conference of 1975, scientists have acknowledged the need for genetic safeguards that enable biocontainment. Sterile technique can limit the spread of genetically modified microorganisms (GMMs), and genetic strategies that prevent lateral transfer of recombinant episomes have been reported, but the problem of genetically containing engineered cells has not been solved. An inducible gene switch permitting regulation of cell viability would diminish the chance of GMM escape and could permit use of engineered microorganisms outside controlled laboratory environments. By bringing essential genes under the control of engineered RNA-based post-transcriptional regulators, we have built a collection of E coli strains capable of growth only in the presence of synthetic small molecule inducers. When grown with these inducers, our biocontained strains show little or no fitness defect compared to wildtype cells. The frequency of escape from biocontainment among these strains is already 10-8. We hypothesize that co-incorporation of several riboregulated essential genes will increase the Hamming distance between contained and escaped genomes causing the escape frequency to be reduced further still. To prevent mutation from occurring in the first place, we are also constructing a strain background genetically optimized for biocontainment. The E coli chromosome encodes nonessential transcription factors, polymerases, mobile genetic elements, and signaling cascades that increase the rate of mutation during stress. A modifiable mutation rate helps bacteria mount a response to selective pressures while preserving genomic integrity at other times. We are deleting these elements and characterizing the basal mutation rate of mutants to find a genetic background whose capacity for stress-induced mutagenesis is ablated. By combining multiple riboregulated essential genes in a genetically stabilized background we hope to build a biocontained strain that would permit introduction of GMMs into animal hosts as living therapeutics or into polluted environments for bioremediation.