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Mycoplasmas  have exceptionally streamlined genomes and are strongly adapted to their many hosts, which provide them with essential nutrients. Owing to their relative genomic simplicity,  Mycoplasmas  have been used to develop chassis for biotechnological applications. However, the dearth of robust and precise toolkits for genomic manipulation and tight regulation has hindered any substantial advance. Herein we describe the construction of a robust genetic toolkit for  M. pneumoniae , and its successful deployment to engineer synthetic gene switches that control and limit  Mycoplasma  growth, for biosafety containment applications. We found these synthetic gene circuits to be stable and robust in the long-term, in the context of a minimal cell. With this work, we lay a foundation to develop viable and robust biosafety systems to exploit a synthetic  Mycoplasma  chassis for live attenuated vectors for therapeutic applications.

A genetic toolkit and gene switches to limit Mycoplasma growth for biosafety applications