Fig. 4

Comprehensive combination of biosafety mechanisms in E. coli. Proposed combination of orthologous biosafety mechanisms. Auxotrophies (blue), TA-systems (red), replication control mechanisms (purple) and self-destruction systems (yellow) could be combined to achieve a high-quality biosafety system. Furthermore, the proposed combination of systems includes physical containment (grey) and a two-component system (green) to enhance the reliability even further. To create artificial auxotrophies, alr, dadX and cysE were deleted in the genome and must be replaced with plasmid-bound gene copies. CcdB and Holin serve as toxins, but their toxicity will only effect wildtype cells. The toxicity of CcdB can be avoided through a single point mutation within the gyrA gene. To neutralize the toxicity of holin, an antiholin-encoding gene is present in the genome of the desired host. By moving the rep gene from the plasmid to the genome, the plasmid can only replicate if Rep is provided in trans. Incorporation of artificial bases into the plasmid (Xeno-DNA) prevents wildtype cells without the corresponding tRNA/tRNA-synthetase to produce any of the encoded proteins. To destroy the plasmid DNA if taken up by wildtype cells, self-destruction systems like barnase and EcoRI are included. Only the desired host possesses the corresponding inhibitors Barstar and EcoRI methylase and hence can counteract the toxicity