A genetic circuit is generated ensuring that genetically modified E.coli cannot survive outside of the required conditions. This genetic circuit is based on the availability of a non-natural amino acid (nnAA) which continuously has to be added to the medium. A reporter protein called mVenus is expressed as the level of nnAA in the medium decreases, signalizing the low nnAA concentration. If the level decreases further, colicin will no longer be inhibited, which leads to the complete destruction of the bacteria’s DNA and eventually their death. In order to keep the metabolic burden of our system as low as possible, we engineered a minimalized Colicin which consists of the DNase domain only. In addition to that, we employed a method to easily measure the metabolic burden by integrating GFP into the genome and even generated the possibility to integrate our entire safety approach into the genome to reduce the metabolic burden again.

• BBa_K1976000