Part:BBa_K2384014

In our project, to increase the robustness of these biocontainment states, the circuit uses a genetic ‘toggle switch’ architecture in which reciprocal repression by the LacI and TetR transcription factors form transcription states that are maintained by the circuit’s linked feedback loops. To create a circuit in which the death state is dominant in the absence of the survival signal, the ribosome binding site (RBS) strengths of LacI and TetR to favor TetR expression was altered in a single-copy plasmid. In the resulting monostable circuit, the presence of the TetR inhibitor anhydrotetracycline (ATc) is required to maintain the circuit in the subordinate LacI+ survival state. Incorporation of toxin genes into the TetR+ state creates a kill switch where the presence of ATc is required to block toxin expression and cell death. The circuit included additional palindromic LacI operator sites in the toxin gene promoter to minimize leaky toxin expression and introduced a transcriptional terminator upstream of the promoter to insulate the gene from spurious transcription. To accelerate the circuit’s switching dynamics, a degradation tag that is specifically recognized by mf-Lon20, a heterologous protease under control of a LacI-dependent promoter was fused to the C terminus of LacI. Upon removal of ATc, TetR repression of LacI allows expression of mf-Lon, which targets LacI for degradation to create a positive feedback loop that accelerates the switch to the TetR+ state.