Previously, we and others have shown that the inclusion of an antitoxin and the careful balancing of toxin and antitoxin expression levels across relevant conditions can mitigate this effect, resulting in a kill switch that is evolutionarily stable over biologically relevant periods

demonstrate that adapting a strain to the intended growth condition increases fitness and in turn improves the stability of the engineered function over hundreds of generations

a few simple design principles: high expression of genetic circuits comes with the cost of low evolutionary stability, avoid repeated sequences, and the use of inducible promoters increases stability. Inclusion of an antibiotic resistance gene within the circuit does not ensure evolutionary stability.