We have strict strain storage regulation and rigorous experimental records. In addition to conventional laboratory safety requirements, we design a kill switch for practical application. It will express MazF and make the engineering bacteria die automatically when the EE2 is completely degraded. The complex of EE2 and estrogen receptor (ER) binds to estrogen response element(ERE) in the promoter and initiate the expression of cⅠDN and cⅠ. CⅠDN, a mutant of CI repressor, increased the affinity to non-specific DNA binding site. When the transcription starts, more CⅠDN molecules are present compared to CI molecules. Therefore, most of the monomers will form heterodimers with CⅠDN, and there will be no CI2 to activate PRM promoter. When EE2 concentration changes, the degradation tag added with CⅠDN to ensure quick degradation of the monomeric proteins and CI2 will activate PRM promoter leading to the expression of mazF.
