Synthetic Auxotrophs with Ligand-Dependent Essential Genes for a BL21(DE3) Biosafety Strain

Synthetic auxotrophs are organisms engineered to require the presence of a particular molecule for viability. We show that these organisms can be generated by engineering ligand-dependence into essential genes. We demonstrate a method for generating a Synthetic auxotroph based on a Ligand-Dependent Essential gene (SLiDE), using six essential genes as test cases: pheS, dnaN, tyrS, metG, orn, and adk. We show that a single SLiDE strain can have a 1 x 108-fold increase in viability when chemically complemented with the ligand benzothiazole. The optimized SLiDE engineering protocol required less than one week and $100 USD. We combined multiple SLiDE strain alleles into the industrial E. coli strain BL21(DE3), yielding an organism that exceeds the biosafety criteria with an escape frequency below the limit of detection of 3 x 10-11.