Synthetic auxotrophs with ligand dependent essential genes for biosafety

Abstract

Synthetic auxotrophs with one or more ligand-dependent essential gene functions and methods of production that can be used for biosafety. The ligand-dependent function of an essential gene product can be produced by a series of mutations in the ORF of an essential gene; N, C, or insertional fusions of ligand-binding domains with essential genes or an engineered ligand-dependent intein splicing to alter essential gene function. A positive and/or negative selection can be used to identify auxotrophs from created mutant libraries. The positive selection is performed by growing a mutant library in conditions where growth or viability depends on the function of mutagenized essential genes. The negative selection eliminates constitutively growing cells that do not require a ligand for growth by growing the library in the absence of complementing ligand and in conditions where growing cells are eliminated. Desirable phenotypes are collected after the selections.