Construction and functional verification of a Zn2+ sensitive mutant of Escherichia coli

Abstract:

[Background] Owing to the key roles of Zn²⁺ in cell detoxification and physiological processes, Zn²⁺ transporters have attracted increasing attention. In Escherichia coli, zntA and zitB are two key genes responsible for Zn²⁺ efflux. [Objective] This study aims to construct a Zn²⁺ sensitive mutant of E. coli and verify its function. [Methods] Using E. coli DH5α as the original strain, we knocked out zntA via a λ Red recombination system by using the homologous recombinant fragment carrying a kanamycin resistance gene. On the basis of the single-gene knockout mutant, zitB was further knocked out via the homologous recombinant fragment carrying a gentamicin resistance gene. In this way, a mutant with the knockout of both zntA and zitB was obtained and designated as KZAB04. The functional complementation experiment was conducted to test the sensitivity of the mutants and the original strain (negative control) under different Zn²⁺ concentrations. [Results] KZAB04 exhibited higher Zn²⁺ sensitivity than the original strain. [Conclusion] A Zn²⁺ sensitive mutant of E. coli was successfully constructed. The construction of this mutant is the prerequisite for the functional study of zntA and zitB and lays a foundation for the functional identification of other Zn²⁺ transporter genes.

Key words:gene knockout;λ Red recombination;Zn²⁺ transporter;zntA;zitB