Construction of an insertional mutant library of Mycobacterium tuberculosis variant bovis

Abstract:

[Background] Mycobacterium tuberculosis variant bovis is a zoonotic pathogen capable of causing tuberculosis in both humans and animals. [Objective] To construct a mutant library of M. tuberculosis var. bovis with M. tuberculosis var. bovis C68001 as the maternal strain and the MycoMar T7 transposon system, providing a technical platform for further investigation of the gene functions and pathogenic mechanisms of M. tuberculosis var. bovis. [Methods] The whole genome sequence of M. tuberculosis var. bovis C68001 was systematically analyzed. The temperature-sensitive phages were screened and the conditions of phage proliferation and infection were optimized. Finally, a mutant library with high storage capacity for M. tuberculosis var. bovis C68001 was obtained. [Results] Based on Nanopore third-generation and second-generation sequencing technology, the whole genome of C68001 was 4.342 Mb in size and contained 4 093 genes. A temperature-sensitive phage with a titer higher than 1×10¹¹ PFU/mL was successfully screened. After infecting C68001 at MOI=10 for 3 h, 9 216 monoclonal transposon mutants were obtained. Seven clones were randomly selected for PCR, which confirmed that transposons were successfully inserted into C68001. [Conclusion] The mutant library of M. tuberculosis var. bovis C68001 was successfully constructed.

Key words:Mycobacterium tuberculosis variant bovis;MycoMar T7;mutant library;phage