Construction And Application Of A Mutation Library Of The Single Stranded DNA Binding Protein Of Mycobacterium Tuberculosis

Mycobacterium tuberculosis, a unique pathogen, is seriously harmful to human health. It presents multiple growth patterns such as an active growing state, dormant state, and recovery state. Therefore, the DNA metabolisms in M. tuberculosis would be rigorously regulated. Single stranded DNA binding protein (SSB) is involved in many biochemical processes such as DNA replication, recombination and repairing, but the characteristics of M. tuberculosis’s SSB, for example, its essential amino acids, have not been studied enough clearly.In the current study, a point mutation library of the M. tuberculosis’s SSB has been successfully established based on a frequency-controlled random mutagenesis method, as the M. tuberculosis’s genome contains high GC contents. Among the 830 randomly selected mutants,814 mutants are successfully sequenced, which contained 123 single-residue mutants,107 double-residue mutants,63 tri-mutants,44 tetra-mutants and 13 C-terminus deletion mutants. All these mutants are involved in 141 amino acids of SSB, and the coverage rate for mutation is 93.4%(141/151).22 mutants were selected to further peform Native-PAGE, and the bacterial two-hybrid experiments,8 amino acids sites of SSB were characterized as the potential residues for affecting the polymerization of SSB.We have successfully established a high covreage of amino acids’mutation library for the M. tuberculosis’s SSB, which would provide an opportunity for further understanding the relationship between its function and structure. Meanwhile, the successful establishment and practice of the mutation become an important platform for studying the high-content GC genes in M. tuberculosis.