Open AccessDevelopment of a new host vector system in mycobacteria
Author(s) -
Goto Yoshitaka,
Taniguchi Hatsumi,
Udou Takezo,
Mizuguchi Yasuo,
Tokunaga Tohru
Publication year - 1991
Publication title -
fems microbiology letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.899
H-Index - 151
eISSN - 1574-6968
pISSN - 0378-1097
DOI - 10.1111/j.1574-6968.1991.tb04477.x
Subject(s) - plasmid , kanamycin , electroporation , transformation (genetics) , shuttle vector , cloning vector , biology , microbiology and biotechnology , escherichia coli , multiple cloning site , strain (injury) , vector (molecular biology) , recombinant dna , dna , genetics , gene , antibiotics , anatomy
The hybrid plasmid pYT72/pYT92 constructed from an Escherichia coli plasmid pACYC177 and mycobacterial plasmid pMSC262 isolated from Mycobacterium scroflaceum strain W262 transformed both E. coli and BCG. Phage‐sensitive mutants S‐10 and S‐20 isolated from BCG Tokyo strain showed higher frequency of transformation than the wild‐type strain. Frequency of transformation was dependent on age of the culture and the electroporation condition used. Several deletion mutants were generated from pYT72/92 to determine the minimum region for the replication in the mycobacteria. A 2.3‐kb fragment of pMSC262 was found to contain an essential region. Using this fragment and pACYC177, a small shuttle vector pYT937 containing two drug‐resistance markers, kanamycin‐ and ampicillin‐resistance, was constructed. pYT937 contains Aat II, Bam HI, Bbv II, Gsu I, Hinc II, Pst I, Sca I and Xba I cloning sites.