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• W1983802182 endingPage "326" @default.
• W1983802182 startingPage "320" @default.
• W1983802182 abstract "Molecular tools for Gram-positive bacteria such as Mycobacterium are less well-developed than those for Gram-negatives such as Escherichiacoli. This has slowed the molecular-genetic characterisation of Mycobacterium spp, which is unfortunate, since this genus has high medical, environmental and industrial significance. Here, we developed a new Mycobacterium shuttle vector (pMycoFos, 12.5kb, Km(R)) which combines desirable features of several previous vectors (controllable copy number in E. coli, inducible gene expression in Mycobacterium) and provides a new multiple cloning site compatible with large inserts of high-GC content DNA. Copy number control in E. coli was confirmed by the increased Km(R) of cultures after arabinose induction and the greater DNA yield of vector from arabinose-induced cultures. Measurement of beta-galactosidase activity in pMycoFos clones carrying the lacZ gene showed that in Mycobacterium smegmatis mc(2)-155, expression was inducible by acetamide, but in E. coli EPI300, the expression level was primarily determined by the vector copy number. Examination of protein profiles on SDS-PAGE gels confirmed the beta-galactosidase assay results. Construction of a fosmid library with the new vector confirmed that it could carry large DNA inserts. The new vector enabled the stable cloning and expression of an ethene monooxygenase gene cluster, which had eluded previous attempts at heterologous expression." @default.
• W1983802182 created "2016-06-24" @default.
• W1983802182 creator A5052892221 @default.
• W1983802182 creator A5056141653 @default.
• W1983802182 creator A5059815482 @default.
• W1983802182 creator A5062668075 @default.
• W1983802182 date "2011-09-01" @default.
• W1983802182 modified "2023-09-27" @default.
• W1983802182 title "Construction and evaluation of pMycoFos, a fosmid shuttle vector for Mycobacterium spp. with inducible gene expression and copy number control" @default.
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• W1983802182 doi "https://doi.org/10.1016/j.mimet.2011.06.005" @default.
• W1983802182 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/21689690" @default.
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