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Volume 132, Issue 3

Molecular Cloning into Tn and Integration in the chromosome: a Tool for Heterologous Gene Expression Free

Abstract

SUMMARY: The DNA primase gene of the promiscuous IncP-1 conjugative plasmid RP1, encoding two polypeptides of 118 and 80 kDa, was inserted into the transposon Tn5 in The derivative transposon, Tn2523, was then transposed to a temperature-sensitive replication mutant of the promiscuous IncP-1 conjugative plasmid R68 at permissive temperature and the plasmid transferred to strain PAO. The latter strain was then grown at non-permissive temperature to identify transposition of Tn2523 into the chromosome. Immunological and enzymic analysis showed the expression of functional primase polypeptides in the constructed strain. This strain also restored wild-type conjugational transfer proficiency, by complementation, to mutants of the IncP-1 plasmid R18 affected in transfer from to or to due to transposon Tn7 insertion mutations in the primase gene. This strategy of cloning into a transposon and integration into the bacterial chromosome should facilitate genetic manipulation and studies of gene expression in a range of Gram-negative bacteria.

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© Society for General Microbiology 1986
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1986-03-01
2024-04-18
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Molecular Cloning into Tn5 and Integration in the Pseudomonas aeruginosa chromosome: a Tool for Heterologous Gene Expression
Microbiology 132, 707 (1986); https://doi.org/10.1099/00221287-132-3-707
/content/journal/micro/10.1099/00221287-132-3-707
/content/journal/micro/10.1099/00221287-132-3-707
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