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Volume 147, Issue 9

Construction of random mutagenesis libraries using Tn transposition complexes Free

Abstract

The ability to generate tagged mutants of spp. will facilitate a deeper understanding of this medically and commercially important genus. The absence of efficient transposon systems in these organisms has here been overcome by the use of Tn-based DNA–protein transposition complexes which can transpose at high efficiency. To achieve this, electroporation efficiencies and antibiotic selection were optimized. A CW25 Tn insertion library of 1500 mutants was created. Southern blotting of 23 representative mutants demonstrated random insertion. A number of auxotrophic mutants were isolated and the disrupted regions involved were identified by inverse PCR and subsequent sequencing. Transposition of Tn was confirmed by the presence of 9 bp direct repeats of DNA flanking the transposon insertion site. To further test this system, a Tn insertion library was constructed in a wild-type soil isolate of spp. This is the first viable transposon knockout system reported for

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Keyword(s): Gram-positive , iPCR, inverse PCR , kanamycin , Mycobacterium , Nocardia and transposon
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2001-09-01
2024-04-26
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Construction of Rhodococcus random mutagenesis libraries using Tn5 transposition complexes
Microbiology 147, 2529 (2001); https://doi.org/10.1099/00221287-147-9-2529
/content/journal/micro/10.1099/00221287-147-9-2529
/content/journal/micro/10.1099/00221287-147-9-2529
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