Download PDF by John N.Abelson, Melvin I.Simon: Advanced Bacterial Genetics

By John N.Abelson, Melvin I.Simon

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When M. xanthus is infected with a lysate of P1::Tn5 with selection for kanamycin resistance (KanR) encoded by the Tn5, the resulting KanR colonies are results of the transposition of Tn5 from the disabled phage onto the chromosome. , the transposon is carried on a suicide plasmid). The plasmid can be transferred from the permissive host to the nonpermissive host by conjugation, transformation, or electroporation, with selection for an antibiotic resistance encoded by the transposon. 14 transposons [2] Overexpression of Transposase in trans The frequency of transposition can often be improved by increasing the concentration of transposase in the cell.

10. Special transposons can be used to construct operon or gene fusions. , 2002). Transposase from the commonly used transposons Tn5 and Tn10 show clear DNA sequence specificity for the insertion site, but the site recognized is sufficiently permissive to allow the isolation of insertions in essentially any gene. The transposase from Mu also demonstrates less DNA sequence specificity for the insertion site. Transposase mutants can reduce the sequence specificity of insertion. For example, mutations in the Tn10 transposase gene tpnA have been isolated with relaxed site specificity (Bender and Kleckner, 1992).

For biosynthetic pathways comprised of unlinked genes, linkage to known markers near specific genes is usually required. Also, PCR with primers designed to different loci can be used to determine the gene location of different auxotrophic MudJ insertions. Insertions defective in carbon or nitrogen source utilization must be screened for according to the specific carbon or nitrogen source. Tn10dTc‐araCþ: Plasmid pDH10 was transformed into Salmonella strain SL4213 (a restriction‐deficient Salmonella strain).

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Advanced Bacterial Genetics by John N.Abelson, Melvin I.Simon

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