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Conjugative Mating Assays for Sequence-specific Analysis of Transfer Proteins Involved in Bacterial Conjugation
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Genetics
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JoVE Journal Genetics
Conjugative Mating Assays for Sequence-specific Analysis of Transfer Proteins Involved in Bacterial Conjugation

Conjugative Mating Assays for Sequence-specific Analysis of Transfer Proteins Involved in Bacterial Conjugation

DOI:
10.3791/54854-v

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10:41 min

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January 04, 2017

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, , , , ,
1Department of Chemistry,York University, 2The Centre for Research on Biomolecular Interactions,York University

Chapters

  • 00:05Title
  • 00:59Amplification of the Chloramphenicol Acetyltransferase (CAT) Cassette from pBAD33 Plasmid Using Oligomers Designed for Homologous Pairing
  • 02:38Generation of pOX38-Tc Δgene::Cm Strains
  • 04:26Conjugative Mating Assay from XK1200 Donors to MC4100 Recipients
  • 08:38Results: Transfer Gene Knockout and Recovery of Conjugative DNA Transfer
  • 09:45Conclusion

Summary

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Here, we present a protocol to knockout a gene of interest involved in plasmid conjugation and subsequently analyze the impact of its absence using mating assays. The function of the gene is further explored to a specific region of its sequence using deletion or point mutations.

Tags

Conjugative Mating AssaysTransfer ProteinsBacterial ConjugationType Four Secretion ComplexHomologous RecombinationPBAD33 PlasmidCat CassettePCR AmplificationElectrocompetent DY330R CellsPOX38-Tc Plasmid

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