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Markerless Gene Deletion by Floxed Cassette Allelic Exchange Mutagenesis in Chlamydia trachomatis
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Markerless Gene Deletion by Floxed Cassette Allelic Exchange Mutagenesis in Chlamydia trachomatis

Markerless Gene Deletion by Floxed Cassette Allelic Exchange Mutagenesis in Chlamydia trachomatis

DOI:
10.3791/60848-v

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10:35 min

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January 30, 2020

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,
1Department of Microbiology, Immunology, and Molecular Genetics,University of Kentucky

Chapters

  • 00:04Introduction
  • 00:27Design and Assembly of pSUmC-4.0 with Homology Arms Specific to the Gene of Interest
  • 02:20Transformation of C. trachomatis with pSUmC 4.0 + Homology Arms for Gene Deletion by Allelic Exchange
  • 05:54Clonal Isolation of Green-only C. trachomatis Deletion Mutant Containing the loxP Flanked Selection Cassette by Limiting Dilution
  • 07:38Transformation of C. trachomatis FRAEM Mutant with pSU-Cre to Initiate Removal of loxP Flanked Selection Cassette
  • 08:42Results: FLAEM Enables Markerless Gene Deletion in C. trachomatis and alleviates Cassette-induced Polar Effects
  • 09:47Conclusion

Summary

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Described here is a method for targeted, markerless gene deletion in Chlamydia trachomatis using floxed cassette allelic exchange mutagenesis, FLAEM.

Tags

Markerless Gene DeletionFloxed CassetteAllelic Exchange MutagenesisChlamydia TrachomatisHomologous RecombinationPCRDNA AssemblyE. Coli TransformationMcCoy CellsChlamydia Infection

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