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Because the DNA segments are cut and reorganized in a direction-specific manner, site-specific recombination has emerged as an efficient genetic engineering technique. Flippase and Cyclization recombinases or Flp and Cre, respectively, are two members of the tyrosine recombinase family derived from bacteriophages, that are used to mediate site-specific DNA insertions, deletions, and targeted expression of proteins in mammalian cell lines.
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Cells are sometimes infected by more than one virus at once. When two viruses disassemble to expose their genomes for replication in the same cell, similar regions of their genomes can pair together and exchange sequences in a process called recombination. Alternatively, viruses with segmented genomes can swap segments in a process called reassortment.
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The basic reaction of homologous recombination (HR) involves two chromatids that contain DNA sequences sharing a significant stretch of identity. One of these sequences uses a strand from another as a template to synthesize DNA in an enzyme-catalyzed reaction. The final product is a novel amalgamation of the two substrates. To ensure an accurate recombination of sequences, HR is restricted to the S and G2 phases of the cell cycle. At these stages, the DNA has been replicated already and the...
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Organisms are capable of detecting and fixing nucleotide mismatches that occur during DNA replication. This sophisticated process requires identifying the new strand and replacing the erroneous bases with correct nucleotides. Mismatch repair is coordinated by many proteins in both prokaryotes and eukaryotes.
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Preparation of the Mgm101 Recombination Protein by MBP-based Tagging Strategy
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Mycobacterial recombineering.

Kenan C Murphy1, Kadamba Papavinasasundaram, Christopher M Sassetti

  • 1Microbiology and Physiological Systems, University of Massachusetts Medical School, 368 Plantation Street, Worcester, MA, 01605, USA.

Methods in Molecular Biology (Clifton, N.J.)
|March 18, 2015
PubMed
Summary
This summary is machine-generated.

Recombineering technology enables efficient gene knockout and modification in Mycobacterium tuberculosis. This method offers higher success rates and faster results compared to previous gene editing techniques.

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Area of Science:

  • Microbiology
  • Molecular Biology
  • Genetics

Background:

  • Precise gene manipulation in Mycobacterium tuberculosis is crucial for understanding its growth and pathogenicity.
  • Previous gene knockout methods in M. tuberculosis relied on host recombination systems, leading to lower efficiency.
  • Recombineering, a powerful gene editing technology, has been adapted for mycobacterial systems.

Purpose of the Study:

  • To describe the application of recombineering using the mycobacterial Che9c phage RecET system for genetic manipulation in M. tuberculosis.
  • To evaluate the efficiency and versatility of this recombineering system for various genetic modifications.

Main Methods:

  • Utilized the Che9c phage RecET recombination system for gene editing in Mycobacterium tuberculosis and M. smegmatis.
  • Applied recombineering for gene knockouts, reporter fusions, promoter replacements, and single base pair modifications.
  • Compared the efficiency of recombineering with established M. tuberculosis gene knockout protocols.

Main Results:

  • The RecET recombineering system facilitated gene knockouts, reporter fusions, promoter replacements, and single base pair modifications at very high frequencies.
  • Recombineering demonstrated significantly higher success rates and required less time compared to previously described M. tuberculosis gene knockout methods.
  • Successful genetic modifications were achieved in both M. tuberculosis and M. smegmatis chromosomes.

Conclusions:

  • The mycobacterial Che9c phage RecET recombineering system is a highly efficient and versatile tool for genetic manipulation in M. tuberculosis.
  • Recombineering offers a substantial improvement over traditional methods for gene knockout and modification in M. tuberculosis, accelerating research.
  • This technology provides a robust platform for future functional genomics studies of M. tuberculosis.