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Related Concept Videos

Conservative Site-specific Recombination and Phase Variation02:53

Conservative Site-specific Recombination and Phase Variation

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.
The recognition sites for Cre recombinase called LoxP...
Viral Recombination00:57

Viral Recombination

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.
Homologous Recombination02:31

Homologous Recombination

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...
Homologous Recombination02:31

Homologous Recombination

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...
Mismatch Repair01:20

Mismatch Repair

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.
The Mutator Protein Family Plays a Key Role in DNA Mismatch Repair
The human genome has more than 3 billion base pairs of DNA per cell. Prior to cell division, that vast amount of genetic...
Mismatch Repair01:36

Mismatch Repair

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Related Experiment Video

Updated: Jul 6, 2026

Preparation of the Mgm101 Recombination Protein by MBP-based Tagging Strategy
11:40

Preparation of the Mgm101 Recombination Protein by MBP-based Tagging Strategy

Published on: June 25, 2013

Mycobacterial recombineering.

Julia C van Kessel1, Graham F Hatfull

  • 1Pittsburgh Bacteriophage Institute and Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA, USA.

Methods in Molecular Biology (Clifton, N.J.)
|March 29, 2008
PubMed
Summary
This summary is machine-generated.

Mycobacterial recombineering, using phage Che9c proteins, enhances genetic manipulation in Mycobacterium tuberculosis by improving DNA recombination. This method simplifies the creation of gene replacement mutants in slow-growing mycobacteria.

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Subcloning Plus Insertion (SPI) - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors
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Subcloning Plus Insertion (SPI) - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors

Published on: January 8, 2015

Related Experiment Videos

Last Updated: Jul 6, 2026

Preparation of the Mgm101 Recombination Protein by MBP-based Tagging Strategy
11:40

Preparation of the Mgm101 Recombination Protein by MBP-based Tagging Strategy

Published on: June 25, 2013

Subcloning Plus Insertion (SPI) - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors
09:02

Subcloning Plus Insertion (SPI) - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors

Published on: January 8, 2015

Area of Science:

  • Molecular Biology
  • Genetics
  • Microbiology

Background:

  • Mycobacterial genetics, especially for Mycobacterium tuberculosis, faces challenges like poor DNA uptake, slow growth, and high illegitimate recombination rates.
  • Recombineering in Escherichia coli uses phage-encoded functions to boost homologous recombination, offering a model for improved genetic manipulation.

Purpose of the Study:

  • To adapt the recombineering technique for efficient genetic manipulation in mycobacteria.
  • To identify and utilize mycobacteriophage-encoded recombination proteins to overcome genetic manipulation hurdles in Mycobacterium tuberculosis.

Main Methods:

  • Identification of RecE and RecT homologs in the mycobacteriophage Che9c.
  • Transient expression of Che9c recombination proteins using an inducible system in both slow- and fast-growing mycobacteria.
  • Utilizing linear DNA substrates for allelic exchange and gene replacement.

Main Results:

  • Expression of Che9c recombination proteins significantly elevated recombination frequencies in mycobacteria.
  • The developed mycobacterial recombineering system facilitated straightforward allelic exchange using linear DNA.
  • This approach proved effective for constructing gene replacement mutants in both M. smegmatis and Mycobacterium tuberculosis.

Conclusions:

  • Mycobacterial recombineering, enabled by Che9c phage proteins, provides a simplified and efficient method for genetic modification.
  • This technique overcomes previous limitations in mycobacterial genome manipulation, paving the way for easier construction of gene replacement mutants.