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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...
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In vitro Mutagenesis01:16

In vitro Mutagenesis

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Related Experiment Video

Updated: Jun 10, 2026

Generation of Marked and Markerless Mutants in Model Cyanobacterial Species
11:45

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Published on: May 29, 2016

En passant mutagenesis: a two step markerless red recombination system.

B Karsten Tischer1, Gregory A Smith, Nikolaus Osterrieder

  • 1Institut für Virologie, Freie Universität Berlin, Berlin, Germany. k.tischer@fu-berlin.de

Methods in Molecular Biology (Clifton, N.J.)
|August 3, 2010
PubMed
Summary

Researchers developed a scarless Red recombination technique for bacterial artificial chromosome (BAC) modification in E. coli. This method efficiently generates mutations, deletions, and insertions without leaving unwanted DNA sequences in BAC clones.

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Generation of Marked and Markerless Mutants in Model Cyanobacterial Species
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Published on: May 29, 2016

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07:18

Gene-targeted Random Mutagenesis to Select Heterochromatin-destabilizing Proteasome Mutants in Fission Yeast

Published on: May 15, 2018

Area of Science:

  • Molecular Biology
  • Genetics
  • Microbiology

Background:

  • Bacterial artificial chromosomes (BACs) are crucial for maintaining and modifying large DNA sequences in Escherichia coli.
  • Red recombination is a common method for sequence modification in BACs.
  • Unwanted foreign sequences, like antibiotic resistance genes or site-specific recombination sites, can contaminate BAC clones.

Purpose of the Study:

  • To develop a scarless Red recombination technique for precise BAC sequence modification.
  • To enable the generation of point mutations, deletions, and insertions without residual foreign DNA.
  • To create a convenient and efficient mutagenesis system in E. coli.

Main Methods:

  • A novel Red recombination strategy was employed using a sequence duplication insertion.
  • In vivo I-SceI cleavage was utilized for selection marker excision.
  • A single E. coli strain (GS1783) with chromosomally encoded inducible Red and I-SceI expression was engineered.

Main Results:

  • The developed Red-based technique allows for scarless generation of various genetic modifications in BACs.
  • The method efficiently introduces point mutations, deletions, and insertions of varying sizes.
  • The engineered E. coli strain facilitates convenient and highly efficient mutagenesis without auxiliary plasmids.

Conclusions:

  • This Red recombination technique offers a significant advancement for scarless BAC engineering.
  • The system streamlines the modification of BACs, crucial for synthetic biology and genetic research.
  • The engineered E. coli strain provides a robust platform for efficient BAC mutagenesis.