Related Experiment Video
Updated: Aug 9, 2025
14:23
Recombineering Homologous Recombination Constructs in Drosophila
Published on: July 13, 2013
19.4K
Recombineering 101: Making an in-Frame Deletion Mutant.
Nara Figueroa-Bossi1, Roberto Balbontín2, Lionello Bossi3
1Université Paris-Saclay, CEA, CNRS, Institut de Biologie Intégrative de la Cellule (I2BC), 91190 Gif-sur-Yvette, France.
Cold Spring Harbor Protocols
|February 22, 2023
Summary
DNA recombineering enables precise bacterial gene editing using PCR-generated DNA fragments and phage lambda Red recombination functions. This method efficiently creates gene knockout mutants and deletions in bacteria like E. coli and Salmonella.
Area of Science:
- Microbiology
- Molecular Biology
- Genetic Engineering
Background:
- DNA recombineering leverages bacteriophage lambda (λ) Red recombination machinery.
- It facilitates the integration of polymerase chain reaction (PCR)-generated DNA fragments into bacterial chromosomes.
Purpose of the Study:
- To describe a streamlined protocol for generating bacterial mutants using DNA recombineering.
- To detail the construction of knockout mutants and gene deletions in *Salmonella enterica* and *Escherichia coli*.
Main Methods:
- Utilizes PCR primers with 5' extensions homologous to target insertion sites.
- Employs antibiotic-resistance cassettes for gene replacement, often flanked by FRT sites for subsequent excision.
- Optimized primer design maintains reading frame integrity to prevent polarity effects.
Main Results:
- Successfully generates knockout mutants of nonessential genes.
- Enables the construction of precise gene deletions by replacing target sequences with resistance cassettes.
- FRT-flanked cassette excision minimizes genomic perturbations, leaving a scar sequence.
Conclusions:
- DNA recombineering provides an efficient method for bacterial genetic manipulation.
- Careful primer design and template selection are crucial for successful gene editing and avoiding polar effects.
- The protocol is highly effective for *Salmonella enterica* and *Escherichia coli*.
More Related Videos
Related Concept Videos
02:53Conservative Site-specific Recombination and Phase Variation
6.1K
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...
The recognition sites for Cre recombinase called LoxP...
6.1K
02:32Exon Recombination
3.7K
The evolution of new genes is critical for speciation. Exon recombination, also known as exon shuffling or domain shuffling, is an important means of new gene formation. It is observed across vertebrates, invertebrates, and in some plants such as potatoes and sunflowers. During exon recombination, exons from the same or different genes recombine and produce new exon-intron combinations, which might evolve into new genes.
Exon shuffling follows “splice frame rules.” Each exon...
Exon shuffling follows “splice frame rules.” Each exon...
3.7K
01:16In-vitro Mutagenesis
14.1K
To learn more about the function of a gene, researchers can observe what happens when the gene is inactivated or “knocked out,” by creating genetically engineered knockout animals. Knockout mice have been particularly useful as models for human diseases such as cancer, Parkinson’s disease, and diabetes.
14.1K
02:31Homologous Recombination
50.8K
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...
50.8K