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Related Concept Videos

In-vitro Mutagenesis01:16

In-vitro Mutagenesis

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.
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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Related Experiment Video

Updated: Jul 22, 2026

Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli
09:01

Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli

Published on: March 16, 2011

One-step, highly efficient site-directed mutagenesis by toxic protein selection.

W Xu1, Y Zhang, L Y Yeh

  • 1Dana-Farber Cancer Institute, Boston, MA 02115, USA.

Biotechniques
|June 21, 2002
PubMed
Summary

This study introduces a rapid site-directed mutagenesis technique using the pTPS19 plasmid. This method achieves high mutagenesis efficiency by leveraging the toxic CcdB protein for stringent selection of desired gene mutations.

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07:04

Site-Directed Mutagenesis for In Vitro and In Vivo Experiments Exemplified with RNA Interactions in Escherichia Coli

Published on: February 5, 2019

Area of Science:

  • Molecular Biology
  • Genetic Engineering
  • Biotechnology

Background:

  • Site-directed mutagenesis is crucial for protein engineering and functional studies.
  • Existing methods can be time-consuming and lack efficiency.
  • A need exists for streamlined mutagenesis protocols with high success rates.

Purpose of the Study:

  • To develop a fast and efficient site-directed mutagenesis method.
  • To utilize the toxic CcdB protein for selection of mutants.
  • To achieve high mutagenesis efficiency using a novel plasmid system.

Main Methods:

  • Construction of the pTPS19 plasmid expressing the toxic CcdB protein.
  • Introduction of desired mutations using two primers: one for mutation, one for CcdB inactivation.
  • Direct selection of mutants on LB plates containing IPTG to induce CcdB toxicity.

Main Results:

  • Achieved 90%-100% mutagenesis efficiency.
  • Efficient elimination of wild-type parental plasmids via CcdB toxicity.
  • Successful mutant selection after a single round of transformation.

Conclusions:

  • The developed method is highly efficient and rapid for site-directed mutagenesis.
  • The pTPS19 plasmid system provides stringent selection for desired mutations.
  • This technique offers a significant improvement for genetic manipulation studies.