5 years ago

Direct and Inverted Repeat stimulated excision (DIRex): Simple, single-step, and scar-free mutagenesis of bacterial genes

Joakim Näsvall

by Joakim Näsvall

The need for generating precisely designed mutations is common in genetics, biochemistry, and molecular biology. Here, I describe a new λ Red recombineering method (Direct and Inverted Repeat stimulated excision; DIRex) for fast and easy generation of single point mutations, small insertions or replacements as well as deletions of any size, in bacterial genes. The method does not leave any resistance marker or scar sequence and requires only one transformation to generate a semi-stable intermediate insertion mutant. Spontaneous excision of the intermediate efficiently and accurately generates the final mutant. In addition, the intermediate is transferable between strains by generalized transductions, enabling transfer of the mutation into multiple strains without repeating the recombineering step. Existing methods that can be used to accomplish similar results are either (i) more complicated to design, (ii) more limited in what mutation types can be made, or (iii) require expression of extrinsic factors in addition to λ Red. I demonstrate the utility of the method by generating several deletions, small insertions/replacements, and single nucleotide exchanges in Escherichia coli and Salmonella enterica. Furthermore, the design parameters that influence the excision frequency and the success rate of generating desired point mutations have been examined to determine design guidelines for optimal efficiency.

Publisher URL: http://journals.plos.org/plosone/article

DOI: 10.1371/journal.pone.0184126

You might also like
Discover & Discuss Important Research

Keeping up-to-date with research can feel impossible, with papers being published faster than you'll ever be able to read them. That's where Researcher comes in: we're simplifying discovery and making important discussions happen. With over 19,000 sources, including peer-reviewed journals, preprints, blogs, universities, podcasts and Live events across 10 research areas, you'll never miss what's important to you. It's like social media, but better. Oh, and we should mention - it's free.

  • Download from Google Play
  • Download from App Store
  • Download from AppInChina

Researcher displays publicly available abstracts and doesn’t host any full article content. If the content is open access, we will direct clicks from the abstracts to the publisher website and display the PDF copy on our platform. Clicks to view the full text will be directed to the publisher website, where only users with subscriptions or access through their institution are able to view the full article.