A Rapid Targeted Gene Inactivation Approach in Sulfolobus islandicus

Changyi Zhang, Serina M. Taluja, Emily N. Hallett, Rachel J. Whitaker

Research output: Chapter in Book/Report/Conference proceedingChapter

Abstract

Homologous recombination–based gene targeting is a powerful and classic reverse genetics approach to precisely elucidate in vivo gene functions in the organisms across all three domains of life. Gene function studies in Archaea, particularly for those flourishing in inhospitable natural environments that are anaerobic, usually hot, and acidic, have been a great challenge; however, this situation was recently overturned with the increasing availability of genetic manipulation systems in several cultivable archaeal species. In the present chapter, we describe a detailed procedure to rapidly generate gene disruption mutants in the hyperthermophilic crenarchaeon Sulfolobus islandicus via a recently developed Microhomology-Mediated Gene Inactivation (MMGI) approach. We highlight crucial experimental details required to be carefully considered when using the MMGI approach for genetic manipulations. We hope this highly reproducible procedure can supplement existing genetic tools for studying the biology of archaeal order Sulfolobales.

Original languageEnglish (US)
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages145-162
Number of pages18
DOIs
StatePublished - 2022

Publication series

NameMethods in Molecular Biology
Volume2522
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029

Keywords

  • Homologous Recombination
  • Hyperthermophilic Archaea
  • One-Step Gene Inactivation
  • Sulfolobus islandicus

ASJC Scopus subject areas

  • Genetics
  • Molecular Biology

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