Genetic Methods and Construction of Chromosomal Mutations in Methanogenic Archaea

Johanna Thomsen; Katrin Weidenbach; William W Metcalf; Ruth A Schmitz

doi:10.1007/978-1-0716-2445-6_6

Genetic Methods and Construction of Chromosomal Mutations in Methanogenic Archaea

Methods Mol Biol. 2022:2522:105-117. doi: 10.1007/978-1-0716-2445-6_6.

Authors

Johanna Thomsen¹, Katrin Weidenbach¹, William W Metcalf², Ruth A Schmitz³

Affiliations

¹ Institute For General Microbiology, Christian-Albrechts-University, Kiel, Germany.
² Department of Microbiology, University of Illinois, Urbana, IL, USA.
³ Institute For General Microbiology, Christian-Albrechts-University, Kiel, Germany. rschmitz@ifam.uni-kiel.de.

PMID: 36125745
DOI: 10.1007/978-1-0716-2445-6_6

Abstract

Genetic manipulation through markerless exchange enables the modification of several genomic regions without leaving a selection marker in the genome. Here, a method using hpt coding for hypoxanthine phosphoribosyltransferase as a counter selectable marker is described. For Methanosarcina species a chromosomal deletion of the hpt gene is firstly generated, which confers resistance to the purine analogue 8-aza-2,6-diaminopurine (8-ADP). In a second step, the reintroduction of the hpt gene on a plasmid leads to a selectable loss of 8-ADP resistance after a homologous recombination event (pop-in). A subsequent pop-out event restores the 8-ADP resistance and can generate chromosomal mutants with frequencies of about 50%.

Keywords: Chromosomal mutant; Genetic manipulation; Genetic system; Markerless exchange; Methanogenic archaea; Pop-in and pop-out.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Archaea*
Hypoxanthine Phosphoribosyltransferase* / genetics
Mutation
Purines

Substances

Hypoxanthine Phosphoribosyltransferase
Purines