In this chapter, we describe the reverse genetics methodology behind generating a targeted gene deletion or
replacement in archaeal species of the genus Haloferax, which are renowned for their ease of manipulation.
Individual steps in the method include the design of a gene-targeting vector, its use in transforming
Haloferax to yield “pop-in” and “pop-out” clones, and techniques for validating the genetically manipulated
strain. The vector carries DNA fragments of 500–1000 bp that flank the gene of interest (or a mutant
allele), in addition to the pyrE2 gene for uracil biosynthesis (Bitan-Banin et al. J Bacteriol 185:772–778,
2003). The latter is used as a selectable marker for the transformation of Haloferax, wherein the vector
integrates by homologous recombination at the genomic locus to generate the “pop-in” strain; this is also
known as allele-coupled exchange. Culturing of these transformants in nonselective broth and subsequent
plating on 5-fluoroorotic acid (5-FOA)-containing media selects for excision of the vector, yielding either
wild type or mutant “pop-out” clones. These 5-FOA-resistant clones are screened to confirm the desired
mutation, using a combination of phenotypic assays, colony hybridization and Southern blotting. The
pop-in/pop-out method allows for the recycling of the pyrE2 marker to enable multiple gene deletions to
be carried out in a single strain, thereby providing insights into the function of multiple proteins and how
they interact in their respective cellular pathways.
Dattani, A., Harrison, C., Allers, T. (2022). Genetic Manipulation of Haloferax Species. In: Ferreira-Cerca, S. (eds) Archaea. Methods in Molecular Biology, vol 2522. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2445-6_3
Link to chapter: https://link.springer.com/protocol/10.1007/978-1-0716-2445-6_3