Cloning vectors for gene delivery, integration and expression in Campylobacter jejuni

Gaylen A Uhlich; Lori Bagi; Nereus W Gunther 4th

doi:10.2144/btn-2021-0096

Cloning vectors for gene delivery, integration and expression in Campylobacter jejuni

Biotechniques. 2022 Jun;72(6):255-262. doi: 10.2144/btn-2021-0096. Epub 2022 Apr 13.

Authors

Gaylen A Uhlich¹, Lori Bagi¹, Nereus W Gunther 4th¹

Affiliation

¹ Molecular Characterization of Foodborne Pathogens Research Unit, Eastern Regional Research Center, Agricultural Research Service, US Department of Agriculture, 600 East Mermaid Lane, Wyndmoor, PA 19038, USA.

PMID: 35416085
DOI: 10.2144/btn-2021-0096

Abstract

Campylobacter genetics research is negatively impacted by a shortage of molecular tools for expressing DNA elements. A previous technique coupled an antibiotic resistance gene and its promoter to a gene of interest, inserting this expression unit into a conserved chromosomal location. Here the authors describe two new plasmids for construction and gene integration utilizing aspects of the previous type of expression unit. pBlueKan+cysM^Pro allows for the assembly of amplified DNA targets behind a kanamycin resistance marker and a constitutively transcribed cysM promoter. Transfer of the transcription unit to plasmid pCJR01 adds flanking regions of Campylobacter rRNA homology for recombination into conserved rRNA regions. System utility was demonstrated by restoring function of a flaAB deletion (RM3194ΔflaAB::tet) with a flaA gene or flaA/flaB combination.

Keywords: Campylobacter; fimbriae; gene expression; plasmids; restored function.

Publication types

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

MeSH terms

Anti-Bacterial Agents
Campylobacter jejuni* / genetics
DNA, Bacterial / genetics
Gene Transfer Techniques
Genetic Vectors / genetics
Plasmids / genetics

Substances

Anti-Bacterial Agents
DNA, Bacterial