pNEB193-derived suicide plasmids for gene deletion and protein expression in the methane-producing archaeon, Methanosarcina acetivorans

Authors

Mitchell T. Shea, University of Nebraska-Lincoln
Mary E. Walter, University of Nebraska-Lincoln
Nikolas Duszenko, University of Nebraska-Lincoln
Anne-Lise Ducluzeau, University of Nebraska-Lincoln
Jared Aldridge, University of Nebraska-Lincoln
Shannon K. King, University of Nebraska-Lincoln
Nicole R. Buan, University of Nebraska-LincolnFollow

Document Type

Article

Date of this Version

3-1-2017

Citation

2016 Authors

Comments

Plasmid. 2016 ; 84-85: 27–35. doi:10.1016/j.plasmid.2016.02.003.

Abstract

Gene deletion and protein expression are cornerstone procedures for studying metabolism in any organism, including methane-producing archaea (methanogens). Methanogens produce coenzymes and cofactors not found in most bacteria, therefore it is sometimes necessary to express and purify methanogen proteins from the natural host. Protein expression in the native organism is also useful when studying post-translational modifications and their effect on gene expression or enzyme activity. We have created several new suicide plasmids to complement existing genetic tools for use in the methanogen, Methanosarcina acetivorans. The new plasmids are derived from the commercially available E. coli plasmid, pNEB193, and cannot replicate autonomously in methanogens. The designed plasmids facilitate markerless gene deletion, gene transcription, protein expression, and purification of proteins with cleavable affinity tags from the methanogen, Methanosarcina acetivorans.