title: pNEB193-derived suicide plasmids for gene deletion and protein expression in the methane-producing archaeon, Methanosarcina acetivorans.
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 Escherichia 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, M. acetivorans.
authors.affiliation: Redox Biology Center, Department of Biochemistry, University of Nebraska-Lincoln, Lincoln, NE, United States.
authors.forename: Mitchell T
authors.initials: MT
authors.lastname: Shea
authors.affiliation: Redox Biology Center, Department of Biochemistry, University of Nebraska-Lincoln, Lincoln, NE, United States.
authors.forename: Mary E
authors.initials: ME
authors.lastname: Walter
authors.affiliation: Redox Biology Center, Department of Biochemistry, University of Nebraska-Lincoln, Lincoln, NE, United States.
authors.forename: Nikolas
authors.initials: N
authors.lastname: Duszenko
authors.affiliation: Redox Biology Center, Department of Biochemistry, University of Nebraska-Lincoln, Lincoln, NE, United States.
authors.forename: Anne-Lise
authors.initials: AL
authors.lastname: Ducluzeau
authors.affiliation: Redox Biology Center, Department of Biochemistry, University of Nebraska-Lincoln, Lincoln, NE, United States.
authors.forename: Jared
authors.initials: J
authors.lastname: Aldridge
authors.affiliation: Redox Biology Center, Department of Biochemistry, University of Nebraska-Lincoln, Lincoln, NE, United States.
authors.forename: Shannon K
authors.initials: SK
authors.lastname: King
authors.affiliation: Redox Biology Center, Department of Biochemistry, University of Nebraska-Lincoln, Lincoln, NE, United States. Electronic address: nbuan@unl.edu.
authors.forename: Nicole R
authors.initials: NR
authors.lastname: Buan
mesh_terms: D003001:Cloning, Molecular / Q000379:methods*; D017353:Gene Deletion; D015870:Gene Expression; D041722:Genes, Transgenic, Suicide / Q000235:genetics*; D008697:Methane / Q000096:biosynthesis; D017020:Methanosarcina / Q000235:genetics* / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics*
chemical_list: D008697:Methane
keywords: Archaea; Methanogen; Methanosarcina; Protein expression