Genetic techniques for studies of methyl-coenzyme M reductase from Methanosarcina acetivorans C2A.

Nayak, Dipti; Metcalf, William W.

doi:10.1016/bs.mie.2018.10.012

Title: Genetic techniques for studies of methyl-coenzyme M reductase from Methanosarcina acetivorans C2A.

Journal Article · Fri Nov 23 00:00:00 EST 2018 · Methods in Enzymology

DOI:https://doi.org/10.1016/bs.mie.2018.10.012· OSTI ID:1596707

Nayak, Dipti ^[1]; Metcalf, William W. ^[1]

Univ. of Illinois at Urbana-Champaign, IL (United States). Carl R. Woese Inst. for Genomic Biology and Dept. of Microbiology

Methanogenic archaea generate methane as a by-product of anaerobic respiration using CO₂, C₁ compounds (like methanol or methylated amines), or acetate as terminal electron acceptors. Methanogens are an untapped resource for biotechnological advances related to methane production as well as methane consumption. Yet, key biological features of these organisms remain poorly understood. One such feature is the enzyme methyl-coenzyme M reductase (referred to as MCR), which catalyzes the last step in the methanogenic pathway and results in methane formation. Gene essentiality has limited genetic analyses of MCR thus far. Therefore, studies of this important enzyme have been limited to biochemical and biophysical techniques that are especially laborious and often reliant on sophisticated instrumentation that is not commonly available. In this chapter, we outline our recently developed CRISPR–Cas9-based genome editing tools and describe how these tools have been used for the introduction of a tandem affinity purification tag at the chromosomal mcr locus in the model methanogen, Methanosarcina acetivorans C2A. Moreover, we report a protocol for rapid affinity purification of MCR from M. acetivorans C2A that will enable high-throughput studies of this enzyme in the future.

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Research Organization:: Univ. of Illinois at Urbana-Champaign, IL (United States)

Sponsoring Organization:: USDOE Office of Science (SC), Basic Energy Sciences (BES). Chemical Sciences, Geosciences, and Biosciences Division; Simons Foundation

Contributing Organization:: Carl R. Woese Institute

Grant/Contract Number:: FG02-02ER15296

OSTI ID:: 1596707

Journal Information:: Methods in Enzymology, Vol. 613; ISSN 0076-6879

Publisher:: ElsevierCopyright Statement

Country of Publication:: United States

Language:: English

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Cited by: 5 works

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Title: Genetic techniques for studies of methyl-coenzyme M reductase from Methanosarcina acetivorans C2A.

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