Abstract
Methanogenic archaea generate methane as a by-product of anaerobic respiration using CO 2 , C 1 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. However, 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. We also 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.
| Original language | English (US) |
|---|---|
| Title of host publication | Methods in Enzymology |
| Editors | Fraser Armstrong |
| Publisher | Academic Press Inc. |
| Pages | 325-347 |
| Number of pages | 23 |
| ISBN (Print) | 9780128163610 |
| DOIs | |
| State | Published - 2018 |
Publication series
| Name | Methods in Enzymology |
|---|---|
| Volume | 613 |
| ISSN (Print) | 0076-6879 |
| ISSN (Electronic) | 1557-7988 |
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Keywords
- Cas9
- Genome editing
- Methane
- Methanogens
- Methanosarcina
- Methyl-coenzyme M reductase
ASJC Scopus subject areas
- Biochemistry
- Molecular Biology
Cite this
Genetic techniques for studies of methyl-coenzyme M reductase from Methanosarcina acetivorans C2A. / Nayak, Dipti D.; Metcalf, William W.
Methods in Enzymology. ed. / Fraser Armstrong. Academic Press Inc., 2018. p. 325-347 (Methods in Enzymology; Vol. 613).Research output: Chapter in Book/Report/Conference proceeding › Chapter
}
TY - CHAP
T1 - Genetic techniques for studies of methyl-coenzyme M reductase from Methanosarcina acetivorans C2A
AU - Nayak, Dipti D.
AU - Metcalf, William W
PY - 2018
Y1 - 2018
N2 - Methanogenic archaea generate methane as a by-product of anaerobic respiration using CO 2 , C 1 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. However, 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. We also 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.
AB - Methanogenic archaea generate methane as a by-product of anaerobic respiration using CO 2 , C 1 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. However, 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. We also 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.
KW - Cas9
KW - Genome editing
KW - Methane
KW - Methanogens
KW - Methanosarcina
KW - Methyl-coenzyme M reductase
UR - http://www.scopus.com/inward/record.url?scp=85057049706&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85057049706&partnerID=8YFLogxK
U2 - 10.1016/bs.mie.2018.10.012
DO - 10.1016/bs.mie.2018.10.012
M3 - Chapter
C2 - 30509472
AN - SCOPUS:85057049706
SN - 9780128163610
T3 - Methods in Enzymology
SP - 325
EP - 347
BT - Methods in Enzymology
A2 - Armstrong, Fraser
PB - Academic Press Inc.
ER -