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Superiority of the PCR-based approach for cloning the acetate kinase gene of Clostridium thermocellum
Date
1998-09-01
Author
Özcengiz, Gülay
Kim, JH
Lin, WR
Ozcengiz, E
Westenberg, D
Lynd, LR
Demain, AL
Metadata
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This work is licensed under a
Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License
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Cloning of Clostridium thermocellum acetate kinase (ack) and/or phosphotransacetylase (pta) genes in Escherichia coli by functional complementation of ack and/or pta mutants was complicated by an alternative acetate assimilation pathway involving acetyl-CoA synthetase (ACS). In addition to the problems encountered with the complementation approach, cloning of these genes was not readily achieved using heterologous probing with corresponding genes from Escherichia coli and Methanosarcina thermophila due to the lack of sufficient homology. The use of a PCR-based approach, on the other hand, yielded a specific C. thermocellum gene fragment which showed significant sequence identity to the ack gene for which primers were designed. The subcloned ack fragment was then successfully used as a probe for the isolation of the corresponding gene and restriction analysis of that region.
Subject Keywords
Clostridium thermocellum
,
Acetate kinase
,
Phosphotransacetylase
,
Thermophilic bacteria
,
PCR
,
Gene cloning
URI
https://hdl.handle.net/11511/40808
Journal
JOURNAL OF INDUSTRIAL MICROBIOLOGY & BIOTECHNOLOGY
DOI
https://doi.org/10.1038/sj.jim.2900567
Collections
Department of Biology, Article