Superiority of the PCR-based approach for cloning the acetate kinase gene of Clostridium thermocellum

Date

1998-09-01

Author

Özcengiz, Gülay
Lin, WR
Ozcengiz, E
Westenberg, D
Lynd, LR
Demain, AL

Metadata

Show full item record

This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.

Item Usage Stats

100
views

0
downloads

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

Suggestions

OpenMETU
Core

Molecular cloning, characterization, and homologous expression of an endochitinase gene from Bacillus thuringiensis serovar morrisoni Okay, Sezer; Özcengiz, Gülay (2011-01-01) The endochitinase gene (chi3023) of Bacillus thuringiensis (Bt) serovar morrisoni strain 3023 was amplified via polymerase chain reaction (PCR) and cloned in Escherichia coli. The ORF of chi3023 (GenBank Accession Number: DQ993175) consists of 2031 nucleotides encoding a 676-residue protein with a calculated molecular mass of 74.5 kDa and a pI value of 6.0. The amino acid sequence of Chi3023 was compared with previously sequenced Bt chitinases and the phylogenetic relationships among them were determined. T...
SYNTHESIS, CLONING AND EXPRESSION OF PUCCINIA STRIIFORMIS F.SP.TRICITI CANDIDATE EFFECTOR Akkaya, Mahinur; Andaç, Ayşe(2013-12-31) SYNTHESIS, CLONING AND EXPRESSION OF PUCCINIA STRIIFORMIS F.SP.TRICITI CANDIDATE EFFECTOR
Cloning and expression of the Clostridium thermocellum L-lactate dehydrogenase gene in Escherichia coli and enzyme characterization Ozkan, M; Yilmaz, EI; Lynd, LR; Özcengiz, Gülay (Canadian Science Publishing, 2004-10-01) The structural gene for L-lactate dehydrogenase (LDH) (EC.1.1.1.27) from Clostridium thermocellum 27405 was cloned in Escherichia coli by screening the Lambda Zap 11 phage library of C. thermocellum genomic DNA. In one positive clone, an open reading frame of 948 base pairs corresponded to C. thermocellum ldh gene encoding for the predicted 315-residue protein. The ldh gene was successfully expressed in E. coli FMJ39 (ldh mutant) under the lac promoter. The recombinant enzyme was partially purified from E. ...
Cloning, sequencing and expression of L-lactate dehydrogenase gene from clostridium thermocellum and isolation, characterization and transformation of various cellulolytic, thermophilic, ethanol-producing bacteria Özkan, Melek; Özcengiz, Gülay; Ögel, Zümrüt B.; Department of Biotechnology (2002) In this study, the structural gene for L-lactate dehydrogenase (LDH; EC. 1.1. 1.27) was cloned and characterized for the first time from Clostridium thermocellum 27405 in Escherichia coli. A 357 bp PCR product of predicted size was obtained with degenerate primers designed for conserved regions of Idh genes of different organisms. This amplicon was used as a probe to screen a Lambda Zap II phage library of C. thermocellum genomic DNA. One positive clone contained an insert of 2.5 kb which included an open r...
Employing DNA barcoding as taxonomy and conservation tools for fish species censuses at the southeastern Mediterranean, a hot-spot area for biological invasion Karahan, Arzu; PAZ, Guy; STERN, Nir; Kıdeyş, Ahmet Erkan; Goren, Menachem; RİNKEVİCH, Baruch (2017-04-01) This study evaluates the utility of DNA barcoding (mitochondrial cytochrome oxidase subunit I; COI) as a biodiversity and conservation applied tool for identifying fish fauna from the southeastern Mediterranean (the continental coast of Israel), a hot-spot area for biological invasion, also with an eye to establish the foundation for follow-up studies that will use environmental DNA (eDNA) tracks of native and invasive species, and for the application of eDNA concepts and methodologies in nature conservatio...

Citation Formats

G. Özcengiz, W. Lin, E. Ozcengiz, D. Westenberg, L. Lynd, and A. Demain, “Superiority of the PCR-based approach for cloning the acetate kinase gene of Clostridium thermocellum,” JOURNAL OF INDUSTRIAL MICROBIOLOGY & BIOTECHNOLOGY, pp. 145–149, 1998, Accessed: 00, 2020. [Online]. Available: https://hdl.handle.net/11511/40808.