Cloning and expression of a haloacid dehalogenase fromPseudomonas sp. strain 19 S |
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Authors: | Semra Kocabiyik Bugraer Aslan Rudolf Müller |
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Institution: | (1) Department of Biological Sciences, Middle East Technical University, 06531 Ankara, Turkey;(2) Arbeitsbereich Biotechnologie II, Technische Universität Hamburg-Harburg, Germany |
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Abstract: | A dehalogenase gene specifying the utilization of a variety of haloacids byPseudomonas sp. Strain 19S has been cloned and expressed inE. coli. Our cloning strategy employed specific amplification of a fragment homologous toPseudomonas dehalogenase gene by Polymerase Chain Reaction (PCR). The PCR amplicon successfully acted as a probe to detect the dehalogenase gene in the Southern Blot of the digestedPseudomonas total DNA. Corresponding fragments were cloned into pUC 18 vector and amplified inE. coli MV 1190. One clone with a substantial dehalogenation activity carried a recombinant plasmid containing a 5.5 kb insert.Abbreviations 2-CPA
2-chloropropionate
- MCA
monochloro acetate
- IPTG
isopropyl-1-thio- -D-galactoside
- NBT
nitroblue tetrazolium salt
- PCR
polymerase chain reaction
- X-gal
5-bromo-4-chloro-3-indolyl- -D-galactoside
- X-phosphate
5-bromo-4-chloro-3-indolyl phosphate |
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Keywords: | haloacid dehalogenase cloning Pseudomonas sp 19S |
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