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Construction of Edwardsiella ictaluri fhuCD inframe deletion mutant.
Proceedings of the 5th Global Fisheries and Aquaculture Research Conference, Faculty of Agriculture, Cairo University, Giza, Egypt, 1-3 October 2012 • 2012
Publication Information
Authors
Abdelhamed, H.; Liu, J. J.; Dahali, N.; Shaheen, A. A.; Amany, A. A.; Lawrence, M. L.; Karsi,
Keywords
Key word: Edwardsiella ictaluri; fhuCD.
Journal
Proceedings of the 5th Global Fisheries and Aquaculture Research Conference, Faculty of Agriculture, Cairo University, Giza, Egypt, 1-3 October 2012
Publisher
Not Available
Volume
Issue
Not Available
Pages
170-184
publication.type
International
Paper Link
Not Available
Supplementary Materials
Not Available
Abstract
A number of different strategies have been developed to achieve Site-directed mutagenesis.
One of these methods is in-frame deletion technique which efficiently creates mutations at specific sites
by deletion a number of nucleotides from a bacterial chromosome. The fhuCD operon consists of two
genes (fhuC and fhuD) which are involved in the uptake of ferric hydroxamate siderophores across the
bacterial membranes. In the present study, a construction of the EiΔfhuCD mutants was conducted.
EiΔfhuCD mutant was achieved by performing the in-frame fhuCD fragment through overlapping extension PCR then digested the in-frame fragment by SacI and XbaI restriction enzyme and ligated to
the suicide plasmid pMEG-375. The recombination plasmid (pEiΔfhuCD) was transformed into E. ictaluri wild type strain by conjugation and integrated into the genomic DNA through two steps of homologous recombination. The colony PCR and DNA sequencing were used to genotypically confirm the deletion. Besides, it was successfully deleted a 1635bp/545aa from a 1707-bp of the fhuCD operon.
One of these methods is in-frame deletion technique which efficiently creates mutations at specific sites
by deletion a number of nucleotides from a bacterial chromosome. The fhuCD operon consists of two
genes (fhuC and fhuD) which are involved in the uptake of ferric hydroxamate siderophores across the
bacterial membranes. In the present study, a construction of the EiΔfhuCD mutants was conducted.
EiΔfhuCD mutant was achieved by performing the in-frame fhuCD fragment through overlapping extension PCR then digested the in-frame fragment by SacI and XbaI restriction enzyme and ligated to
the suicide plasmid pMEG-375. The recombination plasmid (pEiΔfhuCD) was transformed into E. ictaluri wild type strain by conjugation and integrated into the genomic DNA through two steps of homologous recombination. The colony PCR and DNA sequencing were used to genotypically confirm the deletion. Besides, it was successfully deleted a 1635bp/545aa from a 1707-bp of the fhuCD operon.