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Chemical and electroporated transformation of Edwardsiella ictaluri using three different plasmids

Permanent URL:
http://handle.nal.usda.gov/10113/36104
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Abstract:
Transfer of DNA by conjugation has been the method generally used for genetic manipulation of Edwardsiella ictaluri because, previously, attempts to transform E. ictaluri by the uptake of naked DNA have apparently failed. We report here the successful transformation of seven strains of E. ictaluri using electroporation and two different chemical procedures [conventional calcium chloride (CaCl₂) and 'one-step' (polyethylene glycol, dimethyl sulfoxide and MgSO₄) protocols]. Seven strains of E. ictaluri were transformed using three different plasmids [pZsGreen, pUC18 and pET-30a(+)]. The highest transformation efficiency was achieved by electroporation (5.5±0.2 x 10⁴ transformants ng⁻¹ plasmid DNA) than with the CaCl₂ (8.1±6.1 x 10⁻¹ transformants ng⁻¹ plasmid) and the 'one-step transformation' protocol (2.5±2.7 transformants ng⁻¹ plasmid). An efficient transformation by electroporation required only 0.2 ng of plasmid compared with 200 ng required for the CaCl₂ and one-step protocols. The plasmids were stably maintained in E. ictaluri grown in the presence of antibiotic for 12 or more passages. The results of this study show that transformation of E. ictaluri by electroporation can be routinely used for the molecular genetic manipulation of this organism, and is a quicker and easier method than transformation performed by conjugation.
Author(s):
Russo, Riccardo , Panangala, Victor S. , Wood, Raphael R. , Klesius, Phillip H.
Subject(s):
Edwardsiella ictaluri , plasmids , plasmid vectors , genetic transformation , electroporation , conjugation , microbial genetics
Format:
p. 105-110.
Note:
Includes references
Source:
FEMS microbiology letters 2009 Sept., v. 298, no. 1
Language:
English
Publisher:
Oxford, UK : Blackwell Publishing Ltd
Year:
2009
Collection:
Journal Articles, USDA Authors, Peer-Reviewed
Rights:
Works produced by employees of the U.S. Government as part of their official duties are not copyrighted within the U.S. The content of this document is not copyrighted.