An improved method for the transformation of lactobacilli strains using electroporation

M-Q Wei; C M Rush; J M Norman; L M Hafner; R J Epping; Peter Timms

doi:10.1016/0167-7012(94)00038-9

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An improved method for the transformation of lactobacilli strains using electroporation

Journal article

Peer reviewed

An improved method for the transformation of lactobacilli strains using electroporation

M-Q Wei, C M Rush, J M Norman, L M Hafner, R J Epping and Peter Timms

Journal of Microbiological Methods, Vol.21(1), pp.97-109

1995

DOI: https://doi.org/10.1016/0167-7012(94)00038-9

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Abstract

Microbiology

Medical Microbiology

electroporation

cell-wall modification

lactobacillus

plasmid

transformation

Because of their widespread industrial and medical importance, there is considerable interest in the manipulation and improvement of Lactobacillus strains using modern genetic engineering techniques. However, most reports have focused on industrial strains and often have resulted in non-reproducible transformation efficiencies. We have developed an optimised protocol for electroporating foreign plasmid DNA into clinical strains of lactobacilli. Treatment of the recipient lactobacilli with either lysozyme, glycine or penicillin improved electrotransformation efficiencies up to 480-fold. A critical step in achieving efficient and reproducible electrotransformation of clinical lactobacilli with the plasmid pSA3 was the requirement for a post-pulse recovery time of 2-3 h, combined with the use of sub-inhibitory concentrations of antibiotics in the selective plates. While pNZ17 transformants also benefited from a post-pulse recovery period, good transformation efficiencies could be achieved when plated directly onto selective concentrations of chloramphenicol. We also observed significant differences in electrotransformation efficiencies between our guinea pig vaginal Lactobacillus isolates (maximum of 4.8 × 104 transformants/μg pNZ17 DNA) and the human L. casei strain ATCC 393 (3.7 × 106 transformants/μg pNZ17 DNA). An optimised procedure for the electroporation of plasmid DNA into lactobacilli is described.

Details

Title: An improved method for the transformation of lactobacilli strains using electroporation
Authors: M-Q Wei (Author) - University of Washington, United States
C M Rush (Author) - Universita di Siena, Italy
J M Norman (Author) - Queensland University of Technology
L M Hafner (Author) - Queensland University of Technology
R J Epping (Author) - Queensland University of Technology
Peter Timms (Author) - Queensland University of Technology
Publication details: Journal of Microbiological Methods, Vol.21(1), pp.97-109
Publisher: Elsevier BV
Date published: 1995
DOI: 10.1016/0167-7012(94)00038-9
ISSN: 0167-7012
Organisation Unit: University of the Sunshine Coast, Queensland; Centre for Bioinnovation
Language: English
Record Identifier: 99449568902621
Output Type: Journal article

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