Title
Efficient transformation system for Propionibacterium freudenreichii based on a novel vector
Author
Centraal Instituut voor Voedingsonderzoek TNO TNO Voeding
Jore, J.P.M.
van Luijk, N.
Luiten, R.G.M.
van der Werf, M.J.
Pouwels, P.H.
Publication year
2001
Abstract
A 3.6-kb endogenous plasmid was isolated from a Propionibacterium freudenreichii strain and sequenced completely. Based on homologies with plasmids from other bacteria, notably a plasmid from Mycobacterium, a region harboring putative replicative functions was defined. Outside this region two restriction enzyme recognition sites were used for insertion of an Escherichia coli-specific replicon and an erythromycin resistance gene for selection in Propionibacterium. Hybrid vectors obtained in this way replicated in both E. coli and P. freudenreichii. Whereas electroporation of P. freudenreichii with vector DNA isolated from an E. coli transformant yielded 10 to 30 colonies per ug of DNA, use of vector DNA reisolated from a Propionibacterium transformant dramatically increased the efficiency of transformation (≥108 colonies per ug of DNA). It could be shown that restriction-modification was responsible for this effect. The high efficiency of the system described here permitted successful transformation of Propionibacterium with DNA ligation mixtures. Molecular Sequence Numbers: GENBANK: AF291751, X53217; Chemicals/CAS: DNA Restriction-Modification Enzymes
Subject
DNA Restriction-Modification Enzymes
Electroporation
Escherichia coli
Genetic Vectors
Molecular Sequence Data
Plasmids
Propionibacterium
Sequence Analysis, DNA
Transformation, Bacterial
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DOI
https://doi.org/10.1128/aem.67.2.499-503.2001
TNO identifier
56977
ISSN
0099-2240
Source
Applied and Environmental Microbiology, 67 (67), 499-503
Document type
article