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Journal Article

Site-directed and transposon-mediated mutagenesis with pfd-plasmids by electroporation of Erwinia amylovora and Escherichia coli cells

MPS-Authors
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Metzger,  Marianne
Research Group Prof. Dr. Geider, Max Planck Institute for Medical Research, Max Planck Society;

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Bellemann,  Peter
Research Group Prof. Dr. Geider, Max Planck Institute for Medical Research, Max Planck Society;

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Schwartz,  Thomas
Emeritus Group Biophysics, Max Planck Institute for Medical Research, Max Planck Society;

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Geider,  Klaus
Research Group Prof. Dr. Geider, Max Planck Institute for Medical Research, Max Planck Society;

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https://academic.oup.com/nar/article-pdf/20/9/2265/3786545/20-9-2265.pdf
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https://doi.org/10.1093/nar/20.9.2265
(Any fulltext)

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Citation

Metzger, M., Bellemann, P., Schwartz, T., & Geider, K. (1992). Site-directed and transposon-mediated mutagenesis with pfd-plasmids by electroporation of Erwinia amylovora and Escherichia coli cells. Nucleic Acids Research (London), 20(9), 2265-2270. doi:10.1093/nar/20.9.2265.


Cite as: https://hdl.handle.net/11858/00-001M-0000-0019-AB81-5
Abstract
The suicide plasmid pfdA31-Tn5 was constructed to mutagenize Erwinia amylovora and Escherichia coli strains by electorporation. This vector carries the bacteriophage fd replication origin, a beta-lactamase gene and the transposon Tn5. For propagation the plasmid depends on host cells producing fd gene-2 protein. Electroporation of E.amylovora or E.coli cells with plasmid pfdA31-Tn5 yielded more than 10(4) transposition events per micrograms DNA. We have produced and characterized transposon mutants of E.amylovora affecting either galactose metabolism or the synthesis of the phytotoxin (L)-2,5-dihydrophenylalanine. A Tn5-insertion in a gene, involved in exopolysaccharide synthesis of E.amylovora strain Ea7/74, was subcloned into vector pfdA31 and used to mutagenize E.amylovora strain Ea1/79 by site-directed recombination.