期刊
PLASMID
卷 59, 期 3, 页码 193-201出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.plasmid.2008.01.004
关键词
copy number mutants; gene expression; plasmid incompatibility
资金
- NIGMS NIH HHS [R01 GM069628, R01 GM069628-02, R01 GM069628-03] Funding Source: Medline
Mutations that increase the copy number of the pSC101 replicon have been used for construction of new cloning vectors. Replacement of glutamate at position 93 in RepA yields plasmids that replicate at medium (27 copies/cell) and high (similar to 240 copies/cell) copy numbers. Based on the crystal structure of RepE, a structurally similar replication initiator protein from the F factor, the pSC101 repA mutants are predicted to be defective in dimerization. The cloning vectors permit increased expression of gene products along with the advantages of pSC101-derivative plasmids, including stable maintenance and compatibility with ColE1 plasmids. The plasmids also allow blue/white screening for DNA inserts and impart resistance to ampicillin, chloramphenicol and kanamycin. The vectors were used in a genetic assay to suppress temperature-sensitive mutants of ffh, encoding the protein component of the Escherichia coli signal recognition particle, by overproduction of 4.5S RNA. While expression of 4.5S RNA from a wild type pSC101-derivative plasmid was not sufficient for suppression, use of the new vectors did suppress the temperature-sensitive phenotype. (c) 2008 Elsevier Inc. All rights reserved.
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