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Tightly regulated vectors for the cloning and expression of toxic genes.
J Microbiol Methods. 2004 Aug; 58(2):243-50.JM

Abstract

A series of low-copy expression vectors that permits the stable maintenance and regulated expression of highly toxic gene products has been developed. These vectors utilize the lactose promoter/operator system, and protect against read-through transcription from other promoters on the plasmid by placement of the rrnB T1T2 terminators upstream of the lactose promoter. For additional regulatory control, the vectors utilize low-copy origins of replication. Either the pMPP6 origin (pSC101-derived) is used for cloning into Escherichia coli or related species, or the broad-host-range RK2 origin of replication is utilized for cloning into the majority of Gram-negative bacteria. The resulting plasmids have no detectable leaky expression. To test these vectors, the genes for the bacteriocidal colicins D, E3, and E7 were cloned and stably maintained in the absence of their immunity genes. Upon induction with isopropyl-beta-D-thiogalactopyranoside (IPTG), cell death was observed, indicating expression of each colicin. These low-copy expression vectors will be useful for the cloning and expression of toxic genes in bacterial systems.

Authors+Show Affiliations

ConjuGon, University Research Park, 505 South Rosa Road, Suite 29, Madison, WI 53719, USA. lanthony@conjugon.comNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, U.S. Gov't, Non-P.H.S.

Language

eng

PubMed ID

15234522

Citation

Anthony, Larry C., et al. "Tightly Regulated Vectors for the Cloning and Expression of Toxic Genes." Journal of Microbiological Methods, vol. 58, no. 2, 2004, pp. 243-50.
Anthony LC, Suzuki H, Filutowicz M. Tightly regulated vectors for the cloning and expression of toxic genes. J Microbiol Methods. 2004;58(2):243-50.
Anthony, L. C., Suzuki, H., & Filutowicz, M. (2004). Tightly regulated vectors for the cloning and expression of toxic genes. Journal of Microbiological Methods, 58(2), 243-50.
Anthony LC, Suzuki H, Filutowicz M. Tightly Regulated Vectors for the Cloning and Expression of Toxic Genes. J Microbiol Methods. 2004;58(2):243-50. PubMed PMID: 15234522.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Tightly regulated vectors for the cloning and expression of toxic genes. AU - Anthony,Larry C, AU - Suzuki,Hideki, AU - Filutowicz,Marcin, PY - 2004/01/14/received PY - 2004/03/30/revised PY - 2004/04/06/accepted PY - 2004/7/6/pubmed PY - 2004/9/21/medline PY - 2004/7/6/entrez SP - 243 EP - 50 JF - Journal of microbiological methods JO - J. Microbiol. Methods VL - 58 IS - 2 N2 - A series of low-copy expression vectors that permits the stable maintenance and regulated expression of highly toxic gene products has been developed. These vectors utilize the lactose promoter/operator system, and protect against read-through transcription from other promoters on the plasmid by placement of the rrnB T1T2 terminators upstream of the lactose promoter. For additional regulatory control, the vectors utilize low-copy origins of replication. Either the pMPP6 origin (pSC101-derived) is used for cloning into Escherichia coli or related species, or the broad-host-range RK2 origin of replication is utilized for cloning into the majority of Gram-negative bacteria. The resulting plasmids have no detectable leaky expression. To test these vectors, the genes for the bacteriocidal colicins D, E3, and E7 were cloned and stably maintained in the absence of their immunity genes. Upon induction with isopropyl-beta-D-thiogalactopyranoside (IPTG), cell death was observed, indicating expression of each colicin. These low-copy expression vectors will be useful for the cloning and expression of toxic genes in bacterial systems. SN - 0167-7012 UR - https://www.unboundmedicine.com/medline/citation/15234522/Tightly_regulated_vectors_for_the_cloning_and_expression_of_toxic_genes_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0167701204000958 DB - PRIME DP - Unbound Medicine ER -