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Controlling basal expression in an inducible T7 expression system by blocking the target T7 promoter with lac repressor.
J Mol Biol. 1991 May 05; 219(1):45-59.JM

Abstract

Effects of placing a lac operator at different positions relative to a promoter for bacteriophage T7 RNA polymerase were tested. Transcription can be strongly repressed by lac repressor bound to an operator centered 15 base-pairs downstream from the RNA start, but T7 RNA polymerase initiates transcription very actively from this T7lac promoter-operator combination in the absence of repressor, or in the presence of repressor plus inducer. Sequence changes in the transcribed region were found to make transcription from some T7 promoters, including the T7lac promoter, more sensitive to inhibition by T7 lysozyme. The pET-10 and pET-11 series of plasmid vectors have been constructed to allow target genes to be placed under control of the T7lac promoter and to be expressed in BL21(DE3) or HMS174(DE3), which carry an inducible gene for T7 RNA polymerase. These vectors carry a lacI gene that provides enough lac repressor to repress both the T7lac promoter in the multicopy vectors and the chromosomal gene for T7 RNA polymerase, which is controlled by the lacUV5 promoter. Very low basal expression of target genes is achieved, but the usual high levels of expression are obtained upon induction. Addition of T7 lysozyme can reduce basal expression even further and still allow high levels of expression upon induction. Genes that are very toxic to Escherichia coli can be maintained and expressed in this system.

Authors+Show Affiliations

Biology Department, Brookhaven National Laboratory, Upton, NY 11973.No affiliation info available

Pub Type(s)

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

Language

eng

PubMed ID

1902522

Citation

Dubendorff, J W., and F W. Studier. "Controlling Basal Expression in an Inducible T7 Expression System By Blocking the Target T7 Promoter With Lac Repressor." Journal of Molecular Biology, vol. 219, no. 1, 1991, pp. 45-59.
Dubendorff JW, Studier FW. Controlling basal expression in an inducible T7 expression system by blocking the target T7 promoter with lac repressor. J Mol Biol. 1991;219(1):45-59.
Dubendorff, J. W., & Studier, F. W. (1991). Controlling basal expression in an inducible T7 expression system by blocking the target T7 promoter with lac repressor. Journal of Molecular Biology, 219(1), 45-59.
Dubendorff JW, Studier FW. Controlling Basal Expression in an Inducible T7 Expression System By Blocking the Target T7 Promoter With Lac Repressor. J Mol Biol. 1991 May 5;219(1):45-59. PubMed PMID: 1902522.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Controlling basal expression in an inducible T7 expression system by blocking the target T7 promoter with lac repressor. AU - Dubendorff,J W, AU - Studier,F W, PY - 1991/5/5/pubmed PY - 1991/5/5/medline PY - 1991/5/5/entrez SP - 45 EP - 59 JF - Journal of molecular biology JO - J. Mol. Biol. VL - 219 IS - 1 N2 - Effects of placing a lac operator at different positions relative to a promoter for bacteriophage T7 RNA polymerase were tested. Transcription can be strongly repressed by lac repressor bound to an operator centered 15 base-pairs downstream from the RNA start, but T7 RNA polymerase initiates transcription very actively from this T7lac promoter-operator combination in the absence of repressor, or in the presence of repressor plus inducer. Sequence changes in the transcribed region were found to make transcription from some T7 promoters, including the T7lac promoter, more sensitive to inhibition by T7 lysozyme. The pET-10 and pET-11 series of plasmid vectors have been constructed to allow target genes to be placed under control of the T7lac promoter and to be expressed in BL21(DE3) or HMS174(DE3), which carry an inducible gene for T7 RNA polymerase. These vectors carry a lacI gene that provides enough lac repressor to repress both the T7lac promoter in the multicopy vectors and the chromosomal gene for T7 RNA polymerase, which is controlled by the lacUV5 promoter. Very low basal expression of target genes is achieved, but the usual high levels of expression are obtained upon induction. Addition of T7 lysozyme can reduce basal expression even further and still allow high levels of expression upon induction. Genes that are very toxic to Escherichia coli can be maintained and expressed in this system. SN - 0022-2836 UR - https://www.unboundmedicine.com/medline/citation/1902522/Controlling_basal_expression_in_an_inducible_T7_expression_system_by_blocking_the_target_T7_promoter_with_lac_repressor_ L2 - https://linkinghub.elsevier.com/retrieve/pii/0022-2836(91)90856-2 DB - PRIME DP - Unbound Medicine ER -