Controlling basal expression in an inducible T7 expression system by blocking the target T7 promoter with lac repressor.
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.
Dubendorff
J W
JW
Biology Department, Brookhaven National Laboratory, Upton, NY 11973.
Studier
F W
FW
D001483:Base Sequence; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D015967:Gene Expression Regulation, Viral; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D010957:Plasmids; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis; D012367:RNA, Viral / Q000235:genetics; D012097:Repressor Proteins / Q000235:genetics* / Q000378:metabolism; D015183:Restriction Mapping; D013604:T-Phages / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D015345:Oligonucleotide Probes; D012367:RNA, Viral; D012097:Repressor Proteins; D012321:DNA-Directed RNA Polymerases; D001616:beta-Galactosidase