Highly repressible expression system for cloning genes that specify potentially toxic proteins
Open Access
- 30 September 1987
- journal article
- research article
- Published by American Society for Microbiology in Journal of Bacteriology
- Vol. 169 (10), 4457-4462
- https://doi.org/10.1128/jb.169.10.4457-4462.1987
Abstract
A highly repressible expression vector system that allows the cloning of potentially deleterious genes has been constructed. Undesired expression of a cloned gene was prevented (i) at the level of initiation of transcription, by the presence of the strong but highly repressible leftward promoter of bacteriophage lambda, lambda pL, and (ii) at the level of transcript elongation or translation, through synthesis of antisense RNA complementary to the mRNA of the cloned gene. The system was tested by measuring the inhibition of expression of traT, the gene for the TraT major outer membrane lipoprotein. Direct detection and functional assays indicated that an essentially complete inhibition of traT expression was obtained. As a further test of the system, the gene encoding the EcoRI restriction endonuclease was cloned in the absence of the gene of the corresponding protective EcoRI modification methylase. Transformants harboring this construct were only viable when both repression controls were operational.This publication has 40 references indexed in Scilit:
- Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mpl8 and pUC19 vectorsGene, 1985
- Inhibition of thymidine kinase gene expression by anti-sense RNA: A molecular approach to genetic analysisCell, 1984
- Construction of improved M13 vectors using oligodeoxynucleotide-directed mutagenesisGene, 1983
- Translational control of IS10 transpositionCell, 1983
- Expression of the EcoRI restriction-modification system and the construction of positive-selection cloning vectorsGene, 1982
- The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primersGene, 1982
- Convergent transcription in bacteriophage λ: Interference with gene expressionJournal of Molecular Biology, 1979
- Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.Proceedings of the National Academy of Sciences, 1979
- Construction of plasmid cloning vehicles that promote gene expression from the bacteriophage lambda promoterGene, 1979
- Cloned seventeen-nucleotide-long synthetic lactose operator is biologically activeGene, 1978