Highly repressible expression system for cloning genes that specify potentially toxic proteins.
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.
D001425:Bacterial Outer Membrane Proteins / Q000096:biosynthesis / Q000235:genetics*; D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes / Q000235:genetics; D015246:Deoxyribonuclease EcoRI; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins; D005786:Gene Expression Regulation; D005798:Genes, Bacterial; D005822:Genetic Vectors; D007118:Immunoassay; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012313:RNA / Q000235:genetics; D016372:RNA, Antisense; D012329:RNA, Bacterial / Q000235:genetics; D012097:Repressor Proteins; D013696:Temperature; D014158:Transcription, Genetic
D001425:Bacterial Outer Membrane Proteins; D029968:Escherichia coli Proteins; D016372:RNA, Antisense; D012329:RNA, Bacterial; D012097:Repressor Proteins; C036297:TraT protein, E coli; D012313:RNA; D004262:DNA Restriction Enzymes; D015246:Deoxyribonuclease EcoRI