Escherichia coli plasmid vectors containing synthetic translational initiation sequences and ribosome binding sites fused with the lacZ gene.
The construction of a series of Escherichia coli plasmid vectors suitable for assaying the effects of gene control signals fused with the E. coli lacZ gene is reported. A synthetic deoxyoligonucleotide dodecamer 5'-CATGAATTCATG GTACTTAAGTAC-5' containing two translation initiation codons (ATG) separated by an EcoRI site was ligated with a lacZ gene derivative which lacks the codons for the first eight amino acids in plasmid pMC1403 (Casadaban et al., 1980). Two ribosome-binding sequences were synthesised and inserted into the EcoRI site before an ATG, and the effects of these sequences on lacZ gene expression in vivo measured by assaying beta-galactosidase activity. The E. coli ribosomal RNA gene (rrnB) promoter, the tetracycline resistance gene promoter, and a lambda phage promoter were cloned using these plasmids. The plasmids are 9.9 kb in size, have ampicillin resistance as a selectable marker and are generally useful for the detection and in vivo assay of gene control regions.
D001483:Base Sequence; D004262:DNA Restriction Enzymes; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005696:Galactosidases / Q000235:genetics*; D005796:Genes; D005798:Genes, Bacterial; D007763:Lac Operon; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis; D010442:Peptide Chain Initiation, Translational; D010957:Plasmids; D012270:Ribosomes / Q000378:metabolism*; D001616:beta-Galactosidase / Q000235:genetics*
D009838:Oligodeoxyribonucleotides; D004262:DNA Restriction Enzymes; D005696:Galactosidases; D001616:beta-Galactosidase