Use of the rep technique for allele replacement to construct new Escherichia coli hosts for maintenance of R6K gamma origin plasmids at different copy numbers.
Escherichia coli hosts were constructed for maintenance of vectors containing the gamma replication origin of the R6K plasmid (oriRR6K gamma) at different copy numbers (15 or 250/cell). Such vectors require the trans-acting II protein (the pir gene product) for replication. New hosts carry pir+ or pir-116 on the chromosome within uidA, the E. coli gene encoding beta-glucuronidase. They were made using the rep technique for allele replacement and KmR M13 delta uid A::pir+ or M13 delta uidA::pir-116 phage. Because M13 cannot replicate in a rep mutant, KmR transductants arose by integration into the chromosomal uidA locus. Segregants lacking M13 sequences (which were selected as deoxycholate-resistant (DocR) ones) frequently contained delta uidA::pir+ or delta uidA::pir-116 on the chromosome. In principle, this procedure could be used for the introduction of any foreign gene into any nonessential gene on the E. coli chromosome. The delta uidA::pir+ and delta uidA::pir-116 loci were subsequently transferred to a variety of E. coli strains. One such strain is a suppressor-negative one that is especially useful for transposon (Tn) mutagenesis. This strain has an integrated RP4 derivative for conjugative transfer of oriRR6K gamma plasmids also containing oriT from RP4. In addition, new oriRR6K gamma, oriT+ vectors carrying the TcR-encoding genes tetAR from Tn10 are described. These can be used for allele replacement by conjugative transfer of an oriRR6K gamma, oriT+, tetAR plasmid containing a mutated gene into a non-pir recipient and by subsequent selection for Tc-sensitive exconjugants.
Metcalf
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Department of Biological Sciences, Purdue University, West Lafayette, IN 47907.
Jiang
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Wanner
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D000483:Alleles; D017104:Bacteriophage M13 / Q000235:genetics; D001483:Base Sequence; D002876:Chromosomes, Bacterial; D003001:Cloning, Molecular / Q000379:methods*; D003227:Conjugation, Genetic; D004265:DNA Helicases; D017931:DNA Primers; D004261:DNA Replication; D004268:DNA-Binding Proteins; D004926:Escherichia coli / Q000235:genetics*; D005798:Genes, Bacterial; D005822:Genetic Vectors; D005838:Genotype; D005966:Glucuronidase / Q000235:genetics; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D010448:Peptide Initiation Factors / Q000235:genetics / Q000378:metabolism; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods; D015183:Restriction Mapping; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism; D014161:Transduction, Genetic
D017931:DNA Primers; D004268:DNA-Binding Proteins; D010448:Peptide Initiation Factors; D015534:Trans-Activators; C045264:replication initiator protein; D005966:Glucuronidase; D004265:DNA Helicases