Phasmid vectors for identification of genes by complementation of Escherichia coli mutants.
A bacteriophage lambda cloning vector was designed to facilitate the isolation of genes from procaryotic organisms by complementation of Escherichia coli mutants. This vector, lambda SE4, was constructed by attaching a very-low-copy-number replication system (from the plasmid NR1) and a spectinomycin resistance gene to the left arm of lambda 1059 (Karn et al., Proc. Natl. Acad. Sci. U.S.A. 77:5172-5176, 1980). This phasmid cloning vector is capable of growing lytically as a phage in a nonimmune host or lysogenically as a phasmid in an immune host. This phasmid utilizes the Spi- selection for insertions of DNA into the vector and has the ability to accept 2- to 19-kilobase Sau3A1, BamHI, BglII, BclI, or XhoII fragments; recombinants lysogenize immune hosts as single-copy-number selectable plasmids at 100% frequency. An E. coli library was constructed by using the initial vector lambda SE4, and clones of a number of representative genes were identified. A typical clone, lambda ant+, was shown to be readily mutagenized by a mini-Tn10 transposon. A general method for transferring cloned DNA segments onto bacteriophage lambda was developed. The method involves the use of in vivo recombination with a selection and was used to construct two derivatives of lambda SE4. Possible uses of these vectors and of the method for transferring cloned DNA onto phage lambda are discussed.
Elledge
S J
SJ
Walker
G C
GC
D010582:Bacteriophage lambda / Q000235:genetics*; D004251:DNA Transposable Elements; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D005818:Genetic Engineering / Q000379:methods; D005822:Genetic Vectors; D009154:Mutation; D010957:Plasmids; D011815:R Factors; D000198:Spectinomycin / Q000633:toxicity
D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D000198:Spectinomycin