Kit 8C

l 1059 Library of Salmonella DNA

The Vector:

The vector l 1059 was constructed by Karn et al, 1980 (Proc. Natl. Acad. Sci. US 71:5172). This is a vector for large fragments which can be inserted into the BamHI site. Replacement of the "stuffer" region (which contains the red and gam genes) with foreign DNA causes the phenotype of the phage to change from Spi(+) (unable to grow on a P2 lysogen) to Spi(-) (able to grow on a P2 lysogen). The presence of the pBR322 origin of DNA replication allows the vector to be propagated as a plasmid (phasmid) in l lysogens. However, the presence of this pBR322 sequence can make it difficult to screen libraries by using as probes fragments derived from recombinant plasmids. Even after gel purification, such fragments contain pBR322 sequence that hybridize to plaques of non-recombinant phages (Maniatis et al, 1982, Molecular Cloning, Cold Spring Harbor, p.43).

Construction of the Library:

R. Maurer (Dept of Molecular Biology and Microbiology, Case Western Reserve Univ., Cleveland, Ohio) constructed a Sau3A partial-digest library of S. typhimurium DNA in l 1059 following the procedures of Karn et al (1980). The construction of this library is described in Maurer et al (1984, Genetics 108:1-23) and was as follows.

Bacterial DNA was isolated from strain S. typhimurium LT2 DB9005, Sau3A digested, fractionated to collect 15Kb fragments, ligated into BamHI digested l 1059, packaged in vitro, and 5 portions of the packaged mixture, each containing 5000 recombinants, were amplified separately on strain DB4740. This procedure ensured that overlapping clones isolated from the different amplified portions were of independent origin.

Maintenance of the Library in the SGSC:

In 1982 l 1059 phage from the 5 separate pools containing Salmonella DNA was provided by Dr. Maurer to the Salmonella Genetic Stock Centre. Each pool was independently propagated on E. coli LE392 by the confluent lysis method in a soft agar overlay plate on L-agar, and the phage were suspended in LB broth. Four of the five pools were thus propagated, all to titers of approximately 10(10)pfu/ml.

Pools Available from the SGSC:

Pool 1, B130; Pool 2, B131; Pool 3, B126; Pool 5, B127.

Success in Isolation of Salmonella Genes from the Library:

1. Maurer et al. (1984) used a complementation system to isolate l 1059 clones which complement strains of E. coli or of S. typhimurium which have mutations in the dna genes. (Derivatives of S. typhimurium TS736, made l -sensitive by addition of the E. coli lamB gene, were used.)

2. Sirisena, D.M. et al . (1992, J. Biol. Chem.,267(26):18874-84) have isolated Salmonella genes from the l 1059 library by probing the library with P(32)- labelled Salmonella DNA probes. These genes were isolated from the pools which were amplified in the SGSC.

 

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