Kit 9B

Transduction in E. coli with Salmonella Phage P22

 

The laboratory of Peter Reeves (Dept of Microbiology, University of Sydney, Australia) has constructed a system which allows the propagation of P22 in E. coli. This allows the use of P22 for transduction in E. coli, and potentially in other systems. The work is reported in the following manuscript:

P22 has a number of advantages over P1, including higher frequency of transduction, and greater stability of the phage stocks.

The strains required for use in this technique comprise P4729 and P4730, both of which carry pPR1347 (a pSC101-derived plasmid which carries the genes rfb and rfc from S. typhimurium which allows E. coli K-12 to express the lipopolysaccharide side chain which is the receptor for P22). P4729 is based on the restriction-minus strain JM109, and is suitable for preparation of E. coli K-12 modified stocks of P22, and also for the many P22 derivatives available. P4730, a derivative of the K-12 strain SMR10, is a lysogen for a temperature-inducible lambda phage which lacks a cos site; on induction the cosmid pPR1347 is packaged in vivo and the lysate can be used to transduce pPR1347 to any strain with the lam receptor. pPR1347 is a large plasmid and this means of transfer can be advantageous. Transfer of pPR1347 and selection for Km(R) gives recombinants which express the LPS of S. typhimurium, and will form plaques with P22 phage. Phage propagated on E. coli can be used to transduce other E. coli strains. Transductants are obtained, though they are not as frequent as expected, only 10(-7) to 10(-8).

For more information, see Neal et al.

Strains* Included in Kit 9B:

Strain

Number

SGSC

Number

Genotype

Phenotype

P4729

2442

recA1 endA1 gyrA96 thi hsdR17 supE44 relA1 lambda(-) (del)(lac-proAB)/pPR1347 (rfb+rfc of Salmonella group B, 43 Kb)

Rec(-); End(-); Gyr(-); Thi(-); restriction deficient; SupE(+); Rel(-); Lambda(-); Lac(-); Tra(-); Km(R), P22 (Sens)

P4730

2443

lambda packaging strain for pPR1347 (rfb+rfc of Salmonella group B, 43 Kb)

Km(R), P22 (Sens)

**Note: Grow at 30 degrees.

* If required, P22 phage can also be supplied by the SGSC.

 

Transfer of cosmid pPR1347 into other strains

1. Transfer can be done by electroporation as described by Neal et al (1993, J. Bacteriol. 175:7115). The frequency of transfer by this method is not high, because the cosmid is large (43 Kb).

2. Transfer can also be achieved by F-factor mediated conduction. This was done by Andrew Hessel and Neal Mattatall in the lab of K. Sanderson. The strain SAB4793 was constructed by electroporation of pPR1347 into a strain of the following genotype: del lacX74 trp(-) l (+)/F42finp301 lac(+). This strain is able to act as a donor of Flac(+), and will also transfer the cosmid. To obtain transconjugants, mix cells of SAB4793 with cells of the desired recipient, and select for Trp(+)Km(R).

 

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