Kit 10

Displacement of pSLT from Salmonella typhimurium

pKZ1 is an F-mobilizable, non-conjugative plasmid in the same compatibility group as the virulence plasmid of S. typhimurium, pSLT (Sanderson et al., 1983, Can. J. Microbiol.).

Plasmid pLL6 was derived from pKZ1 (mediating kanamycin resistance) by mutagenesis with hydroxylamine, and is temperature sensitive (42 degrees C) for replication/segregation (R. Kelln, and Lintott LG., 1990. Mol Gen Genet. Jul;222(2-3):438-40.). The temperature effect is not 100% in that some colonies still retain Km(R) following cycling at 42 degrees C. The plasmid is approximately 55 kbp and therefore few Km(R) transformants are typically obtained, but it is possible (at least with galE recipients). Plasmid pKZ1, and hence pLL6, are non-conjugative but can be mobilized to a recipient by F factor, and this allows for an alternative means of transfer. Both pKZ1 and pLL6 are incompatible with pSLT but the incompatibility is weak in that heteroplasmid strains predominate upon initial transfer (at least if transformation is used).

Procedure:

1. Introduce pLL6 to recipient at 30 degrees C by either conjugation or transformation, selecting Km(R) (30 ug/ml). For transfer by conjugation mix KR1561 plus an Flac(+) strain (SA3269) with the recipient, select for Km(R) strain.

2. Choose a single colony isolate, and inoculate 1-2 ml of broth (Luria) containing Km (30 ug/ml). Culture at 30 degrees C until visible growth and then use a sterile wire to inoculate a second liquid culture. Incubate at 30 degrees C and streak for isolated colonies on an appropriate (e.g. Luria) medium.

3. Choose 8 to 10 colonies and carry a small scale rapid lysis procedure. Also include two control strains; the recipient and the recipient containing pLL6.

4. Digest the rapid lysates with EcoRI; if pSLT has been displaced, a unique characteristic band at approximately 4 kbp will not be present.

5. Choose an isolate devoid of pSLT and inoculate a broth streaking for isolates at 42 degrees C Score the isolates for loss of the Km(R) phenotype.

6. Do a rapid lysate on a Km(S) isolate to ensure that no plasmid DNA is present.

(The above controls do not check for the possibility that pSLT has integrated into the chromosome. This is expected to be an infrequent event.)

The plasmid pLL6 was constructed by R.Kelln and additional information can be obtained from Kelln and Lintott, 1990 as noted above.

Strains Included in Kit 10:

Strain

Number

SGSC

Number

Genotype

Phenotype

KR1561

1532

met rpsL xyl galE hsr H1-b H2-e,n,x val / pLL6 (derived from pKZ1, mediating kanamycin resistance, by mutagenesis with hydroxylamine, and is temperature sensitive (42C) for replication/segragation)

Met(-); Str(R); Xyl(-); Hsr(-); H1(-); H2(-); Val(-) / pLL6 (displaces pSLT), Kan(R).

NOTE - GROW AT 30C.

SA3269

-

hisF1009 trpC2 metA22 xylR1 rpsL201 rfaJ971 / F'lac

His(-); Trp(-); Met(-); Xyl(-); Rfa(-); Lac(+)

 

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