Kit 1B

Transposons Used to Insert Specific Mutations

Some strains in this transposon kit carry mutations which are linked by P22 transduction to the transposon such as Tn10 which has a selectable marker; thus this strain can be used to transfer the mutation into new backgrounds. There are many such examples, including strains which can be used to transfer galE or recA alleles.

Insertion of galactose epimerase, galE(-) mutations into strains:

A) By genetic transfer of an existing mutation

1. Strain TN1117 (SGSC647) with genotype galE(-), zbi-812::Tn10; is used as transduction donor, 90% of Tc(R) transductions should be Gal(-), zbi-812::Tn10.

2. (i) Strain TA3426 (SGSC264) with genotype proC90, hspL, hspS, hisD2253, dhuA1, purF145, bio-203::Tn10, galE503; is used as transduction donor, Tc(R) transductants should all be Bio(-). (ii) Those Bio(-) recombinants are used as recipients with P22 phage grown on SL1306 (galE503), Bio(+) transductants are selected, all of these should be Tc(S) since Tn10 is removed and part should be galE503, because of galE-bio.

B) By selection of a new GalE(-) mutation

1. From a smooth strain which is sensitive to phage Felix-O (FO) select FO-resistance. 10-20% of these will be Gal(-); part of these will be switchable to smooth phenotype by growth in 1% glucose plus 1% galactose.

2. Selection for resistance to 2-deoxygalactose (Alper and Ames. 1975. J. Bacteriol. 122: 1081-1090).

 

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