KIT 5C

Mudlac (MudJ) Delivery Systems

Material provided by John Roth, University of Utah. This complementation method uses P22 to transduce a defective Mu element which has the operon fusion lacZ reporter gene (Hughes, K.T., Roth, J.E. 1988. Genetics 119:9-12.) It was developed to permit simple isolation of single mutants due to insertion of defective elements. The method as described can be applied to any strain sensitive to infection by phage P22 and does not require removal of the new insertion from the strain of origin.

The Mud sequences in the double mutants in this strain kit are such that when the transducing phage P22 is grown on the double mutant, a single virion can package the entire MudJ (or MudK) element present in the hisD gene and the transposition functions of the nearby Mud I helper prophage. P22 packages 44 kb of chromosome. MudI is 37 kb in length while the MudJ and MudK prophages are 11.3 and 9.7 kb in length, respectively. Roughly 4 kb of his sequence (including the hisH, B and C genes) lies between the two insertion sites. Thus, P22 cannot transduce both inseted elements simultaneously, but transduced fragments that carry MudJ or K frequently include the transposition functions at the end of the nearby MudI element.

Strains:

 Strain #

 SGSC #

Species

 Genotype

 Phenotype
 TT10288  1603 Salmonella typhimurium LT2 hisD9953::MudJ hisA9944::Mud1 HisD(-), Kan(R), Lac(+), HisA(-), Amp(R), gives 90% hops
 TT10381  1604 Salmonella typhimurium LT2 hisD1284::MudK hisA9944::Mud1 HisD(-), Kan(R), Lac(+), HisA(-), Amp (R)

Method:

General transducing lysates are grown on the double mutants described above. These lysates were used to transduce a wild-type recipient strain selecting for inheritance of kanamycin resistance. This selection demands inheritance of MudJ or K, either by transpositon or by recombination into the chromomsome. Transposition transductants arise only if the helper MudI prophage is present in the donor; no such transposition types arise if the donor carried only the defective hisD::MudJ or K mutation. These transposition types include 4-5% auxotrophs, or many different types and appear to be randomly distributed.

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