KIT 5D

TnphoA Delivery Systems

The use of pRT733 for delivery of TnphoA into gram-negative bacteria.

TnphoA is a derivative of Tn5 which makes gene fusios to the gene for E. coli alkaline phosphatase upon insertion into a target gene. This allows one to obtain gene fusions specifically to secreted proteins since these are the only targets that give phoA(+) gene fusions. TnphoA was developed by Colin Manoil and John Beckwith.

This plasmid is a suicide vector since the oriR6K is functional only in the presence of the pir gene (which is suplemented by lambda-pir prophage). pRT733 is provided in SM10 carrying this prophage. Mobilization of pRT733 into various gram-negative species has given mixed results. Km (R) colonies which are Ap(S) are transpositon events. However, depending upon the organism, we have also seen Km(R) Ap(R) colonies. We believe these are cointegrates but other possibilities exist. If you are lucky your transconjugates will be resistant to only Km.

Strains:

 Strain #

 SGSC #

 Genotype

 Phenotype
 RT733  1379 SM10 (lambda-pir(+))/ pRT733 (oriR6L, Tra(-), Mob(+), Amp(R), Kan(R) The plasmid pRT733 is mobilized into gram-negative species. It is a suicide vector, able to replicate only in the presence of pir(+)
 TA2361  167 PhoN2 Pho (-), non-specific acid phosphatase
 TA2362  168  PhoP12  Pho (-), non-specific acid phosphatase

References:

Hoffmann, C. and Wright, A.. 1985. PNAS 82: 5107-5111

Manoil, C. and Beckwith, J. 1985. PNAS 82: 8129-8133

Taylor et. al.1987. PNAS 84: 2833-2837

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