Lambda-TnphoA Mutagenesis of Cloned Genes in E. Coli

References:

• TnphoA – Manoil and Beckwith. PNAS. 82: 8129-8133. 1985;
• lambda-TnphoA – Gutierrez et. al.. J. Mol. Biol. 195: 289-287. 1987.

Method:

1. Before starting the mutagenesis protocol, ensure the plasmid you wish to mutagenize is in an appropriate E. coli background such as TB1 (C268 in our culture collection). Otherwise, you will only succeed in propagating phage!
2. Grow up your strain carrying your plasmid of interest in 5 ml of LB supplemented with 10 mM MgSO₄ and appropriate antibiotic selection to an O.D.₆₀₀ of 0.5 (approximately 2 x 10⁸ cells/ml).
3. To 1 ml of cells (2 x 10⁸ cells) add 2 x 10⁸ lambda-TnphoA phage particles (multiplicity of infection of 1).
4. Incubate at 30ºC for 15 minutes on a tube roller.
5. Set up 10 tubes containing 100 µl of infected culture with 1 ml of LB and antibiotic selection for the plasmid of interest (i.e. no kanamycin selection yet).
6. Incubate the tubes at 30ºC for 4 hours on a tube roller.
7. Dilute 50 µl of culture into 5 ml of LB with selection for plasmid and 50 µg/ml kanamycin (selection for transposon). Grow overnight at 30ºC.
8. Prepare a separate plasmid preparation from each sample using the alkaline lysis miniprep method. (Protocol in this manual.)
9. Use these plasmid preparations to transform E. coli CC118 (C494 in our culture collection). This strain lacks funtional alkaline phosphatase.
10. Plate transformants on LB containing selection for the plasmid, kanamycin and 20 µg/ml 5-bromo-4-chloro-3-indolyl phosphate (XP). XP is a substrate for alkaline phosphatase. Double resistant transformants represent successful transpositions onto plasmid DNA; blue double resistants are producing functional alkaline phosphatase.

Notes On Protocol:

Avoid using ampicillin for selection as complications may arise from synergistic and antagonistic effects of double selecting with ampicillin and kanamycin later on in the protocol. Otherwise, use 100 µg/ml ampicillin rather than 50 if it is necessary to use ampicillin selection. Stock solution of XP is prepared by dissolving the p-toluidine salt of XP in DMSO; store at -20ºC. XP is light sensitive so stocks and plates should be treated accordingly. Frequency of blue transformants is approximately 1 to 5%. Phage lambda-TnphoA is propagated on C493; working stocks of phage can be stored at 4ºC but reserve stock solutions should be stored at -70ºC. When performing colony immunoblots or Western blots of TnphoA mutants, use the horseradish peroxidase development system rather than the alkaline phosphatase system.