Waldminghaus:Back Door/P1 transduction
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(New page: =P1 transduction= Generalized transducing phage such as P1 package almost randomly cuted segments of the bacterial chromosome into phage particles and inject them into recipient strains. ...)
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Revision as of 08:59, 11 October 2012
Generalized transducing phage such as P1 package almost randomly cuted segments of the bacterial chromosome into phage particles and inject them into recipient strains. P1 transduction is very useful for strain construction and for mapping within intervals smaller than 2 minutes on the E. coli chromosome. To avoid making lysogens of recipient strains it is common to use virulent derivatives of P1 ( p1 vir ), which cannot lysogenize infected cells. To avoid excessive killing of the transductants by reinfection with phage release into the medium, citrate is used to complex the calcium ions required for phage adsorption. P1 is an excellent method for mapping close markers. The probability of two loci being incorporated into the same particle decreases as the distance between te loci increases.
P1 plate lysates
- Prepare 5 ml tubes with 2.5 ml soft agar, containing CaCl2 ( 15 mM ) for each dilution and keep them at 43°C
- To 1 ml of a fresh donor culture grown in LB overnight add CaCl2 to 15 mM ( 15 µl 1M CaCl2)
- Dilute a lysate of p1 vir 102, 103, 104, 105 times in LB in small test tubes to 100 µl.
- Incubate 100 µl of each dilutions at 37°C for 20 min in the hood with open tube lid to evaporate traces of CHCl3 ( P1 lysates contain CHCl3 )
- Add 100 µl donor bacteria to each dilution and incubate the tubes 25 min at 37°C to allow absorbtion of phages.
- Mix the phages-bacteria composite with soft Agar and plate on LB plates at 37°C. Incubate plates face up overnight. The plates should be at the most a day old in order to prevent the soft agar from drying out. If lysates are plated in the morning they can be harvested after 8 hours.
- Harvest the plate that gives the best lysis ( confluent lysis ) by scraping the soft agar into a 50 ml centrifuge tube using a sterile cover glass. Rinse plate in 2 ml LB containing 25 mM MgCl2. Add rinsing liquid to the centrifuge tube together with 5 drops of CHCl3. Homogenize by vortexing. Incubate for 30 min at room temperature.
- Centrifuge 10 min at 5000 rpm
- Transfer supernatant to Eppendorf tube and add 2 drops of CHCl3.
- Store the P1 lysat at 4°C
- Dilute P1 lysate 100,101, 102, 103 times in LB in small test tubes.( 10 µl lysate + 90 µl LB )*Incubate 100 µl of each dilution at 37°C for 20min
- Add 100 µl of a fresh overnight recipient culture supplemented with CaCl2 to 15 mM to each tube.
- Incubate 20 min at 37°C
- Add 1 ml LB with 50 mM Na-Citrate
- Spin down 1 min 14000 rpm, discard supernatant and resuspend in 1ml LB with 50mM Na-Citrate
- Culture cells at 37°C for 1 hour while shaking , to allow expression of resistance marker.
- Spin down 1 min 14000 rpm , discard supernatant and resuspend in 100 µl LB
- Plate on selective plates. Remember to include controls. One plate with only recipient cells ( 100 µl ) and one with only the lysate ( 100 µl ). Incubate plates overnight at 37°C.
You shoud get anywhere from 10 to 2000 colonies. These colonies are growing on a plate that is covered wirh P1 phages. If you simply pick a colony from this plate and prepare a freezerstock, you will most likely have phage contamination that will manifest when a culture is crown up in the absence of calcium chelator. Therefore, prepare a plate spread with the selection antibiotic and 100µl of 100mM citrate (pH 5,5). Then, use toothpick to touch the top of a few colonies and re-streak on the new plate for isolated colonies.