I am trying to carry out gene deletion using P1 phage transduction in Ecoli W3110 Strain. Here I have successfully generated lysate from the donor strain with pfu 1.0E+9/ml via plate method. I am facing problems with the transduction events.
I take overnight culture and inoculate a secondary culture in LB with 5mM CaCl2 till O.D. 2.5, then distribute 0.5ml of culture in 6 tube and then add phage according to MOI 0.1, 0.5, 0.75, 1.0 and 5.0. After 30 mins of incubation (I also tried 20 mins incubation after reading some literature), I add 2ml of 1XAmedia : K2HPO4 1.05gm, KH2PO4 0.45gm, (NH4)2SO4 0.1gm and sodium citrate 0.05gm( I also tried precipitating phages using only 5mM Sodium Citrate). Then I plate it in antibiotic plate with 5mM sodium citrate.
While It sounds very easy to get transductants after reading literatures, I am struggling with this from last 4 months. Please suggest
Dear Sonali, I'm also performing P1 phage transduction currently. From your description it is not clear how you incubate your culture with the Phage. The protocol I am working is fine. I was deleting a gene with Kanamycin cassate from my E. coli strain DH12S. I will share my protocol as such, so you need to take 1.5 ml of Overnight grown preculture of your recepient cell and resupend in 500 uL of LB+CaCl2 (5 mM final concentration). You should prepare three 15 ml Falcon Tube. one with your 100 ul cell suspension+100 ul P1 phage lysate, second and third one is for control, one with oly 100 ul lysate+ 100 ul LB+CaCl2 and the last one is for Bacterial control, take only 100 ul of bacteria with 100 ul of LB+CaCl2. Incubate all three tubes at 37 degree for 30 minutes with out shaking, yes without shaking just to allow your phage to attach with bacteria and inject its DNA. After 30 minutes add 200 ul of Na-Citrate (5 mM final concentration) and incubate in same condition for another 1 hour. After that you can pellet the cells by centrifuging at 4000xg for 2 minutes and resuspend in 100 ul of LB+CaCl2. The spread the contents of each tube in LB+ Na-citrate (5 mM) + The selection antibiotic of your choice. Incubate this at 37 degree overnight. You should see colonies only in Lysate + Bacteria plate, then select single colony and plate in same kind of plate. You have to select several times (3 times at least) in Na citrate plate just to get rid off the phage. Hope this will help.
Hey Hi Mohammad, thank you so much for your valuable inputs. I incubate culture with phage under static condition but at room temperature, so as per your protocol I will try incubating at 37degC.
Two questions for you:
1. What pfu/ml of phage you take and what is the MOI you consider?
2. After Nacitrate addition, incubating at static condition for 1hr and after spinning the culture you suspend in LB+CaCl2? I thought its LB+ Nacitrate because we donot want phage to continue binding with the bacterial culture
Hello Sonali, I used 1010 pfu/ml P1 phage. You are right after incubation we don't want any further infection by phage. Thats why I used Na-cirtrate (5 mM) together with LB when incubating for 1 hour more at 37 degree C. So that all calcium will make complex with citrate. You can potentially use LB+Na-citrate or only LB or alternatively you can also pour all contents of the tube with out any resuspension step and incubate placing the cover on top (I think it is useful when you work with very low phage titre). But i never tried these option because my protocol worked fine so far. A short resuspension in LB+CaCl2 is not a big deal, furthermore in next step you plate in Na-citrate containing LB plates.
By the way, just one point to consider your recipient strain should be F' and recA positive because these are required for phage infection and homologous recombination. All the best.
Adding to Mohammad's point, the 1 hour incubation in LB+citrate after the initial transfection is important so that your strains can express marker. And mechanistically, I believe the citrate chelates the calcium so that it doesn't promote formation of receptors on the E. coli that p1 infects through. I typically perform this outgrowth step with good aeration (tape to bottom of 37 degree shaker).
Thank you guys for your valuable inputs. My phage titer is 1.0E+9 pfu/ml, I hope its not less.
Phage titre of 10E8 will work just perfect. So I think your phage titre is quite enough to perform the transduction. Cheers for your success.
Hey Sonali, I'm also performing the P1 transduction protocol currently. I didn't have any problems with it. This protocol (attached) worked perflectly for me. Hope you find it useful.
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