I've had difficulty producing a lysate from a strain of E. coli in which I have engineered a marked allele, for transfer to another strain.
First, some background on the experiment:
I have used homologous recombination from a linear PCR product to engineer an allele with a chloramphenicol selection marker (cat) on the chromosome of an E. coli W3110 subtype. The homologous recombination system that I used was λ-Red, which is encoded by a defective prophage and expressed from the cI857 temperature-inducible promoter.
The prophage is harbored on the genome of E. coli DY378 (developed by Yu et al., An efficient recombination system for chromosome engineering in Escherichia coli, PNAS 2000), which is a subtype of W3110 whose genotype is E. coli W3110 [λ cI857 Δ(cro-bioA)]. The inserted allele was targeted as a knock-in knock-out (KIKO) at the lacZ locus in the same strain, so that I could visually confirm the insertion by blue-white screening. The recombination was successful, and was confirmed by PCR fragment-analysis (to interrogate the new junctions formed by the KIKO) and Sanger sequencing. I did this twice, producing two different strains with different engineered alleles. The engineered strains have been used successfully in Illumina NGS.
I would now like to move the engineered alleles into new strains for further experiments, and here I run into a problem: I can't get lysates. I grew my engineered strains, added P1:vir, and saw no decrease in turbidity or development of clumped cellular debris in infected cultures relative to an uninfected negative-control culture. I checked at 3 hours, and after overnight growth.
My first thought was that the presence of the λ-prophage may be inhibiting P1:vir insertion. The defective prophage has had its attachment sites (attL and attR) removed, which would be a problem if P1 uses the same attachment site as λ, but I don't think that is the case. I could do another round of recombination or attempt P1 transduction from W3110 and screen for bio+ (DY378 is bio-) by growing on M9 minimal media to remove the prophage, but this shouldn't be necessary. I quote from Standard Protocols in Molecular Biology: Recombineering:
"For the Court laboratory’s phage-based systems, after the desired construct is obtained, the defective prophage carrying the recombination genes can be removed, either by recombination as described in Basic Protocol 2 or by P1 transduction (UNIT 1.17), using a non-lysogenic donor, selecting for growth in the absence of biotin (Bio+). Alternatively, engineered alleles on the chromosome can be moved into a different host by P1 transduction (UNIT 1.17), provided there is a selection for them [emphasis added]."
The problem may simply lie with my P1:vir source, or with my protocol. I obtained P1:vir from Yale's Coli Genetic Stock Center (CGSC). It was stored at 4°C for one month before use. I understand that even if stored over CHCl3, phage stocks take several months to depreciate in titer. I hope there is just a problem in my procedure somewhere. Here it is:
P1 Transduction
Preparing the lysate
Any and all advice would be greatly appreciated!
Brendan -
It seems as if the protocol you're following is appropriate for preparing P1 transducing lysates. I do have a few comments based on your experience and the suggestions of several contributors above.
I am certain that there are several labs at Michigan that have P1vir stocks. Since it is so cheap and easy to propagate phage, it should be no problem to procure an aliquot from an E. coli lab on campus (Matt Chapman's lab in MCDB may be a good place to start).
You're welcome to contact me if you have more questions - I've probably done thousands of P1 transductions in my career and I'm happy to help you troubleshoot as you get your feet wet!
Everything seems fine with your procedure (superficially looking at it). It has been over 3 decades since I did such things but I do recall issues with temperatures. In some cases I used to bring the temp above 37ºC to achieve better yield. Perhaps you should try at least 35ºC. If toxicity is observed at 42ºC, you may be fine at 35 or perhaps even at 37ºC. You should be able to distinguish between lysis due to P1 and Lamda-Red.
I suggest to use a P1 vir, a mutant with enhanced transduction rate. Make sure that you have P1 vir, some time there is some kind of reversion to the Wild-tipe. Additionally, if you perform some passage first with the donor strain to make sure that the infection rate is OK, you should increase the efficiency too. Do no leave the stock with chloroform because phages will decay as time goes by. You can filtrate the lysate through 0.45 um filter in sterile conditions instead. Good luck!!
As mentionned by Patrick and Alexandro, I strongly recommend to get a new P1vir stock. Phages can lose their infectivity over time, especially when the preparation was done with chloroform. Your P1vir phage should kill your bacterial cells in regular medium.
Brendan -
It seems as if the protocol you're following is appropriate for preparing P1 transducing lysates. I do have a few comments based on your experience and the suggestions of several contributors above.
I am certain that there are several labs at Michigan that have P1vir stocks. Since it is so cheap and easy to propagate phage, it should be no problem to procure an aliquot from an E. coli lab on campus (Matt Chapman's lab in MCDB may be a good place to start).
You're welcome to contact me if you have more questions - I've probably done thousands of P1 transductions in my career and I'm happy to help you troubleshoot as you get your feet wet!
Hi all,
Good to read all this and see P1 transduction remains a useful genetic tool! I agree that the problem is probably your starting P1 lysate and that you should try to get a new one (P1-vir). Better check then that the phage plates on your strain by spotting drops of 100 fold dilutions of the lystae on a lawn of your strain (poured in soft agar) and a control strain (one that's known to be P1 sensitive) on LBagar plates.
I'm somewhat surprised that everyone grows now P1 lysates in liquid. We used to do it on plates, from a single plaque resuspended in 0.2 ml of an overnight culture of the strain of interest. After adding 5ml of LB, this was separated into 5 x1 ml aliquots, each of which was plated with 2 ml of 0.7% soft agar on fresh LB plates and incubated at 37C (though 30 is OK too) and watching for confluent lysis to occur (6-8 hrs). The soft agar layer is then scraped into a centrifuge tube to pellet the agar and the bacteria, sterilized with chloroform and recentrifuged before titration. I think it is important to know how much phage yo have in the lysate so that you can adjust the M.O.I not only for transduction but also for growing the lysate in liquid if you choose to do it that way. Hope you're successful soon! Ariane.
Thank you all for your thoughtful responses! They have been quite helpful in troubleshooting my transduction problems. I obtained a new P1:vir stock from a lab at U-M, and grew it in E. coli MG1655. I reduced the initial incubation time to 30 minutes, added 100 μL lysate, and incubated for 3.5 hours. I saw substantial reduction in turbidity relative to an phage-free control.
Next, I added chloroform, vortexed, pelleted cellular debris, and collected the supernatant. Rather than storing over chloroform I would like to filter-sterilize the lysate. We have MCE, CA, and nylon filters in 0.22-μm and 0.45-μm. I've read several protocols that recommend 0.45-μm (including "Standard Protocols in Molecular Biology"), but these protocols don't mention the filter material. Perhaps because it doesn't matter. My instinct is that I would want a low protein-binding filter to decrease the likelihood of phage adsorbing to the filter, which would recommend CA, PVDF, and polysulfone. Is this overkill, or should I be concerned about the potential for phage adsorption to the membrane?
Yes, it is an overkill but polyethersulfone (PES) of either pore size should work fine! ;)
One of the added benefit of having a drop of CHCl3 is that not much can grow in it when the tube is stored at 4ºC.
Completely agree with Tausif Alam... having a drop of CHCl3 is not a problme and even beneficial.
Update:
After acquiring a new, local stock of P1:vir, and making several modifications to my protocol, transduction is now working! The insert was confirmed by PCR, blue-white screen, and Sanger sequencing. Thanks to everyone for their helpful suggestions!
I'm having the same issue. I will try to decrease incubation after 1:100 dilution. Thank you for the tips this was a great resource!
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