I suggest you to contact with Phage Consultants

Make sure that you use prophageless strain to avoid spontaneous phage induction and if the problem remains, I can only recommend you to try to use sodium citrate, which should prevent phage particles from binding to cell surface (or make it much more difficult). But I work with Staphylococcus aureus and I don't know whether it applies also for other bacteria. In our lab, we use UV light instead of ozone to sterilize the laboratories. I think it's better as UV light itself destroys the structural components of phage particles (and of all living organisms too, of course) and ozone, which is produced, does the rest.

It is also a good idea to check the filters from the workbenches as well as air-conditioning.

Is there anyone close to your lab that is using phages ? How do you know that are phages? Are you expressing toxic proteins? The lysis effect by toxic proteins on large E.coli cultures is similar to cell lysis by phages. Anyway never share micropipets with people working with phages and use always tips for PCR . Last thing: throw it all away

Absolutely agree with Paola Di Bonito, it can be effect of toxic proteins. But if it's phage, you need to understand, what phage it is, because for example T1 phage is very resistant to cleaning methods. Even if no one in our University is not working with phages, you could bring them to the lab through the dust on shoes, clothing, etc.

Thanks to every one. We are trying to figure out phage type. Moreover, we are using Rosetta and BL21-Gold competent cells for hyper-expression. I will also try your suggestions, and will keep you update here. Thanks to all participant and to the coming ones.

I do agree with the approach suggested by Ry. I have no experience with E. coli but what we use to do with Lactococcus and Streptococcus is to expose the cells to high phage titers and exploit the capacity of a small percent of the cell population to become spontaneously resistant via modification of the cell surface receptor necessary for phage infection. Acquisition of total insensitivity is reached after a number of phage challenges that varies according to the organism / strain used. You mention that 90% of the population is affected and this suggests that theoretically the remaining 10% could have already spontaneously mutated to become insensitive to phage attack. This is generally achieved by point mutations and does not involve big chromosomal changes that might affect particular cell functions. Therefore, you could either buy the tonA mutants Ry is talking about or create these BIMs in your lab and use them for your expression work.

RY Young: Sir, can you share the behaviour of tonA/B mutant. My lab had purchased this one from sigma in 2009, but unfortunatly it never grow. So, can you share your experience, that is there any problem with tonA/B mutant growth or can be effectivly used for expression purposes?

If you choose the route of creating your own BIMs, Ry suggestion to check adsorption defect is correct (as generally this is the way these BIMs acquire their resistance) but I would add that even more important is to confirm by PFGE (and a couple of restriction enzymes) that the mutants have the same genetic profile as the parent strain and no deletion or major rearrangement has occurred. In my experience, this is rarely the case and simple point mutations occur in 99% of the cases. The approach works very well as this is how we create phage-resistant strains of dairy starter cultures, where it is important to create more robust strains without altering any of their technological characteristics such as acidification rates, flavour profiles etc.

Although this is getting out of the main topic of this discussion, I'm really interested to read that the routes to phage resistance mutations differs in different genera... has this been published ?

And since I'm here, let me share our experience with "flying phages". We did regularly experienced T7 mass infections in our lab (which i addition was a mutant that only grew at 30C), in addition to T1 and M13 of course. Using resistant strains to avoid T1 or T7 can be tricky as the phages seem to stick to the cells in the cultures. Thus I'd insist on Ry recommendation that the TonA/resistant host needs to be used for quite a while!

I suppose these days only disposable vessels are used to store phage lysates. At least for M13 it is essential since it resists autoclaving as normally used to sterilize media, glassware etc.

I personally contaminated with T1 a lab in Los-Angeles by bringing my notebook from Brussels!

We have the same problem!!!!!-and I wonder which expression cells(system) you work with.

Oh, We struglled and still struglling. Changed media type according to some papers but it is not working as well. Diassemble all the shakers and clean them manually, gave UV treatment, O3 treatment is normal thing to my lab, gave formaldehyde treatment, We were using miniprep columns so also we prepared our plasmid again by gel-extraction methods. We failed every where. Our lab used antiphage strains for almost three years to avoid phage infection and nothing is working. Since September 2012 we are unable to carry out normal routine. Astrid we are still working and if we get some percentage of sucess then will share to you for sure.

We can't get rid off the problem as well.

The only thing which helped a little bit was heating up the incubators to 80°C over the weekend-kept us working well for let's say 1 week.

Thanks for keeping me updated.Have a nice weekend.

Astrid can you please share me how you people are keeping your incubator on 80 degree, for how much time, and are you pleople using the internal temperature of incubator or doing some thing else? Next how about big shakers, are you guys giving them some special treatment?

Hi Ashfaq , we heat all incubators and big shakers up to 80°C for over night or best the whole weekend. Good luck.

Hi, Can you suggest anyone  How to Isolate DNA From T7 Phage. Ia any specific kit for Minipreparation . if kit is abailable then this is bester. I having kit for M13 phage, Can I used same kit for T7 phage.

In response to the first question, Ry is correct with respect to T1 phage contamination. I’ve seen it shut down an E. coli lab. Assuming you have a T1 phage contamination using tonA E. coli strains should solve your problem. If you have a T1-like phage that uses a different E. coli receptor you may have to select for a resistant mutant which still has the phenotype you need to conduct your study. That should be relatively easy – make a lysate of your culture (i.e. allow a 20 ml culture of your expression strain to grow in your lab and when it clears you have a lysate). You could test this against a tonA strain by cross-streaking analysis to determine if you have T1 phage contamination. If the tonA mutant is sensitive to the phage in your lysate than you will need to isolate a mutant that is resistant. That should be relatively straight forward by growing a culture of the strain of your choice (in a different lab) and select for a phage resistance by adding your lysate to the non-contaminated strain and plating on LB plates. Pick individual colonies, streak them out (several time) and cross-streak against your lysate. You could also try plating out your lysate.  If the resulting strain still has the phenotype you need it should be resistant to the contaminating phage of your lab. I would also guess Ry would like to sequence it’s genome.

I've been working with E. coli in a building with a chronic phage problem for over 15 years. I don't completely know why this works but here's what we do to almost completely eliminate this problem. First, we iodine treat all our glassware immediately after growing cells. This is before washing and either autoclaving or baking our glassware. This has a huge effect in reducing the problem. I tried this after talking to older microbiologists who had been doing this for years. Second, if we have a culture that gets infected, the usual culprit is the water bath we are using. We clean it completely with soap and water, clean any pipettes used near the culture and we're fine for another 6 months :) Ideally of course we remember to clean out the water bath more regularly to avoid this completely.

Agree with Ry Young's explanation.

We once had a T1 phage contamination problem. Removal of the trash/garbage dumpster behind the lab building resolved the problem.