We have suffered from that a lot at the NKI a few years back. What worked for us was a combination of strategies, that were all mentioned before but I will repeat:
1. Buy T1 phage-resistant strains, and also use an old incubator (and luckily an old building that was available at the time) to 'evolve' more strains that would be resistant. 2. Stop storing bacteria and only store plasmids. Work with bacteria strictly within our 'VMT' lab. 3. Perform 'phage' tests (PCR based) in a few (>20) places in the lab that are labeled on a monthly basis. Any positive result is red-alter and means to stop working and start cleaning. 4. Buy our 'phage buster', a machine that can bleach a closed room with acid, as part of the cleaning procedure (ideas to lock in the same room as being bleached team members that work 'dirty' in the lab have been voiced - but not (yet) implemented) It has costed us months of trouble, and its a serious matter. I would recommend anyone to even close the lab down for a couple of weeks to clean, destroy all glycerol stocks, clean fridges, etc, rather than try to handle it while 'normal operations' are in place. We have tried that and only when we got really serious and changed the way we work, we are now phage-free for a few years in a row and have minor trouble. Tassos On 3 Mar 2013, at 10:09, aidong wrote: > Dear Opher, > > It was also our idea how to get over it since several years ago. We > did it based on a philosophy that any bacteria infected with a phage > will not be infected by the same phage again. Therefore, we let our > BL21/DE3 strain infected in the lab and when most were died, some > cells were survived. We picked these to make the competent cells for > expression constructs. We believe these actually contained phages. > In order to avoid this bomb to blow up, we managed to get a commercial > strain (TonA and TonB deletion) purchased from Sigma USA but found it > useless. I wonder how you screened your phage-resistant strains. > > In addition, it is our regular practice to use bleach to extensively > disinfect all surfaces, including benches, used flasks, instruments > and floors. Would virkon work more efficiently in our case? > > Sincerely, > > Aidong > > > On Mar 3, 2013, at 3:36 AM, Opher Gileadi wrote: > > Hi, > > We had a massive phage infection at the SGC in 2004; all our washing > and sterilization efforts could only solve the problem temporarily. I > then recovered a phage-resistant subclone of BL21(DE3), and prepared > derivative strains with pRARE2 (the tRNA plasmid in Rosetta2) or other > plasmids. We have expressed thousands of protein since and never had a > recurrence of phage problems. Although the resistance is to T1 phages, > it seems these are the most common lab infections. > > Write me if you want the strains. > > Opher > > Aidong Han, Ph.D > > Department of Biomedical Sciences > School of Life Sciences > Xiamen University > 3 South Xiangan Road > Xiangan, Xiamen 361102 > China > Phone: 0592-218-8172 (O) > 0592-218-8173 (L) > Web: http://life.xmu.edu.cn/adhanlab/
