I have been doing large scale expression of this protein of interest name, yqjD. Previously was fine, did a couple times, got some proteins and did several crystal screenings. Now that the proteins had ran out, I started carry the out large scale since last week. First round all cells died overnight, induced at OD 0.8+ (maybe phage, maybe bad iptg), second round cells turned bad, foul smell, slimey pellets which after lysis and centrifuge produced dark-coloured supernatant that could not be filtered, and finally third round the same (incubator was disinfect with UV light prior the third round as some labmates encountered cell death issue previously and suspected phage contamination in incubator).
I really can't figure out the problem here, I had used fresh ampicilin and iptg, disinfect the flasks more and more thorough each time I redo. Also, I find this problem weird and only happen to me, no one in my lab experienced this before, so it could be related to my newbie skills, but of course I have followed protocol tightly and had no problems before. Lastly, my mentor thought it was the glycerol stock contamination when similar things happened few weeks ago and he re-transformed the bacteria and created new glycerol stock.
It sounds like you have got a recurrent contamination problem. I suggest that you check you cultures by looking at them under the microscope and by plating them on the same medium that you use for your liquid culture. This will enable you to reisolate the genuine strain and make a fresh stock if needed. I would also make freshly autoclaved liquid medium and take care of not opening the bottle, e.g. for adding filter-sterilized ampicillin (or, preferably, carbenicillin) until immediately before starting your culture. Preferably use wrapped disposable pipettes or freshly sterilized glass pipettes rather than pipette tips. And handle your cultures and media under a sterile cabinet, if available
Check your IPTG concentration. Sounds like your cells are lysing. The cells lyse if there is too much protein being expressed. You can try reducing the shaker speed and/or temperature. If it is contamination of the stock by phage, start over the transformation, and obtain single colonies on plates. Change all solutions.
Thanks Pierre and Sarita for answering my question! To Pierre, I pretty much did most of the things you have mentioned, perhaps I will plate them to see if my glycerol stock is contaminated. To Sarita, I don't think my IPTG is overdosed to the cells, it was 125 micromolar in final concentration, shaking at about 180 rpm for overnight, maybe about 22 hours since the start of culture. However, I will try to check for contamination.
You said the problem started after you moved up to large scale, right? I agree with Sarita: there is too much protein being expressed. When it happens, there are two reasons that can explain the weird pellets and the dark-coloured supernatant:
1. Too much protein causing cell lyses (as Sarita said)
2. Too much protein being expressed that bacterial machinery is not being able to fold it properly, producing inclusion bodies or just soluble protein but not well-folded.
I know it sounds weird, since everything was fine when small-scaled, but I had the same problem (#2) and had to change conditions/concentrations of large-scale protocol to get good harvests.
Hi Eduardo, thanks for your input! I was thinking along that line too, and has changed my competent cells to a more toxic tolerance part. Think I've failed to mention that the protein I am currently expressing possesses a transmembrane motif, more or less I guess it would pose certain toxicity to the cell while overexpressing. However, what puzzled me was the fact that I had no problem growing the cells and express the protein in the past, and problem usually occur after few times of glycerol stock inoculation. Of course my method and way of doing things are more or less constant, so I really can't figure out why the problem arise. Oh by the way my iptg final concentration is 125 um, is it too high?
Hey Goh, I have similar problems at the moment an just wanted to ask if you figured out what had happend?
Hi Nadine, I am sorry to disappoint you that this problem was not solved with definite knowledge on the cause, but eventually we did not encounter this issue anymore. However, I would like to add that it is important to be able to recognize smell from different cultures, for example different type of strains of bacteria, yeast culture, etc. In addition, make sure glassware are washed thoroughly, rinsed with deionized water and disinfected before use. Anyhow, I would like to push this problem to the cause of contamination, so I would suggest to check the bacteria stock, make sure they are the right ones, and then plate them and pick the single colony for pre-culture. This is my usual routine, slightly more time consuming, but even without the use of biosafety cabinet, my cells are usually healthy. All the best to your experiments!
Hi,
You may try to culture your bacteria at room temperature instead of 37°C after plating. It might reduce inclusion bodies and stress less your bacteria.
Good luck!
Hi y'all,
Having the exact same issue. PI and I decided to be optimistic and assume we over-expressed the protein.
Our plan is to lyse one of our tubes recently harvested and run it on nickel and to also take the supernatant after pelleting and run it on nickel as well. Ideally, anything binding to the nickel should just be our his-tagged protein. Again, we're trying to be optimistic but there's also a huge chance this won't work at all. Will update if it works. If not, I'm probably off crying somewhere.
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