Hi,

what do you mean by "disappear": it grows up to a certain density and then undergoes lysis, or it doesn't grow at all? Is this a new protein? In addition to the phage infection, it can also be that the protein is toxic to bacteria. I would try adding 1% glucose to the culture to additionally repress T7 pol transcription.

It sounds like the pLySs doing. It makes the cells very fragile and prone to lysis. Add minor stress from your protein or some other source, and they're gone. pLySs is unreasonably persistent in the absence of selection. I tried to breed it out of the cells once, but they refused to lose it. It could be a phage too. You could try getting a BL21 strain without the pLySs. Then you know.

Those are great answers! I would also recommend cleaning your glassware and rinsing really well. If you want to get rid of any of those contaminants (including random plasmids or phage) just bake the glassware at 180C for a couple hours and autoclave. We've had some problems that are hard to pin down, but glassware maintenance is an easy solution.

thanks for the advice guys. will try all suggestion.

Hi,

After reading your problem and great answers, it seems likely to be a contamination by a phage, but by a lytic phage other than lambda phage. If contamination came from pRSET plasmid (i.e. your plasmid DNA contains Phage DNA or particles), it willd be hard to get rid from phage by treating glassware, pipettors and tips.

Glucose will not perfome a good catabolite repression to lacUV5 promoter that regulates T7 RNA polymerase expression in DE3 lambda prophage in BL21(DE3) strain. Recombinant T7 lysozyme that is produced from plasmid pLysS is a natural inhibitor of T7 RNA polymerase and is better regulator than glucose, and I thing you should use it when expressing your target gene(s) from the plasmid pRSET.

Here are some of my considerations and suggestions.

1. Thereotically, phage Lambda DE3 can undergo rescue and cause the lysis of bacteria upon bad conditions, where expression of a toxic protein is one of them. However, since there is still some bacteria expressing a CI repressor, you should get a turbidity and a well-grown culture but with some cell debris.

2. Recombinant T7 Lysozyme expressed from plasmid pLysS does not perform degradation of peptidoglycan layer of Gram-negative strain when it is inside the cytoplasm. However, and as it was suggested by others, if you have a bad culturing conditions or your target protein is toxic for BL21 or causes destabilisaton of membranes, some bacteria may be "opened" thus delivering the T7-Lysozyme to the medium. I do not admit to this possibilty due to its low probability.

3. pRSET is not a good expression vector as pET vectors are. First of all, it is a high-copy number plasmid, yet there no Lac operator sequence after T7 promoter and it possesses the Ampicillin resitance gene. I suggest to re-subclone your target gene into pET28 or higher number vectors which are Kan-resistant vectors and also have a Lac operator sequence just after the T7 promoter. If you proceed with pRSET vectors, then I suggest lowering the growth temperture to 30^C since pUC19 origin of replication is temprature sensitive ori ( in 37^C there is a low affinitty of of RNAI to RNAII and to ROP protein).

4. The secret for a good expression in BL21 is NOT performing a starter, but rather perform a fresh transformation, take ALL grown colonies into 0.5 to 2L of LB (not TB) media, grow them to OD600 of 0.8 and then induce with IPTG at lowered temperature (18-20^C) for overnignt.

To conclude:

1. Treate all of your glassware, put pipettors at UV, use only tips with a filter.

2. Try using other plasmid than pRSET. pET28 or 30 are the best candidates. Alternatively, use pRSET with another non-toxic gene as a "positive" control.

3. Grow cells at 30^degrees. Perfrom every time fresh transformation and use all the cells instead of starter.

4. Try using BL21(DE3) without pLysS plasmid.

5. Try titering a contaminating phage, or at least grow BL21 as a lawn (the soft agar is a good option) to see if there indeed a presence of lytic phage.

6. great success!

Hi,

According to your description, I think it is highly likely that your seed culture has encountered phages, which is a tricky thing. I had a similar experience, and it was solved by treating all the experimental materials used in the experimental process. The previous comments have given many specific ways to deal with it, I wish you good luck.

Thank you all for your kind answer. We currently trying to change clean bench's filter(since there was biological dumpster was near it), and throw away all antibiotics and plate that we've been used. I will let you know for further update.

Good luck, Hyunsoo!

I suggest you for every cloning/subcloning experiment using DH5alpha (or TOP10) from NEB or Invitrogen with TonA (now called fhuA) mutation that confers resistance to T1 and sometimes to T5 phages. TonA is a a fascinating protein called for "T one" phage.

If you procceed with your pRSET plasmid, I would suggest you perfroming several single-colony stricking, i.e. innoculate a single colony of DH5alpha onto a fresh LB or 2xYT plate, than on the next day perform a fresh innoculation and so several times. Then perform a new miniprep plasmid purification and transform into new (commercial) BL21(DE3)(pLsysS) vial. All grown BL21 cells use without a starter for protein expression at lowered temperature.

Great success,

Yevgeny

Thanks Yevgeny. My plasmids are already purified from DH5a. Should I do it newly?