Extract the plasmid from the BL21 that doesn't work and sequence it, there could be a point mutation if the gene product is toxic to the cells in small quantities. I understand you said you had a culture of "BL21 codon plus cells, that showed proper protein expression at the required molecular weight and hence had made glycerol stocks from it." but did you make the glycerol stock of the pre-culture with low OD and before inducing), or did you make it from some cells left after induction? If it was the second option, you could have had a mixture of expressing and non expressing cells in the glycerol stock and you were unlucky. The ratio can shift further to non-expressing cells when they grow faster, so it matters how you make your glycerol stocks and when you streaked out to get single colonies. If you have streaked out and went to single colonies after reviving, then instead of trying one colony, simply test 20 different colonies and hope that you have still a few expressing lines left. Make new glycerol stocks before inducing protein expression, not after. If you didn't streak out and revived just by inoculating medium with a little bit of melted glycerol stock liquid, then that would have been very very bad microbiology practice and I strongly recommend against that. But in this case, simply go back to the glycerol stoick and try to find an expressing clone. You always have to work with single colonies.

In general, when cloning into E.coli for the purpose of protein expression, make glycerol stocks from single colonies before you know if they express. Use the same low OD starter culture (2mL) for glycerol stock and for inoculating the big culture for expression, then check which big cultures express, and keep those glycerol stocks that belong to that group, throw the others away. Whenever you revive from glycerol stock, streak on plates again, pick a single colony, and start again. Never inoculate a culture directly from taking liquid glycerol stock.

Extract the plasmid from the BL21 that doesn't work and sequence it, there could be a point mutation if the gene product is toxic to the cells in small quantities. I understand you said you had a culture of "BL21 codon plus cells, that showed proper protein expression at the required molecular weight and hence had made glycerol stocks from it." but did you make the glycerol stock of the pre-culture with low OD and before inducing), or did you make it from some cells left after induction? If it was the second option, you could have had a mixture of expressing and non expressing cells in the glycerol stock and you were unlucky. The ratio can shift further to non-expressing cells when they grow faster, so it matters how you make your glycerol stocks and when you streaked out to get single colonies. If you have streaked out and went to single colonies after reviving, then instead of trying one colony, simply test 20 different colonies and hope that you have still a few expressing lines left. Make new glycerol stocks before inducing protein expression, not after. If you didn't streak out and revived just by inoculating medium with a little bit of melted glycerol stock liquid, then that would have been very very bad microbiology practice and I strongly recommend against that. But in this case, simply go back to the glycerol stoick and try to find an expressing clone. You always have to work with single colonies.

In general, when cloning into E.coli for the purpose of protein expression, make glycerol stocks from single colonies before you know if they express. Use the same low OD starter culture (2mL) for glycerol stock and for inoculating the big culture for expression, then check which big cultures express, and keep those glycerol stocks that belong to that group, throw the others away. Whenever you revive from glycerol stock, streak on plates again, pick a single colony, and start again. Never inoculate a culture directly from taking liquid glycerol stock.

The information by Prof Denecke was quite informative.

Many people told me before not to maintain clones in BL21 or any related expression strains, even if you store them as glycerol stocks. It is hard to expect some strain specific instability at such low temperatures, but practicall it seems an issue. But, what i do not understand is why ur dh5 derived plasmid is not working. Can u check by western to see whether u hav any specific expression in this approach?  I would recomment to work with the dh5 plasmid for further troubleshooting. Hope you find a way out soon.

I agree with Denecke, It is very interesting and informative. 

Clone instability is a major issue with protein expression. With toxic genes we observe clone stability and it is hard to find a protein which is not toxic when over expressed.

We have seen this problem even during  DH5alpha   transformation with plasmids like PQE30. I would like to know which plasmid you are using. 

Though, Denecke is against inoculating melted glycerol stock, that is the method followed in industry for production of biologics (seed lot system: Master and working banks are prepared, stored  characterized and used as it is) 

@Prof Denecke. Thank you very much for your elaborate advice. I used 2ml starting culture (before induction) for making my glycerol stocks. From that , for reviving I did inoculate directly from the glycerol stock in 2ml media which you mentioned is a bad practice. I agree. So those clones did not show any protein expression. So do you think over the time these clones just lost the gene of interest? Is that possible?

So, as recommended by you, now I am going to go back to my glycerol stocks, streak from them, take up at least 10-20 single colonies, inoculate them in 2ml starting culture and then see protein induction. We don't have sequencing facilities so sequencing from BL21 plasmid might not be an option to me although I could extract Plasmid DNA and check  if the vector still has my clone. I hope this is the right direction. Thank you very much . I will surely get back to you.

Have your stocks gone through cycles of freeze/thaw after the creation of the glycerol stocks? Be sure to remove the stocks for as little time s possible as thawing can kill your expression chances.

BL21 is DNase positive, and can degrade the plasmid it carries. My suggestions is, instead of spending real long time to trouble shoot your misbehaving BL21 glycerol stock, just extract plasmid from your DH5a cells, and re-do the Bl21 transformation. That will probably solve your problem.

Yes, I agree with Bo Yang, going back to the DH5alpha and check single colonies is better still, then find positive clones and use those to transform BL21 and check for expression. If you make a glycerol stock before induction (split the cutlure), then this could still be a good way to maintain the line. A typical sign for toxicity is obtaining colonies of different sizes, sometimes you can even get that without induction because no promoter is completely shut down. In those cases your working stock should be the positive DNA prep, and each time you want to express you transform BL21 fresh.

The industry approach of making a large quantity of glycerol stocks in individual aliquots works because the entire aliquot is used up in the process, and the culture from which the glycerol stock was made is from a single colony and has been tested positive. All the aliquots are identical, and if one works, all of them will work because they are kept save in freezers. This is not the same as having a glycerol stock in a University research lab and having people poking the frozen surface repeatedly to inoculate liquid cultures directly.

I hope you can find the original clone back, in principle if you have kept the ligation, you can even re-transform DH5alpha and find the clone again. Let me know if you succeed.

Good luck! 

i think Bo hit the nail on the head!

BL21 are not recA or endA they can therefore rcombine or otherwise ´edit´ your plasmid. Never use them for clone storage or plasmid prepping, always use a recA endA strain like DH5alpha, Omnimax etc. If you have a glycerol in these 'B' strains you might be lucky enough to recover your plasmid by miniprepping but I would strongly recommend re-sequencing before using again for expression.

For reliable and repeatable expression you want to do a fresh transformation into the B or K expression strains every time.

Also you didn´t say what plasmid you are using-if it is T7 (like pET) you will have to use a (DE3) strain or you will not have the appropriate polymerase to transcribe from the T7 promoter.

The addition of 1% glucose to transormation plates, starter and expression cultures will also improve the stability of your plasmid if the gene product has any toxicity (can make an all or nothing difference but is obviously not compatible with auto-induction protocols).

Good luck

Hi Utsav,

Any luck in getting expression after picking single colonies?

I repeated the cloning and got new positive clones. Followed all the precautions advised here and then then successfully got the protein at the expected molecular weight. The glycerol stocks made thus, have been used further for repeated experiments successfully.

Thanks everyone for your help specially @Prof.Denecke