If the protein being expressed upsets the bacterium without being lethal, it will grow but find a way to lose the gene. Plasmid may be retained with mutated gene or gene excised, Kanr may even be incorporated into the genome, possible even in recA negative strains. More likely is that your glycerol stocks have some empty vector cells which come to dominate in culture.

I agree with Pete. If an insert is deleterious to the cell, deletions will occur.

This is best checked by making a plasmid miniprep, cutting with the enzyme used to clone the insert and running an agarose gel. If a deletion has occurred, your insert will be the wrong size - quite often it will turn out to be missing one of the restriction sites as well.

I would advise you to change the way you handle your stocks. A strain will almost always change if subjected to repetitive subculturing - People do in vitro evolution experiments this way. Therefore, keep the frozen glycerol stock from the primary culture and use this to periodically make new stock cultures for use. In order to avoid having to thaw your primary stock, you can add small beeds to the culture. The hole in the beed has enough bacteria to subculture and the beed can be freed from the mass without having to thaw the culture.

I am facing the same problem with my clones. After transformation of E coli with plasmid and incubation overnight, I picked up some colonies and did PCR and later agarose electrophoresis. I had nice bands on the gel. I stored the culture plate at 4 degrees for 1month. I came back to the plate to repeat the experiment but there is no amplification, yet the bacterial grows on medium with antibiotics (used as a selectable marker)

An alternative is to load the purified DNA ( plus DNA staining solution) onto an agarose gel and see if you indeed have the plasmid. Do not forget to a control!!!

There is the possibility of having your clones transformed but not transgenic

With E. coli just about any rearrangement is possible. I had a pUC19 clone flip the orientation of the multiple cloning site on me once which I still scratch my head over. Although I make glycerol stocks, I only use them as a last resort. I always do a new transformation when I need more plasmid. You don't need super competent E.coli, in fact you can do a colony transformation and get plenty of transformants. If you have a particularly unstable plasmid you can also go to a different strain of E. coli like stbl3 or MDS42 which have greatly reduced recombinatory activity. The other thing you can try is growing them at 30 C instead of 37.

Do you have a positive over-expression control plasmid/cell line? If you are using IPTG to induce protein expression, it can go bad, since it is light and temp. sensitive. When I am going to induce protein expression, I make up a IPTG solution (from IPTG powder stored at -20oC) right before I need to add it to the cell culture.

Regarding Dieudonné's post I read a paper (probably in BioTechniques, an issue from 1990s) that it can occur that after transformation procedure cells can be covered with DNA and in case of failed ligation where plasmid was closed but does not include insert a bacteria is transformed and antibiotic resistant, a colony gives positive signal in PCR screening but after replating transformants the insert DNA gets diluted or degraded and colony turns negative.

Best storage material is expression vector. Once the presence of insert of correct size is confirmed in the expression vector, prepare a fresh miniprep from the identified clone and store at -20 dec C or even -80 deg C in Tris-EDTA. You will never lose your insert here if the storage conditions are good. Use this for fresh transformations whenever returning to expression experiments after long gaps.

If you are doing purification on a regular basis, it is also a good idea to store induced cell pellets at 80 deg C - this can go for a bit over a month with no problem. If your protein of interest is in inclusion bodies you van store induced cell pellets even longer.

Bhaskar: If possible run a PCR with the gene of interest specific primer to be sure that you have your insert in the bacteria. And yes bacteria can certainly loose the gene while retaining the plasmid. I will not elaborate as answer to this have been given above.

The bacterial can loose the gene of interest not only because of toxicity of the recombinant protein, but also structural stability of the plasmid. If the plasmid contains direct repeat sequences, the DNA part between the two direct repeat could be removed by homologous recombination.

I agree with Eric that it is best to do a transformation from the recombinant plasmid each time, otherwise you can have this situation (not infrequently). And also with Nausheen-- the best way to keep it is as a plasmid in TE even at 4degrees. And yes if you thaw a stock completely you might as well toss it after. However you don't need to thaw your stocks before streaking- just a quick swipe and back into the freezer. And I abso

Thanks everyone for the suggestions. It was resolved. Other colonies on the same plate gave me expression.

Hi all,

I have a similar problem with my BL21 DE3. They are always transformed fresh before protein expression, but I noticed after colony PCR that a lot of them lost the insert. I cut my plasmid from the stock to see, whether there is a contamination. As expected it's clean and looks right. Also the PCR works.

Now I'm speculating that the gene could be toxic, since it's also hard to get a good amount of protein.

Did someone of you try different strains like C41,C43 or the pLys? Might that solve my problem?

Pete Holt Is there a reference for the phenomenon of KanR integration into the E.coli chromosome? I think I might be experiencing this right now, but I couldn't find any resources

KanR can readily integrate into the chromosome in the form of transposon Tn5, but that is probably not what Pete Holt was referring to.