How does your colonies look like? Are these relatively big and clearly single colonies? Or are they small and hard to distinguish? How high is your ampicillin concentration on the plates and how old are they?

If your ampicillin concentration ist too low and/or your plates are too old, you can grow up false positive clones after a transformation.

As said above, try with a fresh stock of Ampicillin (I use 100ug/ml). What do you mean by debris? (if you see something not E.coli, then it may be contamination from the start!?)

I dont think the promoter in the pcDNA will allow E.coli (DH5a) to express protein.

If your protein is toxic for E.Coli, YES.

Remember that pcDNA3 can express your cDNA in bacteria (T7 promoter after CMV) and also, CMV can be expressed in bacteria as reported in some papers. This is an example:

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1181807/

By the way, the causes can be many.

You said you can see a pellet of debris, this can be a phage contamination. Is there anyone in your lab using phages?

Amp concentration can be high, try using the "conventional" 50 ug/ml.

The alternative could be to change bacterial strain...

Thanks for your answer

The colonies are big and single. I have been told about phage contamination, but than ............why does it not affect my other cultures? Also, 1 colony out of 20 grew in liquid culture and it was a re-ligated pCDNA3.

The paper link is very useful. I think my protein is doing something to Ecoli.

Hopefully I can manage to overcome its expression putting a small intron inside

May be a funny idea, why dont you do colony pcr on the colonies and on the debris too with some primers for the vector you have, if there are products from colnies, it may be that the LB medium is contaminated. If there is product from debris, that may be overgrowth. As I said its funny idea

It doesn't seem likely that it is your protein being toxic, as it grows fine on your plate. Even if it's a case where a healthy growth produces a lot of the protein, and at that point it becomes toxic, the liquid culture should still get cloudy.

Are the plates you are growing the initial transformants on also with amp? And you are using the same dosage of the ampicillin for your liquid media?

What do you mean by "transformed the same batch of plates?" Do you mean you used those same cell line, transformed them with a different vector, and plated them on the same plate, or did you actual re-perforate them (electric/heat shock), then repeated the transformation?

A third issue that may come up is whether you are keeping the liquid vial on a shaker at 37 over night. This is important! They die of starvation without the shaker.

A final thing, like everyone else is saying, is to make sure you autoclave your LB before use and practice clean techniques to prevent contamination (the clump possibly). Also, be sure that all of the ingredients are added (I"ve made this mistake before) and to stir thoroughly.

Good luck!

Western is a great idea, unfortunately there is no antibody for the “toxic protein”. What I am trying to clone is a “new” cDNA (coming from alternative splicing)

Following your suggestions, I’ll try to do some colony PCR; grow the culture without shacking and also make some miniprep from plates.

At one point I will have to use a GFP fusion vector and also synthesise the protein (it is less than 50aa) and raise an antibody. Therefore the original problem will be solved somehow.

What I really would like to know is whether I have found something that can kill bacteria.

Here comes the silly question….. If I synthesise the peptide, how can I test if it affects Mr Coli? I mean, how do I get the peptide inside?

yes Miguel, I think you have made a point.

The original cDNA (polyA tail included) was originally cloned in pGEMTeasy vector and the culture was growing without any promlem.

Considering that pGEM has the T7/SP6 promoters If the cDNA was toxic I should have seen something that time as well.....

I don't agree with your interpretation of the pGEM result. pGEM will yield a GST fusion protein that is relatively large and will remain cytosolic. Your pCDNA3 vector will express a candidate cDNA that is the size of a large peptide, which could have toxic effects on E. coli, especially if it forms pores in the cell membrane. When you grow E. coli on LB plates, the osmotic and shear stresses on the cells are extremely different than in liquid culture. In addition, a toxic secreted peptide may accumulate to higher levels in a liquid culture due to the larger number of E. coli present in a fixed volume, relative to those present on a plate with a low # of colonies.

I suggest you try to clone your cDNA into a different expression vector that lacks a prokaryotic promoter. If you expect this cDNA to be toxic to eukaryotic cells, too, you should try an inducible system, preferably one that uses active repression to prevent basal transcription of your gene of interest.

the clones where both directions. some SP^ and some T7.

However, I am afraid I have started to get the problem with other preps (not so strong as for my cDNA) and other people too...

we are asking around if somebody is using phage and cleaned up everything plus UV-lamp.

sorry for misleading, I was really hoping I had found a toxic protein

As expected, deadly phages.... have fun killing them :)

Miguel- good catch. Sorry for the error in the prior post. Did misinterpret pGEM as pGEX. Michela- hope you are able to resolve this issue after decontamination with for possible phage infection.

Thank you all of you.

I will let you know how it will end

For a while, we had the same problem. Could not figure out what was going wrong. We finally figured out that our new batch of toothpicks which we use to put the colonies into the liquid medium contained some sort of growth inhibitor. If we used a sterile loop or a different brand of toothpicks, all cells grew again.

Dear Raponi, There was a possibility that the copy number of the clone carrying the cDNA insert was reduced than that of the original vector plasmid caused by the inserted cDNA. The concentration (100 micro g per ml) of ampicillin in LB liquid was probably too high for the clone, therefore cells elongated and became a pellet of debris. Did you observe the debris with a microscope? There might be many elongated ghost cells. Please note that the level of ampicillin resistant 'strictly' depends on the copy number of plasmid. In the case of low copy number mini-F plasmid (1 or 2 copies per chromosome), we used 20 micro g per ml of ampicillin in LB liquid.

Dear Raponi. Continue to the answer:

Upon transformation of cloning, there were lots of ampicillin-sensitive recipient cells on the LB agar plate containing 100 micro g per ml of ampicillim, these cells incorporated ampicillin into the cells and elongated, resulting in reduced concentration of ampicillin in the surface of the agar medium. Therefore, the clone with a cDNA insert was barely able to grow on the plate. A lower concentration of ampicillin must be used in liquid LB medium to grow the transformant clone.

Please note that reduction in the copy number of plasmid is often caused by an inserted DNA segment, especially, by a long segment or wrong segment for cell growth and that the level of ampicillin-resistance depends on the copy number of ampicillin-resistant plasmid. Therefore, it is better to use 50 micro g per ml of ampicillin in LB agar medium for the first cloning and 20 micro g per ml in liquid LB medium for growth of transformant cells.

I got the same problem, is working hard to figure it out!