Zeocin Resistance in Nature? - (Jun/21/2013 )

Hello microbiology friends,

I have been struggling with a simple ligation/transformation in my lab for over a month now. The plasmid of interest has a zeocin resitance, but also a "kill" gene where if it comes back on itself during ligation the cell simply dies. The problem is I get growth on zeocin plates&broth, but when I go to cut the plasmid there's nothing there. My PI and I have gone back over my work repeaeldy and all the components look good, and have even made media with 3x the normal aliquot of zeocin and still gotten growth.

It would appear then that I have some sort of contaminant that is zeocin resistant. I am not overly familar with microbiology, but am concerned about the existence of such a contamination. Is there an bacteria in nature that is zeocin resistant that could be plagueing me?

I don't know if this will be helpful, but the colonies seem to be smaller than the usual e.coli colonies on plates. They have no discernable color, but are a bit more "clear" than normal e.coli colonies. They are also more mucous-y than usual.

Thank you for your help in solving this mystery!

A few things:

Are you sure your zeocin is ok and hasn't gone bad?

Are you sure you are using the right concentration (simple dilution errors etc, do happen to the best of us)

Have you checked to make sure your bacteria (minus the plasmid of interest, so just straight up competent cells) are sensitive to the concentration of zeocin you are using?

If you are getting a contaminant- this means you are having problems with your aseptic technique somewhere along the line. Are you certain your competent cells are pure? If not, I would start here. Streak out a new plate, pick a single colony and remake your comp cells from here. If you buy them, the cells are unlikely to be the source, which means you would be introducing a contaminant somewhere along your procedure. So you may want to have a chat with someone with a bit more micro experience to go over the basics with you, to make sure there isn't anything you are missing?

We did a series of controls with the zeocin plates with other strains of e.coli and those that were amp resistant grew on amp and died on zeocin as they should have, empty comp cells also died on both plates as they should have; so I'm fairly confident with my plates. We make the competent cells but I have also used purchased cells and had the same problems. I'm beginning to think the plasmid I'm working with is mutated somehow. I cut an aliqout of it and the size of the insert that gets released is what it should be. The kill gene is supposed to reduce background "noise" from the plasmid coming back on itself if there was incomplete cutting. So the only thing I can think of besides bacterial contamination from the environment is that the kill gene is mutated OR the PacI and AscI sites are somehow coming together during ligation even though they don't match.

Ok.

What "kill" gene is it? I've heard some are more likely to mutate than others. Also, often killing is incomplete and so you will get some smaller "pin prick" negative colonies appearing on your plates.

If this is what is happening, you could disregard those and make sure to only screen the larger colonies for your insert?

How many colonies (roughly) do you screen for insert following transformation?