ccdb toxin - (Jul/30/2014 )

hallo all,

 

just curious: anyone here using this toxin as a selection marker in the gateway system? I have been using it lately but to my surprise I noticed that it is not that effective. I still get lots of cells that harbor the plasmid with this toxin.

 

I am wondering others might have seen this too? 

 

 

Yes, I have a lot of experience with this. The problem is that mutations will occur in the fragment, and they are (of course) selected for. Evolution really wants these cells to survive. We switched away from using it, moving to an RFP cassette, whcih can be used to easily identify parent plasmid. But the thing that really worked for us was to start PCR amplifying the plasmid backbone with minimal template, then DpnI digesting the product, practically eliminating parent plasmid. That, and using a cloning plasmid with diferent antibiotic resistance than the source of the inserts.

Shetty R, Lizarazo M, Rettberg R, Knight TF. Assembly of BioBrick standard
biological parts using three antibiotic assembly. Methods Enzymol.
2011;498:311-26. doi: 10.1016/B978-0-12-385120-8.00013-9. PubMed PMID: 21601683.

Yes, I can see your point.

According to the seller of the system it should not be such a problem. I just noticed it the last few weeks that the system is not that bulletproof!

I also sequenced the gene itself and it still is the original fragment! Perhaps in some colonies it is indeed a problem with mutations.

 

Your system, I remember it from a while ago, seems nice, but for the work I am doing its too much work I am afraid.

 

And the biggest problem is pretty much the fact that the genes I have to use are given to me in a gateway plasmid.

So I am pretty much forced to use it.

 

 

The PCR amplification and digesting , restriction free cloning as I know it, is indeed a very usefull system. I pretty much use it all the time when I can!

Makes me wonder why so many people still keep getting stuck working with restriction enzymes and ligations and so on.

What kind of competent cells did you use? Bugs harbouring the F'-episome survive ccdB selection, e.g. XL1 Blue and derivatives. You should check the genotype of the bugs before giving up on the system altogether 

I am using top10 F' cells from life technologies.. They do contain a part of the F plasmid, but not the ccdB gene (at least it should not contain it!).

 

BioMiha on Wed Aug 6 19:31:26 2014 said:

What kind of competent cells did you use? Bugs harbouring the F'-episome survive ccdB selection, e.g. XL1 Blue and derivatives. You should check the genotype of the bugs before giving up on the system altogether 

 

F' is a huge plasmid and all of its genes are rarely described in the manuals. Essentially any DH5-alpha strain (e.g. OneShot TOP10) should work as long as it doesn't have the F' episome.

I've never tried ccdB with an F' strain. I avoid those strains, since most have additional resistance genes that get in the way of routine cloning.

BioMiha on Thu Aug 7 10:13:03 2014 said:

F' is a huge plasmid and all of its genes are rarely described in the manuals. Essentially any DH5-alpha strain (e.g. OneShot TOP10) should work as long as it doesn't have the F' episome.

The cells I have , top10 F' cells, they only contain a short piece of the F plasmid! It should be fine for ccdb selection (its even mentioned in the website (life technologies) that they are not ment for ccdb propagation.

 

I , personally, would have used the regular top10 cells, not the top10F cells, but for some obscure reason (we don't even work with phages or whatever reason you might need the F episome) they got the Top10F ones...

phage434 on Thu Aug 7 14:53:43 2014 said:

I've never tried ccdB with an F' strain. I avoid those strains, since most have additional resistance genes that get in the way of routine cloning.

Tetracycline is indeed coded on that F' episome, but for me its not a problem, but you are right, I would avoid the F' ones too, but I have to work with I have been given at the moment.

In the future I might change to a different strain.

ccdB is the toxin and ccdA is the antitoxin. So if ccdA is present on the F' episome they won't be selected against even if they haven't taken up your insert. That's why you see background.