Hi hive,
We are trying to use ccdb as a counter selection marker. We are using the cassette from this paper, which should be highly expressed: Article A new method to customize protein expression vectors for fas...
Both BL21 and DHaalpha are growing happily with plasmids carrying this plasmid, even though they should definitely be dead. When we sequence the plasmids after transformation into these strains we find no mutations in the gene or its promoter/rbs.
We plate out on normal LB and use chemically competent cells. Has anyone encountered this/ are we missing something totally obvious?
Cheers,
Georg
Hi Georg,
I've struggled with Ccdb for quite a while and came to the conclusion that it doesn't work 100%, full stop. In my opinion a small fraction of bacteria always find a way to evade counters election by either mutating their genome of selecting plasmids with mutation that inactivate or lower ccdb expression. I've sequenced some of this ccdb inactive colonies which were happily propagating in dh5a or Top10 cells and never found mutations in promoter or CDS regions. Occasionally I've got some mutations upstream the promoter, quite far from it but enough (I think) to alter ccdb expression (apparently).
Interestingly there is a subtle difference between colony shapes which has been reported elsewhere in a blog. Clear colonies tend to contain correct vectors, while opaque colonies most often contain ccdb+ vectors. It could help but it's hard to spot.
I've tried to use stronger promoters for ccdb expression but that only worked for a limited time, then it started happening again. The paper you are citing quotes this problem too, and attributes it to propagation in ccdb survival, therefore they are using PCR to amplify the backbone rather than continuous propagation.
The amount of background I have is often acceptable 5-10%, but if you can work using PCR or linearized vectors to prevent transformation of the backbone I think it's the fastest way to go. At least it worked for me.
Hope it helps
Francesco
I don't think you are missing anything obvious. Either your strain is not correct (but that is unlikely since you have tried 2 different strains) or your plasmid is not actually expressing ccdB. It could be a mutation or it could be that you don't have expression.
Hi Georg,
I've struggled with Ccdb for quite a while and came to the conclusion that it doesn't work 100%, full stop. In my opinion a small fraction of bacteria always find a way to evade counters election by either mutating their genome of selecting plasmids with mutation that inactivate or lower ccdb expression. I've sequenced some of this ccdb inactive colonies which were happily propagating in dh5a or Top10 cells and never found mutations in promoter or CDS regions. Occasionally I've got some mutations upstream the promoter, quite far from it but enough (I think) to alter ccdb expression (apparently).
Interestingly there is a subtle difference between colony shapes which has been reported elsewhere in a blog. Clear colonies tend to contain correct vectors, while opaque colonies most often contain ccdb+ vectors. It could help but it's hard to spot.
I've tried to use stronger promoters for ccdb expression but that only worked for a limited time, then it started happening again. The paper you are citing quotes this problem too, and attributes it to propagation in ccdb survival, therefore they are using PCR to amplify the backbone rather than continuous propagation.
The amount of background I have is often acceptable 5-10%, but if you can work using PCR or linearized vectors to prevent transformation of the backbone I think it's the fastest way to go. At least it worked for me.
Hope it helps
Francesco
We managed to solve this by using a different ccdb cassette from here:
https://www.snapgene.com/resources/plasmid-files/?set=gateway_cloning_vectors&plasmid=pDEST17
Seems like in our initial cassette there must have been some mutations in the regulatory regions.
Hi Georg
I had a look at the sequence of the vectors you've just posted. It's the same cassette I'm using. The problem might appear again, just keep an eye out for it and make a glycerol stock of the original plasmid so you can go back to it if things go wrong again ;)
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