Hello everyone,
I'm currently facing a challenge in my cloning strategy involving the ccdB gene. Despite ordering a gene block with a J13119 and a standard RBS, I've encountered a lack of success after three rounds of cloning using ThermoFisher's ccdB survival strain.
My working hypothesis is that the toxin is being overexpressed, leading to the inability of bacterial cells to metabolize it effectively. To overcome this issue, I'm seeking alternative promoters or sequences that can provide a smaller, more versatile solution than the current chloramphenicol-resistance + ccdb gene from Thermo. Ideally, my sequence should be compatible with both gateway cloning and standard restriction cloning techniques.
Any insights or suggestions regarding suitable promoters or sequences for my specific case would be greatly appreciated.
Thank you for your time,
Juliano
You've eliminated simple things like poor transformation efficiency and or plasmid assembly already right?
Alexandra Johnson Yes! Transformation efficiency and plasmid assembly for all of my other constructs have been pretty good. I also haven't tried inserting the chloramphenicol-resistance + ccdb gene from Thermo because it doesn't really match what I want to do.
Are the competent cells you are using direct from the vendor or did you make your own competent cells? If the latter, then your strain might have lost the F element that confers ccdB resistance. You could try any other strain that carries an F'
Juliano Ribeiro You're probably correct that the promoter is too strong then. I found a comment on this https://blog.addgene.org/plasmids-101-ccdb-the-toxic-key-to-efficient-cloning article which seems to corroborate your hypothesis:
Joanne Kamens
12/15/2016, 11:20:51 AM
We had a good question on Facebook in response to this blog so I thought I would share Mike's answer here. Take Murata asked: I heard that some plasmids kill later developed competent strains other than DB3.1. Is it true? Response: "In an atypical case where the ccdB gene is under a strong enough promoter, competent strains other than DB3.1, such as ccdB Survival™ cells, can be killed. The reason why this does not happen with DB3.1 is because the different strains resist the toxic effects of CcdB through different mechanisms. DB3.1 cells contain a mutant DNA gyrase (the target of CcdB toxin) which prevents the toxic CcdB/gyrase complex from forming, and hence preventing cell death. ccdB Survival™ cells, on the other hand, are resistant to CcdB because they express CcdA, which is an antitoxin. So in the case of ccdB Survival™, the relative amounts of CcdB and CcdA determine whether the cells will die. When larger amounts of CcdB are present, there will not be enough antitoxin available to counteract the poison, and the cells will die."
If you can track down the DB3.1 strain (which doesn't seem to be commercially available anymore), you might not have to redesign your construct.
What is the vector that you're talking about from Thermo with the chloramphenicol promoter by the way?
Alexandra Johnson Thanks for your help. Glad to know that I am not the only one with the same problem. It would be good to know if people have tried different promoter with the Thermo ccdB survival.
Thermo doesn't offer a proper vector but a dna sequence to transform into ccdb resistant (https://www.thermofisher.com/order/catalog/product/11828029?SID=srch-srp-11828029). The problem it is only for gateway cloning and I would like to use for other applications that would use restriction cloning as well. It has a Lac promoter with Cr and ccdb genes, but the ccdB gene is all the way at the back and I am not sure if that is to decrease the levels of ccdB by placing it far away from the promoter.
The other option might be to alter the RBS to make it much less efficient, perhaps by altering the spacing to the ATG
Michael J. Benedik I agree that could work. It would be nice if someone had already figured that out because depending on how the science gods are feeling that could be more than 3 months of optimization :/. I emailed Thermo and hopefully they have a promoter that works better for my case. Thanks for your help!
Hello everyone, I was finally able to clone using the DB3.1 strain. I think the issue is as mentioned on this thread is that the ccdB survival cells cannot handle overexpression of the ccdB gene.
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