I have observed that BbsI (and this phenomenon occurred with both the original conventional form, as well as the High Fidelity; note, enzymes were ordered brand new, handled properly i.e. aliquoted and stored at -80, since these proteins apparently die at -20 in the Stratcooler) AND BpiI (sorry Thermo, your enzyme actually performed even worse; and this was also handled with the same aliquoting and care taken with the NEB materials) can still cut at domesticated BbsI sites when I have only made a single base change. Has anyone else observed this phenomenon, or have any insight on the minimum number of changes that should be made, and which residues are most impactful when making changes to eliminate cutting by this enzyme, which frankly in my opinion should die in the fires of the deepest hell?
I haven't worked with this enzyme but I have observed this with other restriction enzymes with the High Fidelity tag. I think restriction enzymes, by their nature, are a bit more promiscuous than most people think, even the engineered ones.
I don't know what your digestion conditions were but I would recommend against prolonged reactions. My supervisor used to swear by doing overnight digestion to get complete cutting but, in my experience, that only hurt more than helped. Digestion for 1-3 hours is usually high enough efficiency for most applications.
Also try to avoid using too much enzyme. I wouldn't go any higher than 10 units/ug.
Another thing might be the method you're using to keep your reactions at temperature. If you're using a heat-block, be careful because you'll get evaporation and condensation of moisture on the lid, which will over-concentrate your reaction mixture. That could possibly cause some weird chemistry to occur. You could run your reaction in an incubator or a thermocycler.
You took a shot already at the enzymes, but in my experience the NEB worked excellent. I am assume you are using this enzyme to perform GoldenGate cloning reaction? And I assume this why you removed any internal restriction site to prevent cutting of your DNA of interest.
By promiscuity do you mean you get complete digestion of the DNA polymer or a cut of the in your sequence modified BbsI recognition site?
In case of the former I would be highly surprised and indeed unfavorable reaction conditions might have a play in this, however in case of the former: how sure are you that the restriction is gone? The primers used to modify may have been wrong or exonuclease activity could have eaten up the SNP containing part of the primer when poorly positioned.
This last option I have experienced, however in my subsequent cloning, using 2 pieces and a backbone, this - still present - internal restriction site casuad no issues.
By promiscuity I mean that the enzyme is cutting at the domesticated site. I don't do PCR to perform domestication, all of my molecules are synthesized. Since these plasmids are fully sequenced, the molecules are exactly as I have dictated. I have observed BbsI cutting at sites that I have domesticated. I would like to understand which of the six bases that define a BbsI site are the best to change to eliminate/minimize this apparent promiscuity.
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