I only learned about Gibson when I started my postdoc, and it completely changed how I approached science. In some experiments with Ethan when I was in the lab, I was blown away when we realized that you don’t even need Gibson mix to piece a plasmid back together; this is something we were exploring to try to figure out if we could come up with an easier & more economical library generation workflow. I was disappointed but equally blown away when I realized numerous people had repeatedly “discovered” this fact in the literature already; the most memorable of the names given to it was IVA, or In-Vitro Assembly. Ethan had tried some experiments, and had said it worked roughly as well as with Gibson. Of course, I can’t recall exactly what his experiment was at this point (Although probably a 1-piece, DNA recircularization reaction, since this was in the context of inverse PCR-based library building, after all). So the take away I had was that it was a possible avenue for molecular cloning in the future.
We’ve done a fair amount of molecular cloning in the lab already, creating ~ 60 constructs in the first 4 months since Sarah joined. I forgot exactly the circumstances, but something was right where it made sense to try some cloning where we didn’t add in Gibson mix. I was still able to get a number of intended constructs on that first try, so I stuck to not adding Gibson mix for a few more panels of constructs. I’ve been trying to keep very organized with my molecular cloning pipelines and inventories, which included keeping track of how often each set of mol cloning reactions yielded correctly pieced-together constructs. I’ve taken this data, and broken it down based on two variables: whether it was a 1- or 2-part DNA combination (I hardly ever try more than 2 in a single reaction, for simplicities’ sake, and also because properly combined cloning intermediates may still be useful down the line, anyway), and whether Gibson mix was added or not. Here’s the current results:
So this was extremely informative. Some points
1) I’m willing to screen at least 4 colonies for a construct I really want. Thus, I’m counting a success rate > 0.25 as being a “successful” attempt at cloning a construct. In the above plot, that means any area above the dotted red line. Thus, 1-part DNA recircularizations have pretty decent success rates, since the area of the colored curve above the red dotted like >> the area below it. Sure, Gibson mix helps, but it’s not a night-and-day difference.
2) 2-part DNA combinations are a completely different story,. Lack of Gibson means that I have just as many failed attempts at cloning something as successful attempts. Those are not great odds. Adding Gibson mix makes a big difference here, since it definitely pushes things in favor of a good outcome. Thus, I will ALWAYS be adding GIbson mix before attempting any 2-part DNA combinations.
Other notes: I’m using home-grown NEB 10-beta cells, which give me pretty decent transformation rates (high-efficiency 1-part recircularization reactions can definitely yield many hundreds of colonies on the plate from a successful attempt), so there have been relatively few plates where I literally have ZERO colonies, where I’m more likely to have a few colonies that are just hard-to-remove residual template DNA).