In Gavin's class Rob gave a presentation on de novo computational design of a Diels-Alderase. The enzyme produced in this research had relatively low activity when compared to normal enzymes. Improving the activity of this de novo enzyme is limited by the development of screen/selection.
This paper describes a fluorescent sensor for a monitoring a ribozyme Diels-Alderase. They use anthracene attached to a BODIPY fluorophore. This sensor could be a good tool (or a starting point) for improving the activity of other alderases including the de novo enzyme developed by Siegel et al. (Science. 2010 Jul 16;329(5989):309-13).
Well found! However, the paper linked below describes the in vitro selection of a multiple turnover Diels Alderase via droplet based methods. The substrates look a little more forgiving given the longer and more flexible linkers.
ReplyDeletehttp://www.pnas.org/content/102/45/16170.long
That is cool, but what is the effect of having a big piece of DNA on the anthracene and a biotin tag on the dieneophile? Seem likes it would remove a lot of active mutants based on those large/long groups.
ReplyDeleteI think it would be nice to have as little linker as possible. The original paper uses a pretty big fluorophore, but if it could be reduced a similar IVC would be more ideal..? I imagined converting a non-fluorescent substrate to a fluorescent one. Then the evolution could be done with droplets and screened by FACS. This would allow for the other mutants that can not convert the substrates with large linkers.
There would be a trade off with the loss of geneotype-phenotype linkage and it would no longer be a selection....decisions...
What is great about the paper that Gavin posted is that the author only had to use a primary emulsion. For the screen that your talking about Chris, you would have to use a double emulsion. Primary emulsions cannot be screened by FACs. As you can imagine, a lot more problems arise when your working with double emulsions than primary emulsions.
ReplyDeleteObviously you would need a double emulsion, but the problem still remains that the large linker and DNA would prevent a whole population of mutants that can not get the substrate in the enzymatic pocket.
ReplyDeleteAlso the screen doesn't have to be done via IVC. A high throughput method could also be done with the fluorescent screen.
I haven't found a compound that is small and becomes fluorescent after a diels alder reaction, but that would be more ideal. Even if it can't be done in a primary emulsion, I would take the evolution without the linkers.
There are 2 advantages of the primary emulsion selection over a FACS based screen. First, primary emulsions are much easier to make. Second, and most importantly, the primary emulsion selection is only limited by the number of droplets you can make (~10^10-11). You can analyze much vaster regions of sequence space with this method than with FACS (which tops out at ~10^8 mutants).
ReplyDeleteI was very surprised to see that the selection requires multiple turnover. By increasing the concentration of free anthracene over multiple rounds, you could increase the kinetic properties of the enzyme. This circumvents a huge problem with such selections in a very elegant way.
Also, anthracene is fluorescent. The reaction would cause fluorescence quenching.
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