ssDNA gibson and biobricks

[Koeng]

Hey

I was thinking that it might be interesting to be able to bridge the gap between 2 DNA fragments using a ssDNA. Essentially you could cut a DNA fragment with restriction enzymes and then just gibson together the junction using an ssDNA primer.

Anyway, for that you'd probably use lambda exonuclease because it doesn't degrade ssDNA very quickly, unlike T5. It looks like the Hifi mastermix uses it, which is pretty cool. 

I'm more of interested in whether this could be used in assembly of multiple biobricks at once. When I used biobricks for an iGem team last year I found they were quite annoying to use when compared with the awesome power of gibson assembly. 

The beginning part would be cut with EcoRI and SpeI and the last part with XbaI and PstI, kinda like a normal reaction. However, the backbone would be cut with SpeI and XbaI (this is so you don't have to have primers for the ends of your assembly). Then, all parts in between would be cut with XbaI and SpeI and then the junction between those parts would be filled with the ssDNA so you can assemble them in a predefined order.

Although the problem of course would be the ligase in the reaction. Since lambda exonuclease can't digest nicks, it'd be possible to just add the ligase at the end. However, there would still be the chance of a lot of contamination as the SpeI/XbaI sites get ligated together. And then there's also the question of the efficiency of such a method... although I think both those could be solved simply by PCRing your entire assembled construct out and then cloning it again after purification.

Thoughts on the idea? Also feel free to steal it if you're in an iGem team

-Koeng

[Nathan McCorkle]

Are you talking one-pot or sequential reactions? With all the in-the-middle being cut with the same ends.... I don't see how a one-pot would work to assemble in a defined order (the direction of the fragments would all be 'pointing' the same way though).

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[Koeng]

https://www.youtube.com/watch?v=JcYZiGjhqVk

Interesting. And btw, no a 1 pot wouldn't work, it would be sequential

-Koeng