[DIYbio] Gibson vs SLiCE (single part recommendations)

Recently I've begun using SLiCE for cloning and have got some of my labmates to jump aboard. From about 1-2 weeks of trials, here's what I've found with single part SLiCE assembly. In the future, I hope to try 1. PEG8000 to increase SLiCE efficiency and 2. Multipart assembly 3. using homemade comp cells.

Important stuff: SLiCE is approx. 1/3 the efficiency of homemade gibson assembly. Gibson increases efficiency with additional homology while SLiCE decreases. The optimal homology length for gibson is ~40 while the optimal homology length for SLiCE is ~20. SLiCE recommended time is 15 minutes @ 37c while gibson is 1 hour @ 50c. SLiCE mix can be produced cheaply with Triton-X100/Tris-HCl buffer, the only solution required.

Experiments: I produced the SLiCE enzyme mix using the protocol outlined in (1). I took them out at about OD 2.7 because I was getting tired of waiting for the cells to grow. If you put the cells in fairly late at night, you might be able to get them in the morning. An important point that I have not verified is that efficient SLiCE without lambda red requires refrigerated centrifuge during purification.

Immediately after making the mix, I did a single part cloning with ~30bp homology with gave me 1/3 efficiency of 13 colonies (SLiCE) vs 42 colonies (homemade gibson) with both at their optimal temperature of 37c and 50c for 30 minutes. When I sequenced the DNA, I found no mutations within the overlap of either one of the constructs, both being completely correct (only sequenced 2 from each and got proper sequence data)

Some other people in lab tried the SLiCE mix and found that it gave roughly 1/2 to 1/4 and 1/3 the efficiency in 2 separate cloning experiments using the same amount of DNA in both coloning reactions (10µl total for SLiCE and 20µl total for gibson with ~100ng of insert for both) and transformed 5µl of that. That means that they transformed twice as much DNA for SLiCE, (per ng) but still got ~1/3 the efficiency. They also used 30 minutes for SliCE, which is optimal (2), but 1 hour for gibson.

I did another experiment that was more controlled. I used 100ng of vector and 200ng of insert for this cloning. Reaction times were 37c for 15 minutes for SLiCE and 1 hour at 50c for gibson. The reaction volume was 10µl for SLiCE and 20µl for gibson and I transformed 5µl. I was cloning a CDS, and decided to test overlap length. The design would be [vector - [flank 1] - [insert] - [flank 2] - vector]. Flank 1(SLiCE) had a tm of 58c overlap of 19bp, while Flank 2(SLiCE) had an overlap length of 24 and a tm of 55c. Optimizing the other side, I had 2 flanks which simply used up the entire 60bp possible because empirically that increased the efficiency of gibson. Flank 1(gib) had a tm of 71c overlap of 41bp, while Flank 2(gib) had an overlap length of 37 and a tm of 61c. When the SliCE optimized flanks were used for SLiCE, I got 28 colonies while when I used the SLiCE optimized flanks for gibson I got 35 colonies. Remarkably, when I used the gibson optimized overlaps for SLiCE I got only 3 colonies while when I used the gibson optimized overlaps for gibson I got 64 colonies! It seems like going over a certain length can actually hurt your SLiCE reaction. More is not always better. This data matches (2) fairly well. 

I'm going to do more experiments, so I'll keep this post updated in case there is interest. I plan on doing the above experiments soon. Note that I did do all this cloning with lab made comp cells using the zymo kit, https://www.zymoresearch.com/e-coli/transformation-kits-accessories/transformation-kit-buffer-set , so I don't know how good this would be with homemade cells.

-Koeng

(1) http://www.sciencedirect.com/science/article/pii/S2405580815000850

(2) http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4453199/