SLIC cloning (Sequence and Ligation Independent Cloning) theory & workflow

My molecular cloning method of choice is SLIC (Sequence and Ligation Independent Cloning). Instead of the conventional “cut and pasting” with restriction enzymes & DNA ligase, SLIC is more like “copy and stapling.” You use PCR to make lots of copies of the pieces you want to join together, adding on overlapping segments at the end. Then, after a little chewing to get the pieces to stick, you let bacteria stitch them together (the bacteria just sees it as damaged DNA so uses its homologous recombination machinery to “fix” it). SLIC is awesome because it’s really versatile, you don’t need restriction sites, it leaves no trace (it’s scarless), and you don’t have to do any gel purification. Here’s how it works.
blog form: bit.ly/SLICworkflow
note: If you want much more detail one molecular cloning, see: bit.ly/molecularcloningguide, but today I want to dive into the nitty gritty details.
note: text adapted from past post, and I added an uploaded protocol on Google drive: docs.google.com/document/d/1y...
Regardless of what cloning method you choose you’ll need 2 things:
1. plasmid vector you want to stick your gene into (destination)
2. something containing the gene or “insert” you want to stick into that vector - these days, this insert is usually already inserted into a different plasmid vector just not the one you want so what you need to do is SUBCLONE it → move it from one plasmid to another instead of “traditional” cloning where you’d be moving it from its original location (such as chromosomes inside human cells)
The basic idea with SLIC is that we design the insert piece to have bits of the vector piece at the ends, so that when DNA Pol starts copying our gene, it adds on a bit of the vector at the beginning (kinda like adding a few words from the page you want to come before it)- specifically it adds the part of the plasmid that’s flanking where your gene will go. The PCR reaction gives us double-stranded DNA, so the complementarity is “hidden” by the second strand. You therefore have to chew back one of the strands a bit (with an exonuclease) to generate single-stranded “sticky ends” - only the end is chewed & this is the part that matches the vector, so it exposes vector-matching single-stranded DNA that can stick to the vector DNA.
The exonuclease chewing is much less precise than the restriction enzyme cleavage, so you’ll get overhangs of different lengths and when you combine them & they stick together, they’ll leave gaps - but this is ok because the bacteria can fill them in.
But in order for the bacteria to fill it in properly, they need the right template sequence - the original gene doesn’t “know” the sequence of the plasmid - so if you cut off some of the sequence you loose those instructions & the bacteria don’t know how to fill it in → BUT if you add enough of the plasmid sequence to the ends, that’s what gets chewed back - that part will match the other part so you’ll get sticking, & your gene will be there to provide complete template info. So you design primers so that: 1: the ends match & when you chew them back & 2: they’re long enough that you don’t loose the “unique” information (you want your primers to have ~20bp overlap between the end of thing 1 & the beginning of thing 2).
Our lab uses 2 major “go-to” expression systems - bacteria & insect cells, & they use different vectors (a plasmid for the bacteria and a backed for the insect cells). We can’t get around having to do 2 sets of molecular cloning (since we need to stick it into 2 different vectors), but we can save some time with clever primer design. We’ve adapted the plasmids so that they have the same sequences flanking where we want the gene to go, so we can use the same “plasmid bit” overhangs & the same primers to amplify either plasmid.
We use vector-specific (but specific to the generic part) primers to amplify either vector & we use vector-insert chimera primers to amplify the insert. So we only have to design 1 set (of 2) SLIC primers (the hybrid vectorend-insertstart ones) per clone. We use these to get the “gene piece” that we can put in either one. To get the different plasmid vectors, we use the same “vector primers” but a different plasmid template. Most of the vector’s different but that primer binding site is all that matters here, and it’s the same. Even with this time-savingness, they still add up to lots and lots of tubes & boxes). But Most of the SLIC-ing I do is actually site-directed mutagenesis. My gene’s already where I want it I just want to change the sequence. More on this here: bit.ly/sitedirectedmutagenesis
finished in comments

[@thebumblingbiochemist]

And how does it work in practice? Here’s an overview:
* choose a vector
* design primers to copy just the regions of the vector and the insert you want to combine - instead of primers that just match what’s already there, you make “Special” primers - have ~20bp overlap - basically you design the primers to start with the end of the other piece, so that when you make a copy you also copy a few of the words that you want to come before it when you staple them together
* do PCR - you end up with double-stranded DNA (dsDNA) fragments whose ends are hybrids between the vector & the insert - but you still have 2 pieces... and those pieces are pretty content as is… need to chew their ends up a little to create sticky ends like those you’d get from restriction enzymes (now, instead of cleaving in the middle of DNA, which you want ENDOnucleases for you want to chew from the end so you need EXOnucleases
* add another endonuclease, DpnI to selectively degrade any of the original parent vector, as that vector would give you false positives in the selection step because it has the antibiotic resistance gene. DpnI only degrades methylated DNA and methylation (addition of a methyl (-CH₃) group) occurs in bacteria but not PCR, so the PCR-generated DNA is *not* methylated and thus is safe, whereas the parent DNA *is* methylated and thus is at risk so DpnI is able to selectively degrade the parent. more here: bit.ly/reasesvsmtases
* purify the products (easy to do with a spin column kit) - this is important - want to remove unused nucleotides because you’re about to add a polymerase again!
* add T4 polymerase - polymerase? isn’t that what adds the nucleotides? yes - but proofreading polymerases can also remove them - the 3’-5’ exonuclease activity is important for proofreading because it allows the polymerase to “backtrack.” But since you took away the nucleotide train track pieces, the polymerase can’t build track to go forward, so it gets “bored” and starts going the direction it can go - “backwards,” chewing off the ends as it does so and creating single-stranded (ssDNA) overhangs at the 5’ end. You don’t want it to erase all of your (ok, the thermal cycler’s) hard work - you just want it to chew off enough to give you sticky ends, so you only give it ~10 min to do this
* then you stop it by adding a dNTP (DNA letter), but just 1 of the letters - it can add this letter, so it switches to its track-laying (polymerization) mode, but then gets stuck and stalls when it needs to add a different letter
* at this point you have sticky ends that can stick together, but when they do so there may be gaps because the different strands have gotten chewed different amounts - if you stitched it “as is” you’d be deleting letters. Instead, you want to fill those letters back in, which will require more than just the stitcher, so instead of adding DNA ligase to stitch up the ends in a test tube, we just stick it in bacteria and have the bacteria stitch it up for us using its own machinery - the bacteria’s homologous repair machinery (part of its DNA maintenance crew) is well equipped to handle cases like these
A similar method is GIBSON ASSEMBLY - the PCR part’s basically the same but then things change a bit - instead of relying on one enzyme (T4) to do both the chewing & the polymerizing, it uses 2 separate enzymes - T5 exonuclease & Phusion polymerase. It also uses DNA ligase. Gibson is much more expensive because you’re supplying all these products “in vitro”(in a test tube) in their pure form. We don’t need them to be “pure” - we just stick the partial product into bacteria and have them finish the work - their “impure” forms work great! Gibson can be more efficient, but we’ve had great success with SLIC
Don’t confuse “Gibson” with “Golden Gate” which uses PCR to add on restriction enzyme sites then uses those restriction enzyme sites to cut & paste (like copy but add cut sites when you copy -> then cut & paste) and don’t confuse “Golden Gate” with “Gateway” which uses λ integrase - a whole different mechanism
Whatever method you choose, you still need to make sure it worked, same as with restriction cloning. We can use the same techniques to check if a gene we’re interested in got inserted into the plasmid - techniques like diagnostic digest, blue-white screening, and sequencing. bit.ly/cloningcheck
Here’s the original SLIC paper: Li, M.Z., Elledge, S.J. (2012). SLIC: A Method for Sequence- and Ligation-Independent Cloning. In: Peccoud, J. (eds) Gene Synthesis. Methods in Molecular Biology, vol 852. Humana Press. doi.org/10.1007/978-1-61779-564-0_5
Here’s a nice blog post from Addgene on SLIC: Plasmids 101: Sequence and Ligation Independent Cloning (SLIC), By Mary Gearing, 2015 blog.addgene.org/plasmids-101-sequence-and-ligation-independent-cloning
Here’s a comparison of SLIC, Gibson, and a few other methods: J5 manual, The SLIC, Gibson, CPEC, and SLiCE assembly methods (and GeneArt® Seamless, In-Fusion® Cloning) j5.jbei.org/j5manual/pages/22.html
         
more about all sorts of things: #365DaysOfScience All (with topics listed) 👉 bit.ly/2OllAB0 or search blog: thebumblingbiochemist.com

[@chuchu7248]

AMAZING way to explain!!!!

[@thebumblingbiochemist]

thank you!

[@aryarezayi8622]

Thank you so much 💓

[@cies_do]

Thank you for this very informative video! ❤

[@thebumblingbiochemist]

Glad you found it helpful!

[@exez2116]

Hi I have one quesiton from T4 reaction part in this video. When I mix vector and insert PCR product to form plasmid what I want, should I have to add other buffer or dH2O? In other words, just adding insert and vector PCR products can make plasmid what I want?

[@thebumblingbiochemist]

Normally, I just dilute in water

[@exez2116]

@@thebumblingbiochemist so basically the plasmid will be made with just adding insert and vector after T4 polymerase reaction. Did i understand correctly...?

[@thebumblingbiochemist]

The bacteria will do the hard work :) It doesn't actually get stitched together until it's in there

[@exez2116]

@@thebumblingbiochemist THANKS!

[@GordonChao0508]

Does Restriction clone also Homologous Recombination? Why does it need DNA ligase while LIC don't need?

[@thebumblingbiochemist]

It needs ligase because it is not homologous recombination - no matching pieces

[@matejatadei5671]

Isn't this IVA cloning? What are the differences?

[@thebumblingbiochemist]

I hadn't actually heard of IVA, but looking it up, that appears it does multiple PCRs together rather than separate amplifications