Introduction

Here we will look at Sequence and Ligation Independent Cloning (SLIC). In this approach, the overhangs are added to the intended inserts by PCR. The overhangs are much longer than with other methods and the complementary base pairing between the long overhangs (15 to several hundred nucleotides) is sufficient to hold the DNA molecules together without the need for ligase. Both 5′ and 3′ overhangs can be generated depending on the (polymerase!) enzymes selected. As with other methods, primer design is key – you must ensure that the correct sequence is added to the primers.  Getting the orientation of the sequence is essential to ensure that the fusion occurs correctly. That means that the planning process is very important.

A. SLIC cloning:

A-1. Benefits

With sequence and ligation independent cloning:

• The most important benefit is the ability to fuse any sequence with any other sequence.
• The fusion of the two sequences is “seamless” which means no sequence is added  between the two sequences that have been joined (compare to gateway).
• There is no need to “cure” vectors of certain restriction sites, or worry about whether there are internal restriction enzyme sites in your intended insert.
• For example, if you were cloning an insert into an EcoRI site of a vector, you could only use EcoRI sites on the primers to amplify the insert if the insert you were trying to amplify didn’t already have an EcoRI site inside the insert sequence. If it did, when you cut the PCR product, it would be cut in multiple sites. This would make the cloning much more complex.

In this type of cloning, we don’t have the ccdB gene to use for selection.  Blue-white selection is possible, though, depending on which vector you use.

A-2. How it works

For SLIC cloning, as mentioned, you must amplify the insert with primers that recognize the DNA you want to amplify, as well as sequence that matches the vector to either side of the intended cut site. During the initial stages of the PCR, only the gene specific parts of the primers will recognize template.

The overlapping sequence ranges between 15 extra nucleotides on the primers up to hundreds depending on what you are trying to do. This is the crucial stage – it is important to carefully draw out the sequence you plan to join at the site where they will be joined, and ensure it will work as planned. Recently I took a shortcut, thinking that I didn’t need to draw both strands, but because I did not include the lower strand, I made a mistake in the primer design.  Even with some experience it is a mistake to skip the important planning stages.

The PCR product is purified by a column method, to separate it from the unused primers and free nucleotides. These will interfere with the efficiency of the cloning process.

Cut the vector with the selected restriction enzyme (as determined in advance during the primer design phase). It is important to run a gel to ensure that the vector has been fully cut, or as close to fully cut as possible. In order to make sure most/all of the vector is cut, I  have done an overnight digest of the vector with an excess of enzyme, followed by the addition of 5-10 more units of enzyme in the morning and several more hours of digest. The cloning method is quite accurate but only if all of the vector molecules are cut with the enzyme, so extended digest is very important, as is confirming that the digestion went as expected (using gel electrophoresis).

In separate reactions, treat the insert and the vector with a polymerase that has exonuclease activity, either 5’ to 3’ or 3’ to 5’. You would not use an enzyme like DNA polymerase I that has both types of exonuclease activity – this would lead to the chewing back of both strands and therefore no overhangs.  The key to this technique is that when no nucleotides are present to incorporate into newly made DNA, the exonuclease activity of the enzyme is stimulated. The DNA is incubated with the polymerase in the appropriate buffer (and water, of course, to the correct final volume) and no nucleotides.  The approximate speed of the exonuclease activity of each enzyme is known., and the reaction is timed to generate the required single stranded region on the vector and insert – which will be a little longer than the intended overlap.

The reaction is stopped by the addition of a large excess of nucleotides which halts the exonuclease activity and stimulates the DNA polymerase activity of the enzyme. Only one nucleotide is added, for instance, a large amount of dATP (or any other, as long as it is just one nucleotide). Think about why we would not want to add all four nucleotides in the reaction.

In some protocols, the insert and vector are combined and the polymerase added. The advantage of doing this incubation with the insert and vector combined is that the generated overhangs should be the same length. Whether the insert and vector are combined before or after the polymerase treatment, a 2:1 to 4:1 insert to vector ratio is suggested for efficient cloning.

The insert and vector should now have overhangs with a substantial amount of overlap. The two are incubated together for 10 minutes or more on ice, so that the complementary overlaps on the single stranded overhangs can base pair with each other.  The combination is directional because the overhangs on the insert have been designed to match the overhangs that have been generated on the vector in one orientation only.  So long as 15 base pairs – or more – of overlap exist between the two DNA pieces and these find and bind each other, the molecule will form a stable circle that can transform bacteria.  The gaps can be filled in and the molecule sealed with ligase once inside the bacteria.

The transformation procedure uses heat shock or electroporation and works the same way as with any other vector + insert combination.  Selective plating depends on which vector is used. The vector will have one or more antibiotic resistance genes and the transformed cells should be plated on agar containing that antibiotic. And if blue-white selection is to be used, the genotypes of the E. coli cells to be transformed must be checked carefully. If the cells contain a full length lacZ gene, all the colonies will be blue. If the cells have a functional repressor protein, all the colonies will be white unless an inducer such as IPTG is added.  If you don’t remember how the lac operon regulation works, please review this to help you understand. If everything has worked well, and the cells have just the part of the lacZ gene that can complement the part held by the plasmid, blue-white selection will work. But we would still expect a few blue colonies because there will always be some vectors that have not been cut by the restriction enzyme. Note that most SLIC cloning does not use blue-white selection, but it is included here because it can be done.

A-3. Planning your SLIC cloning

Start with the finished product. What restriction site are you going to clone into? How will the insert fuse seamlessly with the vector? What orientation do you want the insert to be in? Draw the sequence at each join point. At each fusion point include 18-20 base pairs of insert specific sequence plus the overhang generated after exonuclease treatment and about 20 base pairs of vector sequence – the 15 nucleotides that will form the overlap plus a few additional nucleotides.

Then underline the sequences that will form the primers. On the top strand on the left side, underline the 15-nucleotide overlap plus 18 nucleotides of insert specific sequence. That is the forward primer. On the bottom strand, on the right side underline the 15 nucleotides of overlap plus the 18 nucleotides of insert specific sequence. That is the reverse primer, which must be written out 5’ to 3’ when you are ordering it.

This seems complicated maybe, but once you’ve done it a couple of times, it is quite straightforward. So long as you’ve correctly written the sequences on both strands you will be able to design this type of primer.

The image below shows the insert with the interior sequence not shown, and a small stretch of vector sequence from the polylinker. The cut site, an EcoRV site, is indicated wth a line. Below that is the vector sequence separated at the cut site, with the insert sequence between. Below that is the same image, but with lines that indicate the sequences of the forward and reverse primers.

You can make 3′ overhangs or 5′ overhangs. If you use T4 DNA polymerase, you will generate a 5′ overhang, via its 3′-5′ exonuclease activity (chews back the 3′ ends of DNA molecules). The T5 DNA polymerase has 5′-3′ exonuclease activity and so will generate a 3′ overhang (chews back at the 5′ ends of the DNA molecule). So long as the insert and vector are treated with the same polymerase, the procedure should work.

There are a number of different versions of SLIC cloning and these vary in the size of the overlaps generated and the type of overhang, 5’ or 3’. An entire artificial bacterial genome was synthesized by Gibson assembly and reported in 2010¹. The chromosome was designed and then synthesized in segments of about 1 kb each. These segments had overlaps of 80 bp between adjacent segments.  These pieces were assembled in groups of 10, i.e. into 10 kb pieces, that were then further assembled into the full 1.08 Mb genome. There is more to it than that; if you are interested, you can check out the reference that describes the entire process of building the artificial genome.

This is a link to the Gibson Assembly song. I think it is important for you to see it :D.

Below is the recording that summarizes SLIC cloning. Click here to download the powerpoint slides.

References

¹Gibson, D.G., Glass, J.I., Lartigue, C., Noskov, V.N., Chuang, R.,  Algire, M.A., Benders, G.A.,  Montague, M.G., Ma, L., Moodie, M.M.,  Merryman, C., Vashee, S., Krishnakumar, R., Assad-Garcia, N., Andrews-Pfannkoch, C., Denisova, E.A., Young, L., Qi, Z., Segall-Shapiro, T.H., Christopher H. Calvey, C.H., Parmar, P. P., Hutchison III, C.A., Smith, H.O., J. Craig Venter, J.C., (2010) Creation of a Bacterial Cell Controlled by a Chemically Synthesized Genome. Science Vol. 329, Issue 5987, pp. 52-56
DOI: 10.1126/science.1190719

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