Plasmids 101: Sequence and Ligation Independent Cloning (SLIC)

Posted by Mary Gearing on Dec 17, 2015 10:30:00 AM


If cloning methods had personalities, SLIC (sequence- and ligation-independent cloning) would be a true rebel. Not only does this system not use site-specific recombination, it also doesn’t require a ligation step! Based on the robust system of homologous recombination found in E. coli, SLIC is a cheap, standardized, and rapid multi-part DNA assembly method - read on to learn how to use it in your research.

The starting point: Ligation-independent cloning

Ligation-independent cloning (LIC) was first developed in the 1990s. While traditional restriction enzyme cloning used short sticky ends, LIC employed the exonuclease activity of T4 DNA polymerase to create longer, “chewed-back” overhangs of about 10-12 bases. Only one type of dNTP would be present in the reaction mix, limiting the exonuclease activity to the first occurrence of that nucleotide. At that position, T4 would perform the favored polymerase reaction and subsequently stall due to the absence of other dNTPs. Once digested separately, the vector and insert could be annealed, forming a circular product with four nicks easily repairable by the bacteria after transformation.

LIC is a reliable cloning method, but it is limited by its sequence constraints. The 10-12 base overhangs must not contain the dNTP present in the reaction, or polymerization will occur at that position, preventing T4 from chewing back the entire 10-12 bases. As such, the use of LIC is often limited to specially-designed plasmids.

A SLIC makeover

In 2007, LIC received an important update, courtesy of Addgene depositor Stephen Elledge. His new method, named sequence- and ligation-independent cloning (SLIC), eliminates many of LIC’s constraints. Key to SLIC is the power of homologous recombination. In E. coli, a robust homologous recombination system allows for the repair of gaps and overhangs based on regions of sequence homology. This process can occur through one of two pathways: RecA-mediated recombination or RecA-independent single-stranded annealing. Elledge realized that he could generate imperfect “recombination intermediates” through PCR and imprecise T4 exonuclease activity, overcoming the requirement for carefully designed DNA overhangs used in LIC.  As long as there was enough sequence homology (20-60 bp) to organize the fragments and hold them together, E. coli would be able to “repair” the plasmid, generating recombinant DNA. Adding purified RecA to the pre-transformation incubation enhances the repair process, allowing SLIC to be used with very small amounts of DNA (e.g. 3 ng). When working with larger amounts of DNA (~100 ng,) RecA is not required.

The practicalities of being SLIC

To start the SLIC cloning process (see figure above), a fragment of interest is PCR amplified to add the specified 5’ and 3’ homology regions. This PCR fragment and linearized plasmid are both partially digested using T4 polymerase in the absence of dNTPs. In the second step, the addition of a single dNTP stops the exonuclease reaction. The PCR fragment and plasmid are then combined, annealed, and transformed into E. coli. To make matters even easier, SLIC is also compatible with incompletely synthesized, or iPCR, fragments (right branch of the figure above). If PCR does not include a final extension step, many of the products will have single-stranded overhangs due to incomplete extension, and these fragments can induce recombination. Mixed PCR, shown in the middle branch of the above figure, can also be used to create an insert. Two PCR products are used to generate a target gene with a 5’ or 3’ overhang. When the products are mixed and annealed, 25% of the resulting DNA will have two single-stranded overhangs that can robustly stimulate recombination.

SLIC is ideal for multicomponent assembly (see figure below), as overlapping sequence homology specifies the order of multiple fragments, and the assembly is scarless. With 40 bp homology regions, a five piece assembly reaction is highly efficient (~80%.) Ten-fragment assembly can also be successful, but at a lower efficiency (~20%).

How does SLIC compare to other cloning methods?

SLIC’s limitations arise from its dependence on single-stranded overhangs. These overhangs must be accessible to allow for complementary base pairing, so SLIC can’t be used if the overhangs would have stable ssDNA secondary structure. One common example is the stem-loop structure of transcriptional terminators. Another potential issue is sequence similarity. If fragments in a multicomponent assembly have 5’ or 3’ sequence homology to each other, they may be assembled incorrectly. To overcome this limitation, one option is to perform a hierarchical assembly, assembling fragments in multiple steps to avoid using multiple fragments that share homology in the same reaction. In these cases, the use of another cloning method, such as Golden Gate assembly, may also be beneficial.

SLIC is most often compared to Gibson assembly, another cloning method based on homologous recombination that will be further detailed in an upcoming Plasmids 101 post. Instead of using T4 DNA polymerase, Gibson assembly requires T5 exonuclease in combination with Phusion polymerase and DNA ligase. This reaction takes place in one step rather than two steps required for SLIC, and ligase may improve the efficiency of multipart assembly. The higher temperature at which Gibson assembly takes place may also limit formation of secondary structures at the ends of fragments. The major advantage of SLIC over Gibson assembly is cost, as T4 polymerase is much less expensive than the enzymes required for Gibson assembly.

SLIC is a standardized method for multi-fragment DNA assembly, and its low cost makes it ideal for researchers doing large amounts of cloning. Assembly is scarless, unlike Gateway cloning, and the method’s flexibility allows it to be used with different types of PCR-generated inserts. By harnessing the power of DNA repair in E. coli, you can assemble multiple fragments without the need for specific restriction sites or DNA ligase!


References

1. Harnessing homologous recombination in vitro to generate recombinant DNA via SLIC. Li MZ, Elledge SJ. Nat Methods. 2007 Mar;4(3):251-6. Epub 2007 Feb 11. PubMed.

2. SLIC: a method for sequence- and ligation-independent cloning. Li MZ, Elledge SJ. Methods Mol Biol. 2012;852:51-9. doi: 10.1007/978-1-61779-564-0_5. PubMed.

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Topics: Plasmid How To, Plasmids 101, Protocols, Plasmid Cloning

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