SLiCE: a novel bacterial cell extract-based DNA cloning method

Abstract

We describe a novel cloning method termed SLiCE (Seamless Ligation Cloning Extract) that utilizes easy to generate bacterial cell extracts to assemble multiple DNA fragments into recombinant DNA molecules in a single in vitro recombination reaction. SLiCE overcomes the sequence limitations of traditional cloning methods, facilitates seamless cloning by recombining short end homologies (≥15 bp) with or without flanking heterologous sequences and provides an effective strategy for directional subcloning of DNA fragments from Bacteria Artificial Chromosomes (BACs) or other sources. SLiCE is highly cost effective as a number of standard laboratory bacterial strains can serve as sources for SLiCE extract. In addition, the cloning efficiencies and capabilities of these strains can be greatly improved by simple genetic modifications. As an example, we modified the DH10B Escherichia coli strain to express an optimized λ prophage Red recombination system. This strain, termed PPY, facilitates SLiCE with very high efficiencies and demonstrates the versatility of the method.

Bricks and blueprints: methods and standards for DNA assembly

“…Uracil-Specific Excision Reagent (USER) assembly does so by producing nicks in the homology regions 46,47 , whereas Gibson assembly instead employs a "chew-back" mechanism on one strand, improving on the previously described Sequence and Ligation-Independent Cloning (SLIC) method by the addition of in vitro DNA repair 2,48 . In these methods, purified repair enzymes can also be replaced by cellular DNA repair systems; for example, the Seamless Ligation Cloning Extract (SLiCE) method can assemble DNA parts in vitro at very low cost by using E. coli cell extracts instead of an enzyme cocktail like that used by Gibson assembly 49 . Other methods take a step further, relying on the repair machinery of live organisms like Bacillus subtilis and Saccharomyces cerevisiae, which are able to take up linear DNA parts with overlaps and assemble them spontaneously in vivo into the desired constructs via native homologous recombination [50][51][52] .…”

Kinetochore assembly and heterochromatin formation occur autonomously in Schizosaccharomyces pombe

“…CNP1 was tagged at the amino terminus with mGFP as described previously (14), CNP3 was tagged at the carboxyl terminus with the PK-9 epitope, Swi6 was tagged with GFP using the EGFPpAL2U plasmid (a gift of Jun-ichi Nakayama, Nagoya City University, Nagoya City, Japan), and Rad 21 was tagged with PK-9 as described previously (17). Tagging constructs were assembled from constituent fragments using SLiCE (25). All tagging and targeting used plasmid constructs, because CBS 2777 transforms ∼50-fold less efficiently than the laboratory strain.…”

ACEMBL Tool-Kits for High-Throughput Multigene Delivery and Expression in Prokaryotic and Eukaryotic Hosts

“…The advent of the polymerase chain reaction (PCR) enormously advanced the field [16], making DNA cloning commonplace in virtually all molecular biology laboratories worldwide. Today, DNA assembly has been further accelerated by new and powerful technologies, including ligation independent cloning methods (LIC, SLIC) [17,18], circular polymerase extension cloning (CPEC) [19] and seamless ligation cloning extract (SliCE) [20], to name a few. For the assembly of very large fragments as precursors of entire synthetic genomes, specific cloning methods have been implemented [21].…”