A simple and efficient seamless DNA cloning method using SLiCE from Escherichia coli laboratory strains and its application to SLiP site-directed mutagenesis

Abstract

Background: Seamless ligation cloning extract (SLiCE) is a simple and efficient method for DNA assembly that uses cell extracts from the Escherichia coli PPY strain, which expresses the components of the λ prophage Red/ET recombination system. This method facilitates restriction endonuclease cleavage site-free DNA cloning by performing recombination between short stretches of homologous DNA (≥ 15 base pairs).

Results: To extend the versatility of this system, I examined whether, in addition to bacterial extracts from the PPY strain, other E. coli laboratory strains were suitable for the SLiCE protocol. Indeed, carefully prepared cell extracts from several strains exhibited sufficient cloning activity for seamless gene incorporation into vectors with short homology lengths (approximately 15-20 bp). Furthermore, SLiCE was applied to the polymerase chain reaction (PCR)-based site-directed mutagenesis method, in a process termed "SLiCE-mediated PCR-based site-directed mutagenesis (SLiP site-directed mutagenesis)". SLiP site-directed mutagenesis simplifies the steps of PCR-based site-directed mutagenesis, as it exploits the capability of the SLiCE method to insert multiple fragments.

Conclusions: SLiCE can be performed in the laboratory with no requirement for a special E. coli strain, and the technique is easily established. This method increases the cloning efficiency, shortens the time for DNA manipulation, and greatly reduces the cost of seamless DNA cloning.

Fig. 1

Overview of SLiCE cloning using Escherichia coli laboratory strains. a Schematic view of the SLiCE cloning protocol. The black boxes and white boxes represent the two homologous regions required for SLiCE cloning. b SLiCE cloning with flanking heterologous sequences in vector sites

Fig. 2

Optimization of the SLiCE reaction. The insert glucose-6-phosphate dehydrogenase 1 (G6PDH1) DNA fragments were amplified using 19-bp overlap primers. The linearized vectors were prepared by PCR amplification. The SLiCE reaction (from JM109 strain of E. coli) was performed at 37 °C. Each value is the mean ± standard deviation of three independent experiments. a Time course of the SLiCE reaction. The insert G6PDH1 DNA fragments and linearized vector were mixed in a molar ratio of 2:1. b The effect of the molar ratio of insert (G6PDH1) DNA fragment to vector on the number of colonies formed. The SLiCE reaction was performed for 10 min

Fig. 3

Outline of PCR-based site-directed mutagenesis using SLiCE. Arrows represent PCR primers. a Original overlap extension method for site-directed mutagenesis. b SLiCE-mediated PCR-based site-directed mutagenesis (SLiP site-directed mutagenesis). The linearized vectors were prepared by PCR amplification or by digesting the vector with restriction enzymes. DpnI was active in the PCR buffers. In the case of linearized vectors prepared by restriction enzymes, DpnI added to the insert DNA fragment mixture was heat-inactivated for 15 min at 80 °C

Fig. 4

Protocol for SLiCE from E. coli laboratory strains. a A “standard protocol” is available for various PCR fragments. b The “rapid protocol” is recommended for PCR fragments amplified as a single band. DpnI treatment efficiently reduces background colony formation. When a restriction enzyme-digested vector is used, DpnI should be inactivated for 15 min at 80 °C