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

Ken Motohashi

doi:10.1186/s12896-015-0162-8

Abstract

BackgroundSeamless 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).ResultsTo 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.ConclusionsSLiCE 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-015-0162-8) contains supplementary material, which is available to authorized users.

Highlights

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
An E. coli PPY strain that expresses an optimized λ prophage Red/ET recombination system circumvents this problem by increasing the colony formation rate during polymerase chain reaction (PCR) fragment cloning [14]
To extend the utility of this method, I prepared SLiCE extracts from several E. coli laboratory strains with some modifications, and estimated the efficiency with which redox-related genes from Arabidopsis could be cloned into expression vectors [27–30] (Fig. 1a)

Summary

Introduction

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). Various restriction endonuclease cleavage site-independent cloning methods, which overcome the limitations associated with the lack of unique restriction enzyme sites, have been described [2–13] These methods are based on homologous recombination, in which overlapping sequences present at the 5′ and 3′ ends of DNA fragments are combined. Cell extracts from DH10B (a common E. coli laboratory strain) did not exhibit efficient cloning activity

Methods

Results

Discussion

Conclusion