A simple and ultra-low cost homemade seamless ligation cloning extract (SLiCE) as an alternative to a commercially available seamless DNA cloning kit

The seamless ligation cloning extract (SLiCE) method is a novel seamless DNA cloning tool that utilizes homologous recombination activities in Escherichia coli cell lysates to assemble DNA fragments into a vector. Several laboratory E. coli strains can be used as a source for the SLiCE extract; therefore, the SLiCE-method is highly cost-effective.The SLiCE has sufficient cloning ability to support conventional DNA cloning, and can simultaneously incorporate two unpurified DNA fragments into vector. Recently, many seamless DNA cloning kits have become commercially available; these are generally very convenient, but expensive. In this study, we evaluated the cloning efficiencies between a simple and highly cost-effective SLiCE-method and a commercial kit under various molar ratios of insert DNA fragments to vector DNA. This assessment identified that the SLiCE from a laboratory E. coli strain yielded 30−85% of the colony formation rate of a commercially available seamless DNA cloning kit. The cloning efficiencies of both methods were highly effective, exhibiting over 80% success rate under all conditions examined. These results suggest that SLiCE from a laboratory E. coli strain can efficiently function as an effective alternative to commercially available seamless DNA cloning kits.

[1]  S. Lovett,et al.  Mechanisms of Recombination: Lessons from E. coli , 2008 .

[2]  A. Kuzminov.  Recombinational repair of DNA damage in Escherichia coli and bacteriophage lambda. , 1999, Microbiology and molecular biology reviews : MMBR.

[3]  Youming Zhang,et al.  RecE/RecT and Redα/Redβ initiate double-stranded break repair by specifically interacting with their respective partners , 2000, Genes & Development.

[4]  D. G. Gibson,et al.  Enzymatic assembly of DNA molecules up to several hundred kilobases , 2009, Nature Methods.

[5]  T. Hisabori,et al.  CcdA is a thylakoid membrane protein required for the transfer of reducing equivalents from stroma to thylakoid lumen in the higher plant chloroplast. , 2010, Antioxidants & redox signaling.

[6]  Y. Meyer,et al.  Resemblance and Dissemblance of Arabidopsis Type II Peroxiredoxins: Similar Sequences for Divergent Gene Expression, Protein Localization, and Activity1 , 2003, Plant Physiology.

[7]  L. Bird,et al.  Codon optimization and factorial screening for enhanced soluble expression of human ciliary neurotrophic factor in Escherichia coli , 2014, BMC Biotechnology.

[8]  W. Edelmann,et al.  SLiCE: a novel bacterial cell extract-based DNA cloning method , 2012, Nucleic acids research.

[9]  F. Horling,et al.  The function of the chloroplast 2-cysteine peroxiredoxin in peroxide detoxification and its regulation. , 2002, Journal of experimental botany.

[10]  W. Brown,et al.  Accuracy and efficiency define Bxb1 integrase as the best of fifteen candidate serine recombinases for the integration of DNA into the human genome , 2013, BMC Biotechnology.

[11]  Masasuke Yoshida,et al.  A novel approach to crystallizing proteins with temperature-induction method: GrpE protein from Thermus thermophilus , 1998 .

[12]  K. Motohashi,et al.  Evaluation of seamless ligation cloning extract preparation methods from an Escherichia coli laboratory strain. , 2015, Analytical biochemistry.

[13]  S. Lovett,et al.  Mechanisms of recombination: lessons from E. coli. , 2008, Critical reviews in biochemistry and molecular biology.

[14]  T. Hisabori,et al.  HCF164 Receives Reducing Equivalents from Stromal Thioredoxin across the Thylakoid Membrane and Mediates Reduction of Target Proteins in the Thylakoid Lumen* , 2006, Journal of Biological Chemistry.

[15]  Margaret C. M. Smith,et al.  A novel Streptomyces spp. integration vector derived from the S. venezuelae phage, SV1 , 2014, BMC Biotechnology.

[16]  K. Motohashi,et al.  Method for enhancement of plant redox-related protein expression and its application for in vitro reduction of chloroplastic thioredoxins. , 2014, Protein expression and purification.

[17]  P. Bjerling,et al.  Multi-fragment site-directed mutagenic overlap extension polymerase chain reaction as a competitive alternative to the enzymatic assembly method. , 2014, Analytical biochemistry.

[18]  H. Taguchi,et al.  Stabilization of FtsH-unfolded protein complex by binding of ATP and blocking of protease. , 2002, Biochemical and biophysical research communications.

[19]  I. Polikarpov,et al.  High-throughput cloning, expression and purification of glycoside hydrolases using Ligation-Independent Cloning (LIC). , 2014, Protein expression and purification.

[20]  Eric Koesema,et al.  Combining the polymerase incomplete primer extension method for cloning and mutagenesis with microscreening to accelerate structural genomics efforts , 2008, Proteins.

[21]  Jingdong Tian,et al.  Circular Polymerase Extension Cloning of Complex Gene Libraries and Pathways , 2009, PloS one.

[22]  S. Elledge,et al.  Harnessing homologous recombination in vitro to generate recombinant DNA via SLIC , 2007, Nature Methods.

[23]  T. Hisabori,et al.  Chloroplast Cyclophilin Is a Target Protein of Thioredoxin , 2003, Journal of Biological Chemistry.

[24]  H. De Greve,et al.  Cloning, expression, and purification of the N-terminal domain of the Flo1 flocculation protein from Saccharomyces cerevisiae in Pichia pastoris. , 2013, Protein expression and purification.

[25]  S. Lovett,et al.  RecA-independent recombination is efficient but limited by exonucleases , 2007, Proceedings of the National Academy of Sciences.

[26]  H. Ohta,et al.  In vitro reconstitution of monogalactosyldiacylglycerol (MGDG) synthase regulation by thioredoxin , 2006, FEBS letters.

[27]  S. Lovett,et al.  Crossing over between regions of limited homology in Escherichia coli. RecA-dependent and RecA-independent pathways. , 2002, Genetics.

[28]  D. Middleton,et al.  Expression and purification of the aortic amyloid polypeptide medin. , 2014, Protein expression and purification.

[29]  S. Smits,et al.  A simple in vitro acylation assay based on optimized HlyA and HlyC purification. , 2014, Analytical biochemistry.

[30]  A. Kuzminov.  Recombinational Repair of DNA Damage inEscherichia coli and Bacteriophage λ , 1999, Microbiology and Molecular Biology Reviews.

[31]  Ken Motohashi,et al.  A simple and efficient seamless DNA cloning method using SLiCE from Escherichia coli laboratory strains and its application to SLiP site-directed mutagenesis , 2015, BMC Biotechnology.

[32]  C. Benning,et al.  Genome-wide analysis of glucose-6-phosphate dehydrogenases in Arabidopsis. , 2004, The Plant journal : for cell and molecular biology.