DNA Fragments Assembly Based on Nicking Enzyme System

A couple of DNA ligation-independent cloning (LIC) methods have been reported to meet various requirements in metabolic engineering and synthetic biology. The principle of LIC is the assembly of multiple overlapping DNA fragments by single-stranded (ss) DNA overlaps annealing. Here we present a method to generate single-stranded DNA overlaps based on Nicking Endonucleases (NEases) for LIC, the method was termed NE-LIC. Factors related to cloning efficiency were optimized in this study. This NE-LIC allows generating 3′-end or 5′-end ss DNA overlaps of various lengths for fragments assembly. We demonstrated that the 10 bp/15 bp overlaps had the highest DNA fragments assembling efficiency, while 5 bp/10 bp overlaps showed the highest efficiency when T4 DNA ligase was added. Its advantage over Sequence and Ligation Independent Cloning (SLIC) and Uracil-Specific Excision Reagent (USER) was obvious. The mechanism can be applied to many other LIC strategies. Finally, the NEases based LIC (NE-LIC) was successfully applied to assemble a pathway of six gene fragments responsible for synthesizing microbial poly-3-hydroxybutyrate (PHB).

[1]  R. Seidel,et al.  Nicking enzyme–based internal labeling of DNA at multiple loci , 2012, Nature Protocols.

[2]  B. Wanner,et al.  One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[3]  H. Kong,et al.  The nicking endonuclease N.BstNBI is closely related to type IIs restriction endonucleases MlyI and PleI. , 2001, Nucleic acids research.

[4]  Guoqiang Chen,et al.  Production of 3-hydroxypropionate homopolymer and poly(3-hydroxypropionate-co-4-hydroxybutyrate) copolymer by recombinant Escherichia coli. , 2011, Metabolic engineering.

[5]  Rolf Müller,et al.  Full-length RecE enhances linear-linear homologous recombination and facilitates direct cloning for bioprospecting , 2012, Nature Biotechnology.

[6]  Huimin Zhao,et al.  DNA assembler method for construction of zeaxanthin-producing strains of Saccharomyces cerevisiae. , 2012, Methods in molecular biology.

[7]  Penghua Zhang,et al.  Discovery of natural nicking endonucleases Nb.BsrDI and Nb.BtsI and engineering of top-strand nicking variants from BsrDI and BtsI , 2007, Nucleic acids research.

[8]  Xiaohua Huang,et al.  Linear nicking endonuclease-mediated strand-displacement DNA amplification. , 2011, Analytical biochemistry.

[9]  B. Wanner,et al.  Use of the rep technique for allele replacement to construct new Escherichia coli hosts for maintenance of R6K gamma origin plasmids at different copy numbers. , 1994, Gene.

[10]  Guo-qiang Chen,et al.  Overexpression of NAD kinase in recombinant Escherichia coli harboring the phbCAB operon improves poly(3-hydroxybutyrate) production , 2009, Applied Microbiology and Biotechnology.

[11]  Shuang-yong Xu,et al.  Engineering strand-specific DNA nicking enzymes from the type IIS restriction endonucleases BsaI, BsmBI, and BsmAI. , 2004, Journal of molecular biology.

[12]  P. D. de Jong,et al.  Ligation-independent cloning of PCR products (LIC-PCR). , 1990, Nucleic acids research.

[13]  Fernando Geu-Flores,et al.  USER fusion: a rapid and efficient method for simultaneous fusion and cloning of multiple PCR products , 2007, Nucleic acids research.

[14]  J. Bitinaite,et al.  DNA Cloning and Engineering by Uracil Excision , 2009, Current protocols in molecular biology.

[15]  G. Kachalova,et al.  Nicking endonucleases , 2009, Biochemistry (Moscow).

[16]  T. Tanabe,et al.  Preparation of long sticky ends for universal ligation-independent cloning: sequential T4 DNA polymerase treatments. , 2009, Journal of bioscience and bioengineering.

[17]  Qingming Luo,et al.  A ligation-independent cloning method using nicking DNA endonuclease. , 2010, BioTechniques.

[18]  Qian-Qian Liu,et al.  Biosynthesis of poly(3-hydroxydecanoate) and 3-hydroxydodecanoate dominating polyhydroxyalkanoates by β-oxidation pathway inhibited Pseudomonas putida. , 2011, Metabolic engineering.

[19]  B. Tsvetanova,et al.  Genetic assembly tools for synthetic biology. , 2011, Methods in enzymology.

[20]  Shuang-yong Xu,et al.  Engineering Nt.BtsCI and Nb.BtsCI nicking enzymes and applications in generating long overhangs , 2009, Nucleic acids research.

[21]  Huimin Zhao,et al.  Rapid characterization and engineering of natural product biosynthetic pathways via DNA assembler. , 2011, Molecular bioSystems.

[22]  Barbara A Halkier,et al.  USER cloning and USER fusion: the ideal cloning techniques for small and big laboratories. , 2010, Methods in molecular biology.

[23]  Jessica S. Dymond,et al.  Assembling DNA fragments by USER fusion. , 2012, Methods in molecular biology.

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

[25]  Tom Ellis,et al.  DNA assembly for synthetic biology: from parts to pathways and beyond. , 2011, Integrative biology : quantitative biosciences from nano to macro.

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

[27]  George M Church,et al.  Towards synthesis of a minimal cell , 2006, Molecular systems biology.

[28]  Ahmad S. Khalil,et al.  Synthetic biology: applications come of age , 2010, Nature Reviews Genetics.

[29]  C. S. Millard,et al.  A family of LIC vectors for high-throughput cloning and purification of proteins. , 2009, Methods in molecular biology.

[30]  Lin-Ping Wu,et al.  Production and characterization of poly(3-hydroxypropionate-co-4-hydroxybutyrate) with fully controllable structures by recombinant Escherichia coli containing an engineered pathway. , 2012, Metabolic engineering.

[31]  Ralf Seidel,et al.  Efficient preparation of internally modified single-molecule constructs using nicking enzymes , 2010, Nucleic acids research.

[32]  A. C. Chang,et al.  Construction of biologically functional bacterial plasmids in vitro. , 1973, Proceedings of the National Academy of Sciences of the United States of America.

[33]  Morten H. H. Nørholm,et al.  Advancing uracil-excision based cloning towards an ideal technique for cloning PCR fragments , 2006, Nucleic acids research.

[34]  Romualdas Vaisvila,et al.  USER™ friendly DNA engineering and cloning method by uracil excision , 2007, Nucleic acids research.