DNA Fragments Assembly Based on Nicking Enzyme System

Rui-Yan Wang,Guo-Qiang Chen,Ying-Ying Guo,Jin-Chun Chen,Zhen-Yu Shi,John R Battista

doi:10.1371/journal.pone.0057943

Rui-Yan Wang, Guo-Qiang Chen + Show 4 more

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https://doi.org/10.1371/journal.pone.0057943

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Abstract

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).

Highlights

The assembly of pathways, controllable systems and whole genomic level manipulation are important in synthetic biology and for certain applications including microbial productions of antibiotics, biofuels, biomaterials and the creation of minimal free living cells [1,2,3,4]
Following polymerase chain reaction (PCR) amplification and DNA purification, all fragments were digested by nicking endonucleases to produce a nick at one single strand of the double-stranded DNA fragments, followed by incubating the digested fragments in a thermo-cycler for formation of single-stranded DNA overlaps via denaturing the double strands DNA at 90uC for 5 min
Nicking Endonucleases (NEases) have been used to generate ss DNA overlaps for cloning DNA fragments into plasmids [27], this study is the first one using NEases in ligation-independent cloning (LIC) for constructing a pathway consisting of multiple genes

Summary

Introduction

The assembly of pathways, controllable systems and whole genomic level manipulation are important in synthetic biology and for certain applications including microbial productions of antibiotics, biofuels, biomaterials and the creation of minimal free living cells [1,2,3,4]. Methods of ligation-independent cloning (LIC) such as LIC based on exonuclease [9,11], sequence and ligation independent cloning (SLIC) [12], improved SLIC (i.e. the one-step thermo-cycled assembly method [13]), and uracil excision-based cloning [14,15,16,17,18], have become popular. All these LIC methods are based on the annealing of complementary single-stranded (ss) DNA [9]. DNA exonucleases such as T4 DNA polymerase or lambda exonuclease, have been used to produce single-stranded overlaps as described in most of the above methods except the uracil excision-based cloning one [19]

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