Abstract
BackgroundWith the rapid development of synthetic biology, the demand for assembling multiple DNA (genes) fragments into a large circular DNA structure in one step has dramatically increased. However, for constructions of most circular DNA, there are two contradictions in the ligation/assembly and transformation steps. The ligation/assembly consists of two different reactions: 1) the ligation/assembly between any two pieces of a linear form DNA; 2) the cyclization (or self-ligation) of a single linear form DNA. The first contradiction is that the bimolecular ligation/assembly requires a higher DNA concentration while the cyclization favors a lower one; the second contradiction is that a successful transformation of a ligation/assembly product requires a relatively high DNA concentration again. This study is the first attempt to use linear plasmid and Cyclization After Transformation (CAT) strategy to neutralize those contradictions systematically.ResultsThe linear assembly combined with CAT method was demonstrated to increase the overall construction efficiency by 3–4 times for both the traditional ligation and for the new in vitro recombination-based assembly methods including recombinant DNA, Golden Gate, SLIC (Sequence and Ligation Independent Cloning) and Gibson Isothermal Assembly. Finally, the linear assembly combined with CAT method was successfully applied to assemble a pathway of 7 gene fragments responsible for synthesizing precorrin 3A which is an important intermediate in VB12 production.ConclusionThe linear assembly combined with CAT strategy method can be regarded as a general strategy to enhance the efficiency of most existing circular DNA construction technologies and could be used in construction of a metabolic pathway consisting of multiple genes.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0204-x) contains supplementary material, which is available to authorized users.
Highlights
With the rapid development of synthetic biology, the demand for assembling multiple DNA fragments into a large circular DNA structure in one step has dramatically increased
Synthetic biology often requires the assembly of many fragments at one time
Since Cyclization after Transformation (CAT) strategy has a higher tolerance to high DNA concentration without formation of byproducts as long as every junction is specific, it should have wide application in multiple-fragments ligation/ assembly
Summary
With the rapid development of synthetic biology, the demand for assembling multiple DNA (genes) fragments into a large circular DNA structure in one step has dramatically increased. The rapid developing synthetic biology has an increasing need for assembling multiple genes or operons into a large DNA fragment consisting of multiple metabolic pathways [1,2,3,4]. The second DNA assembly technique is in vitro recombination method based on single-strand overhangs generated by single-strand exonucleases or incomplete PCR (Polymer Chain Reaction), typically represented by OE-PCR (Overlap Extension-PCR). The method is commonly used in vitro to assemble 0.5 to 5 kb DNA fragment [9]. All three improvements use a proprietary enzyme mixture to assemble any fragment with 15+ bp sequence overlap. The resultant abasic site(s) cleaved by an AP-lyase, leaving 30 overhangs annealed with the complementary overhang of a fragment sharing the same overlap sequence [13]
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
