Abstract
BackgroundRecent development of DNA assembly technologies has spurred myriad advances in synthetic biology, but new tools are always required for complicated scenarios. Here, we have developed an alternative DNA assembly method named AFEAP cloning (Assembly of Fragment Ends After PCR), which allows scarless, modular, and reliable construction of biological pathways and circuits from basic genetic parts.MethodsThe AFEAP method requires two-round of PCRs followed by ligation of the sticky ends of DNA fragments. The first PCR yields linear DNA fragments and is followed by a second asymmetric (one primer) PCR and subsequent annealing that inserts overlapping overhangs at both sides of each DNA fragment. The overlapping overhangs of the neighboring DNA fragments annealed and the nick was sealed by T4 DNA ligase, followed by bacterial transformation to yield the desired plasmids.ResultsWe characterized the capability and limitations of new developed AFEAP cloning and demonstrated its application to assemble DNA with varying scenarios. Under the optimized conditions, AFEAP cloning allows assembly of an 8 kb plasmid from 1-13 fragments with high accuracy (between 80 and 100%), and 8.0, 11.6, 19.6, 28, and 35.6 kb plasmids from five fragments at 91.67, 91.67, 88.33, 86.33, and 81.67% fidelity, respectively. AFEAP cloning also is capable to construct bacterial artificial chromosome (BAC, 200 kb) with a fidelity of 46.7%.ConclusionsAFEAP cloning provides a powerful, efficient, seamless, and sequence-independent DNA assembly tool for multiple fragments up to 13 and large DNA up to 200 kb that expands synthetic biologist’s toolbox.
Highlights
Recent development of DNA assembly technologies has spurred myriad advances in synthetic biology, but new tools are always required for complicated scenarios
DNA sequence assembly, which refers to the precise aligning and merging multiple fragments of DNA, in an end-to-end fashion, into large synthetic circuits and pathways, plays a pivotal role in protein structurefunction, metabolic engineering, and synthetic biology [1,2,3,4,5]
The assembly of DNA fragments with AFEAP cloning into circular plasmid requires four steps (Fig. 1a): (i) In the first-round PCR, several PCRs are carried out in parallel with forward and reverse primers of the first set, i.e., Fw1–1 and Rv1–1, Fw2–1 and Rv2–1, Fw3–1 and Rv3–1,..., and Fwn-1 and Rvn-1, to produce double-stranded DNA fragments, i.e., dsDNA 1, dsDNA 2, dsDNA 3,..., dsDNA n; (ii) In the second-round PCR, two single-primer PCRs run in parallel with each one of the forward and reverse primers of the second set, i.e., Fw1–2 or Rv1–2, Fw2–2 or Rv2–2, Fw3–2 or Rv3–2,..., or Fwn-2 or Rvn-2, using each DNA product generated in the first-round PCR as template
Summary
Recent development of DNA assembly technologies has spurred myriad advances in synthetic biology, but new tools are always required for complicated scenarios. Gate assembly [9], uracil-specific excision reagent cloning (USER) [10], ligase cycling reaction (LCR) [11], DNA assembler [12], twin-primer assembly (TPA) [6], sequence and ligation-independent cloning (SLIC) [13], seamless ligation cloning extract (SliCE) [14], enzyme-free cloning (EFC) [15], polymerase incomplete primer extension (PIPE) [16], in Vivo assembly (IVA cloning) [17], DNA assembly with thermostable exonuclease and ligase (DATEL) [7], and overlap extension PCR and recombination (OEPR Cloning) [18], have been designed and developed (Additional file 1: Table S1), which opened doors to a wide variety of applications These methods differ in both mechanism and scale, providing the effective means to cope with different needs [2, 4]. Despite the advantage of these assembly techniques, to our knowledge, no one
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