Loop assembly: a simple and open system for recursive fabrication of DNA circuits.

High efficiency methods for DNA assembly have enabled routine assembly of synthetic DNAs of increased size and complexity. However, these techniques require customisation, elaborate vector sets or serial manipulations for the different stages of assembly. We have developed Loop assembly based on a recursive approach to DNA fabrication. The system makes use of two Type IIS restriction endonucleases and corresponding vector sets for efficient and parallel assembly of large DNA circuits. Standardised level 0 parts can be assembled into circuits containing 1, 4, 16 or more genes by looping between the two vector sets. The vectors also contain modular sites for hybrid assembly using sequence overlap methods. Loop assembly enables efficient and versatile DNA fabrication for plant transformation. We show construction of plasmids up to 16 genes and 38 Kb with high efficiency (>80%). We have characterized Loop assembly on over 200 different DNA constructs and validated the fidelity of the method by high-throughput Illumina plasmid sequencing. Our method provides a simple generalised solution for DNA construction with standardised parts. The cloning system is provided under an OpenMTA license for unrestricted sharing and open access. This article is protected by copyright. All rights reserved.Support for the authors was provided by Becas Chile and the Cambridge Trust (to B.P.), University of Cambridge BBSRC DTP programme (to M.D.), and the Biotechnology and Biological Sciences Research Council and Engineering and Physical Sciences Research Council [OpenPlant Grant No. BB/L014130/1] (to N.P., F.F. and J.H.). Laboratory automation, nextgeneration sequencing and library construction was delivered via the BBSRC National Capability in Genomics (BB/CCG1720/1) at Earlham Institute. F.F. acknowledges funding from CONICYT Fondecyt Iniciación 11140776. F.F. and R.A.G. acknowledge funding from Fondo de Desarrollo de Areas Prioritarias (FONDAP) Center for Genome Regulation (15090007) and Millennium Nucleus Center for Plant Systems and Synthetic Biology (NC130030)