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

Bernardo Pollak,Fernán Federici,Ariel Cerda,Rodrigo A Gutiérrez,Tomás Moyano,Nicola J Patron,Mihails Delmans,Jim Haseloff,Anthony West,Simón Álamos

doi:10.1111/nph.15625

Abstract

High-efficiency methods for DNA assembly have enabled the routine assembly of synthetic DNAs of increased size and complexity. However, these techniques require customization, 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. Standardized 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 the construction of plasmids up to 16 genes and 38kb 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 generalized solution for DNA construction with standardized parts. The cloning system is provided under an OpenMTA license for unrestricted sharing and open access.

Highlights

Standardised approaches to the assembly of large DNAs have played an important role in the development of systematic strategies for reprogramming biological systems
The design of Loop assembly was inspired by existing assembly methods such as GoldenBraid, MoClo, and standardised Gibson assembly
Type IIS restriction endonuclease (RE) sites are employed in head-to-head configurations, eliminating the requirement for end-linkers used in MoClo systems

Summary

Introduction

Standardised approaches to the assembly of large DNAs have played an important role in the development of systematic strategies for reprogramming biological systems. Assembly techniques that enabled the parallel assembly of multiple components in a single reaction have been established These include methods that utilise long-sequence overlaps (Bitinaite et al, 2007; Li and Elledge, 2007; Zhu et al, 2007; Gibson et al, 2009; Bryksin and Matsumura, 2010; Zhang et al, 2012; Beyer et al, 2015; Jin et al, 2016), systems reliant on in vivo recombination (Ma et al, 1987; Gibson et al, 2008b; Joska et al, 2014), and Golden Gate (Engler et al, 2008) based methods that rely on selective digestion and re-ligation of plasmid DNAs with Type IIS RE (Sarrion-Perdigones et al, 2011; Weber et al, 2011; Sarrion-Perdigones et al, 2013; Engler et al, 2014; Storch et al, 2015; Iverson et al, 2016; Moore et al, 2016). Laboratories that employ Gibson assembly rely on their own set of rules and templates for DNA parts, and there has been no community-wide effort to develop a common standard

Methods

Results

Conclusion