One-pot DNA construction for synthetic biology: the Modular Overlap-Directed Assembly with Linkers (MODAL) strategy

Arturo Casini,Geoff S Baldwin,Paul S Freemont,Georgia Christodoulou,James T Macdonald,Joachim De Jonghe,Tom Ellis

doi:10.1093/nar/gkt915

Abstract

Overlap-directed DNA assembly methods allow multiple DNA parts to be assembled together in one reaction. These methods, which rely on sequence homology between the ends of DNA parts, have become widely adopted in synthetic biology, despite being incompatible with a key principle of engineering: modularity. To answer this, we present MODAL: a Modular Overlap-Directed Assembly with Linkers strategy that brings modularity to overlap-directed methods, allowing assembly of an initial set of DNA parts into a variety of arrangements in one-pot reactions. MODAL is accompanied by a custom software tool that designs overlap linkers to guide assembly, allowing parts to be assembled in any specified order and orientation. The in silico design of synthetic orthogonal overlapping junctions allows for much greater efficiency in DNA assembly for a variety of different methods compared with using non-designed sequence. In tests with three different assembly technologies, the MODAL strategy gives assembly of both yeast and bacterial plasmids, composed of up to five DNA parts in the kilobase range with efficiencies of between 75 and 100%. It also seamlessly allows mutagenesis to be performed on any specified DNA parts during the process, allowing the one-step creation of construct libraries valuable for synthetic biology applications.

Highlights

Synthetic biology is a rapidly growing interdisciplinary field that takes an engineering approach to biosciences research and to the development of new biotechnologies [1,2]
On in the emergence of synthetic biology, a standard for DNA assembly that defined DNA sequences as biological parts known as BioBricks [17], gained significant traction despite recognized shortcomings that include requiring all DNA parts to be absent of four common restriction enzyme recognition sites, and only permitting pairwise assembly [9]
The approach described here brings standardization to the powerful overlap-directed methods described above and enables rapid one-pot modular assembly through the use of designed overlap sequences that are compatible with a variety of long overlapping end assembly techniques. We call this strategy MODAL (Modular Overlap-Directed Assembly with Linkers) and we show here how it can be used to efficiently build and shuffle synthetic constructs made from gene-level parts (i.e. DNA parts encoding everything from promoter through to terminator)

Summary

Introduction

Synthetic biology is a rapidly growing interdisciplinary field that takes an engineering approach to biosciences research and to the development of new biotechnologies [1,2]. On in the emergence of synthetic biology, a standard for DNA assembly that defined DNA sequences as biological parts known as BioBricks [17], gained significant traction despite recognized shortcomings that include requiring all DNA parts to be absent of four common restriction enzyme recognition sites, and only permitting pairwise (two-at-a-time) assembly [9]. Iterations of this standard have since appeared that address some of the limitations of BioBrick assembly and the BioBrick part format [18,19], but these have not been widely adopted. In e7 Nucleic Acids Research, 2014, Vol 42, No 1

Methods

Results

Conclusion