Start-Stop Assembly: a functionally scarless DNA assembly system optimized for metabolic engineering.

George M Taylor,Paweł M Mordaka,John T Heap

doi:10.1093/nar/gky1182

Abstract

DNA assembly allows individual DNA constructs or libraries to be assembled quickly and reliably. Most methods are either: (i) Modular, easily scalable and suitable for combinatorial assembly, but leave undesirable ‘scar’ sequences; or (ii) bespoke (non-modular), scarless but less suitable for construction of combinatorial libraries. Both have limitations for metabolic engineering. To overcome this trade-off we devised Start-Stop Assembly, a multi-part, modular DNA assembly method which is both functionally scarless and suitable for combinatorial assembly. Crucially, 3 bp overhangs corresponding to start and stop codons are used to assemble coding sequences into expression units, avoiding scars at sensitive coding sequence boundaries. Building on this concept, a complete DNA assembly framework was designed and implemented, allowing assembly of up to 15 genes from up to 60 parts (or mixtures); monocistronic, operon-based or hybrid configurations; and a new streamlined assembly hierarchy minimizing the number of vectors. Only one destination vector is required per organism, reflecting our optimization of the system for metabolic engineering in diverse organisms. Metabolic engineering using Start-Stop Assembly was demonstrated by combinatorial assembly of carotenoid pathways in Escherichia coli resulting in a wide range of carotenoid production and colony size phenotypes indicating the intended exploration of design space.

Highlights

In recent years, new types of DNA cloning methods often referred to as DNA assembly have been reported which are much more suitable for combining multiple DNA sequence elements or ‘parts’ in a single step than conventional restriction-ligation cloning [1]
We devised a strategy to mitigate the impact of the scars normally generated by modular multi-part DNA assembly methods by exploiting start and stop codons
Start and stop codons represent natural constraints on DNA sequence design, as they must occur at the beginning and end of every coding sequences (CDSs), sites which are sensitive to scars

Summary

Introduction

New types of DNA cloning methods often referred to as DNA assembly have been reported which are much more suitable for combining multiple DNA sequence elements or ‘parts’ in a single step than conventional restriction-ligation cloning [1] These multi-part DNA assembly methods can mostly be categorized into one of two types: (i) Modular approaches, in which a framework of specified steps, rules and design constraints including predefined module formats allows highly efficient multi-part assembly of individual constructs or designed combinatorial mixtures of constructs. Multi-part DNA assembly has quickly become important in synthetic biology, enabling an increase in the scale, scope and speed of studies [1]

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