Open AccessStart-Stop Assembly: a functionally scarless DNA assembly system optimized for metabolic engineering
Author(s) -
George M. Taylor,
Paweł M. Mordaka,
John Heap
Publication year - 2018
Publication title -
nucleic acids research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 9.008
H-Index - 537
eISSN - 1362-4954
pISSN - 0305-1048
DOI - 10.1093/nar/gky1182
Subject(s) - biology , modular design , metabolic engineering , stop codon , computational biology , synthetic biology , operon , scalability , sequence assembly , dna origami , dna , computer science , gene , genetics , escherichia coli , programming language , gene expression , transcriptome , database
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.