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

Abstract

DNA assembly allows individual DNA constructs or designed mixtures 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 minimising the number of vectors. Only one destination vector is required per organism, reflecting our optimisation of the system for metabolic engineering in diverse organisms. Metabolic engineering using Start-Stop Assembly was demonstrated by combinatorial assembly of carotenoid pathways in E. coli resulting in a wide range of carotenoid production and colony size phenotypes indicating the intended exploration of design space.

Synthetic biology tools for the study of relevant factors in the control of transgene expression

“…Non-modular DNA assembly techniques are closer to conventional approaches, based on the overlapping of sequences and direct assembly (Taylor et al, 2018). In the last years, many DNA assembly techniques have been published such as SLIC (Zhang et al, 2012), SLiCE (Zhang et al, 2012) or AQUA cloning (Beyer et al, 2015), but the most commonly used is the Gibson Assembly strategy, a single-reaction method able to join multiple large overlapping DNA fragments in an isothermal reaction using a 5' exonuclease, a DNA polymerase and a DNA ligase (Gibson et al, 2009).…”

“…The same researchers developed one year later the Golden Gate cloning system (Engler et al, 2009), based on their previous work using type IIS restriction enzymes. In this new strategy, each DNA part is cut with IIS restriction enzymes and is left with unique nonpalindromic cohesive ends that anneal only with its corresponding overhang present in other DNA part, which ends up in directional and efficient assembly, since the concentration of initial or re-ligated plasmids decreases over time due to concurrent restriction/ligations reactions (Engler et al, 2009;Taylor et al, 2018).…”

“…In fact, there are many publications that describe adaptations of these methods to different organisms and the development of toolkits with sets of DNA parts for specific purposes. (Taylor et al, 2018) The use of these systems allows the design and generation of SynBio tools to study any topic of interest. In this thesis, different SynBio tools have been applied for the study of two different problems by applying the same strategy, that consisted on the generation of multiple constructs that just differ in one specific DNA part, which permits to study the effect that those DNA parts have when the rest of the construct remains exactly the same.…”