Advanced DNA assembly strategies and standards for synthetic biology

Abstract

DNA assembly is a fundamental enabling technology for synthetic biology, yet it is also extremely unreliable, expensive and time-consuming. The process usually requires a significant part of total time and effort that can be dedicated to a project, reducing the resources available for the rest of the research, and is also frequently subject to unexpected problems, introducing an undesirable element of unpredictability that might compromise an entire project. This thesis describes the development of three DNA assembly tools that aim to facilitate and speed up synthetic biology research: “MODAL” is a fast and easy to use assembly strategy that brings the advantages of standardisation and modularity to the latest-generation long overlap-based DNA assembly techniques. “Linker” is a software tool that generates DNA sequences specifically optimised to act as high-efficiency homology regions in long overlap-based DNA assembly reactions. Finally with “BASIC” we propose a new DNA assembly standard that incorporates the advances of MODAL and Linker and brings an additional series of improvements in an original assembly workflow. BASIC aims first of all to make DNA assembly significantly more reliable by addressing and/or removing all the unpredictability elements. It also maintains the speed, ease of use and flexibility of MODAL while achieving the same or better efficiency than the best currently available DNA assembly techniques and standards.