Design and implementation of multi-protein expression constructs and combinatorial libraries using start-stop assembly

Start-Stop Assembly is a multi-part, modular, Golden Gate-based DNA assembly system with two key features which distinguish it from previous DNA assembly methods. Firstly, coding sequences are assembled with upstream and downstream sequences via overhangs corresponding to start and stop codons, avoiding unwanted ‘scars’ in assembled constructs at coding sequence boundaries. Scars at these crucial, sensitive locations can affect mRNA structure, activity of the ribosome binding site, and potentially other functional RNA features. Start-Stop Assembly is therefore both functionally scarless (an advantage usually only achieved using bespoke, overlap-based assembly methods) and suitable for efficient, unbiased and combinatorial assembly (a general advantage of Golden Gate-based methods). Secondly, Start-Stop Assembly has a new, streamlined assembly hierarchy, meaning that typically only one new vector is required in order to assemble constructs for any new destination context, such as a new organism or genomic location. This should facilitate more rapid and convenient development of engineered metabolic pathways for diverse nonmodel organisms in order to exploit their applied potential. This chapter explains both design considerations and practical procedures to implement multi-part, hierarchical assembly of multi-protein expression constructs, either individually or as combinatorial libraries, using Start-Stop Assembly.