Seamless Assembly of BiologicalPartsinto FunctionalDevicesand Higher OrderMulti-Device Systems

A new method is described for the seamless assembly of independent, prefabricated and functionally tested blunt-end, double strand nucleic acid parts (DNA fragments) into more complex biological devices (vectors) and higher order multi-device systems . Individual parts include bacterial selection markers, bacterial origins of replication, promoters from a variety of different species, transcription terminators, shuttle sequences and a variety of “N” and “C” terminal solubility/affinity expression tags. Pre-assembly modification of parts with DNA modifying enzymes is not required. Seamless assembly of multiple parts is accomplished in a single step using a specialized thermostable enzyme blend in about 30 minutes. Combinatorial assembly of parts is an inherent feature of the new process, substantially simplifying device and system optimization. To underscore the utility of the new process, parts were assembled into several protein expression devices in order to identify the optimal expression construct for a model target gene, as an example of the utility of the assembly process, and a higher order multi- device system is also described, for the over-expression of a four-enzyme bio-synthetic pathway, and optimized for end-product accumulation in E. coli as a paradigm for how this assembly process could be used to address the assembly of more complex biological pathways.