Abstract
AbstractA new method is described for the seamless assembly of independent, prefabricated and functionally tested blunt-end, double strand nucleic acidparts(DNA fragments) into more complex biologicaldevices(vectors) and higher ordermulti-devicesystems.Individualpartsinclude 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 ofpartswith 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 ofpartsis an inherent feature of the new process, substantially simplifyingdeviceandsystemoptimization. To underscore the utility of the new process,partswere assembled into several protein expressiondevicesin 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 systemis also described, for the over-expression of a four-enzyme bio-synthetic pathway, and optimized for end-product accumulation inE. colias a paradigm for how this assembly process could be used to address the assembly of more complex biological pathways.
Publisher
Cold Spring Harbor Laboratory