Abstract
A new method is introduced allowing seamless assembly of independent, functionally tested, blunt-end double strand nucleic acid parts (DNA fragments not supplied in vectors such as plasmids) into more complex biological devices (e.g. protein expression vectors) and higher order multi-device systems (e.g. biochemical pathways). Individual parts include bacterial selection markers and origins of replication, promoters useful in a variety of species, transcription terminators, shuttle sequences and a variety of “N” and “C” terminal solubility/affinity protein tags. Parts are not subjected to pre-assembly manipulation with nucleic acid modifying enzymes. Instead, they are simply mixed in appropriate pre-defined combinations and concentrations and then seamlessly linked into devices employing a specialized thermostable enzyme blend. Combinatorial assembly of parts is an inherent time-saving feature of the new method, in contrast to hierarchical binary assembly (“one part at a time”) methods. This feature substantially simplifies and speeds optimization of device and system development. The versatility and functionality of the new method was shown by combinatorial assembly of parts into vector devices, one of which optimally expressed protein from a model gene. Also, a four-enzyme biosynthetic pathway system was re-created by combinatorial construction from parts and devices. Concepts discussed in this paper provide synthetic biologists, chemists and bio-engineers with improved and expanded capability to create novel biological molecules and systems.
Highlights
The discipline of synthetic biology has greatly benefitted from key enabling technologies such as DNA synthesis and sequencing becoming accessible to more researchers due to the reduction in the previously prohibitive financial entry point
These are: (1) Functional DNA parts are manufactured and quality controlled at a location away from the assembly site (Agilent Technologies, Inc.); (2) Parts, other requisite assembly materials and detailed assembly instructions are delivered to the site of construction for assembly; (3) from a synthetic biology standpoint, processes (1) and (2) enable rapid and reliable combinatorial assembly of desired devices destined for introduction into E. coli, mammalian and yeast cells
The salient features of this new method were demonstrated by constructing a set of protein expression devices to identify the best device for the expression of a target gene of interest (GOI) (Nedd5)
Summary
The discipline of synthetic biology has greatly benefitted from key enabling technologies such as DNA synthesis and sequencing becoming accessible to more researchers due to the reduction in the previously prohibitive financial entry point. A third enabling technology, molecular cloning, has not kept pace with technological advances made in DNA synthesis and DNA sequencing. One of the most highly recognized collection of techniques and materials developed to improve conventional cloning of biological parts, devices and systems is “BioBricks” [1, 2]. The “bricks”, or parts, of this technology represent cloned DNA sequences possessing defined functions, such as antibiotic resistance and ribosome binding sites. Parts are assembled to create larger devices such as protein expression vectors and several devices are joined into a system.
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