Seamless assembly of DNA parts into functional devices and higher order multi-device systems

RESEARCH ARTICLE Seamless assembly of DNA parts into functional devices and higher order multidevice systems Jeffrey Carl Braman ID*, Peter J. Sheffield* Agilent Technologies, Inc., La Jolla, CA, United States of America * (JCB); (PJS) a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation: Braman JC, Sheffield PJ (2019) Seamless assembly of DNA parts into functional devices and higher order multi-device systems. PLoS ONE 14 (6): e0199653. https://doi.org/10.1371/journal. pone.0199653 Editor: Mark Isalan, Imperial College London, UNITED KINGDOM Received: June 6, 2018 Accepted: June 7, 2019 Published: June 28, 2019 Copyright: © 2019 Braman, Sheffield. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 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. Data Availability Statement: All relevant data are within the paper. Introduction Funding: Agilent internally funded. The authors commercial affiliation did not play a role in the study design, data collection and analysis, decision to publish or preparation of the manuscript and only provided financial support in the form of authors salaries and research materials. More specifically, the funder provided support in the form of salaries and research materials for authors (JCB and PJS) but did not have any additional role in the study design, data collection and analysis, 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. To date however, 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]. Briefly, 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 PLOS ONE | https://doi.org/10.1371/journal.pone.0199653 June 28, 2019 1 / 19 Seamless assembly of DNA parts to form multi-device systems decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section of the online submission form. Competing interests: The authors confirm that our commercial affiliation Agilent Technologies Inc does not alter our adherence to all PLOS ONE policies on sharing data and materials. such as a biosynthetic pathway. BioBrick devices and systems are constructed by “hierarchical binary assembly” of parts, or “one-brick-at-a-time.” More specifically, BioBricks represent functional double strand DNA molecules housed within carrier plasmids flanked by universal and precisely defined upstream and downstream sequences that are technically not part of the BioBrick. These universal sequences contain restriction enzyme recognition sites for one of two closely related enzymes, each having slightly different recognition sequences but upon cleavage generate identical termini (isocaudomers). Linking two BioBricks together requires isolation of the individual parts from their carrier plasmids by specific isocaudomer(s) digestion, end repair in some cases, ligation and finally bacterial transformation. A major drawback to this technique is that BioBrick parts must not contain these restriction enzyme recognition sites within the sequences to be assembled. Also, BioBrick hierarchical binary assembly is time consuming, tedious and not conducive to combinatorial assembly. Current assembly methods that convert parts into devices also rely on the isolation of parts and devices from dedicated BioBrick-like “destination vectors” (BioBricks [1, 2], SLIC [3], Gibson [4], CPEC [5], SLiCE [6], and In-Fusion [http://www.clontech.com/US/Products/ Cloning_and_Competent_Cells/Cloning_Resources/Selec-tion_Guides/In-Fusion_Cloning_ Kits]). In other methods, significant parts manipulation with either one or more Type-II restriction enzymes is required (GoldenGate [7], MoClo [8], GoldenBraid [9]). Alternatively, parts manipulation with T5-exonuclease or a combination of Pfu and Taq DNA polymerases are required for Gibson [4] and DATEL [10] assembly methods, respectively, to create overlaps for subsequent annealing and ligation. In summary, assembly methods are complicated when restriction enzyme specificity must be considered at each stage of parts and devices design. Also, creating small parts between 50 and 250 base pairs with one or more enzymes possessing exonuclease activity is difficult due to the propensity of these enzymes to completely degrade the parts. It is apparent that these limitations curtail combinatorial experimental design and significantly slow the process of identifying optimal devices and systems. Providing functionally validated parts to researchers without the need for retrieval from destination vectors, combined with a seamless protocol conducive to combinatorial assembly of parts into d (...truncated)