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Rapid, Low-Cost, and High-Fidelity DNA Synthesis and Assembly Techniques


OBJECTIVE: Develop a platform based on novel DNA synthesis and assembly techniques that can produce sequence-verified, dsDNA constructs of at least 20,000 bp in length (including A/T- and G/C- rich sequences), at a cost of less than $0.05/bp, and with a turn time of less than one week. DESCRIPTION: Current approaches to engineering biology rely on an ad hoc, laborious, trial-and-error process, wherein one successful project often does not translate to enabling subsequent new designs. As a result, the state of the art development cycle for engineering new biologically based products and capabilities often takes 7+ years and costs tens to hundreds of millions of dollars (e.g. microbial production of artemisinic acid for the treatment of malaria and the non-petroleum-based production 1,3-propanediol). The impact of current approaches is two-fold. First, the number of new entrants and innovators into both the commercial and research space is immediately limited few have the expertise, capital and/or time necessary to develop and engineer a new product. Second, combined with the inherent complexity of biology, an ad hoc approach often results in one-off efforts that are limited to modifying only a small set of genes and constructing simple, isolated systems and devices. Consequently, while progress has been made, we are constrained to producing only a tiny fraction of the vast number of possible chemicals, materials, diagnostics, therapeutics, and fuels that would be enabled by the ability to truly engineer biology. A new approach is needed. Engineering biology with useful complexity requires new approaches for synthesizing, assembling, and manipulating genetic designs rapidly, cheaply, and accurately. The goal is to shift the designers"mindset towards design and experimentation and to facilitate more complex, previously unattainable system designs and architectures. Unlike computer programming, where writing and producing variants of new code is essentially free, the synthesis and assembly of large DNA constructs (the writing of"biological code") is expensive ($0.40 $0.80 per bp), slow (2wks 2mos turn time), error prone (~10-2 10-3), and limited in length and complexity (typically<5 kbp; A/T and G/C rich sequences are challenging or impossible to construct). These limitations restrict biological designers to constructing conservative, evolutionary designs, with little room for multiple design refinements, variants or new ideas. The ability to synthesize, modify and test many new designs (up to the genome scale) with little overhead will help to inform and create the biological design rules and tools that are necessary for the complex design and development of new biologically-based products and devices. This solicitation is focused on development of a platform, based on novel DNA synthesis and assembly techniques, that can produce error-free, 20 kbp lengths of DNA at scale with a reduced cost per base pair (<$0.05/bp) and rapid turn time (<1wk) compared to the state of the art. A successful platform could be readily transitioned to academic, government, and commercial researchers, all of whom are dependent on DNA synthesis for the evaluation of new biological designs. PHASE I: Determine the technical feasibility and projected cost at scale of the new approach for DNA construction. This includes determining the appropriate component processes for oligonucleotide synthesis, error correction, DNA assembly and verification methods, among others. Establish the performance goals of the new approach for cost per base pair, error rate, turn time, and maximum construct length. Perform appropriate analyses (e.g. modeling) to determine the limits to base pair length, error rate, cost, and turn time as well as limitations on A/T and G/C rich sequences for this approach. Develop an initial concept design and model key elements to transition this approach from benchtop to production at scale. Phase I deliverables will include: a technical report of experiments supporting the feasibility of this approach; defined milestones and metrics for cost per base pair, error rate as a function of base pair length, maximum construct length, and turn time; and a detailed design of proposed manufacturing system with estimated production rate. Also included with the Phase I deliverables is a Phase II proposal that outlines plans for the development, fabrication, and validation of a DNA synthesis and assembly platform. This proposal should include a detailed assessment of the potential path to commercialization, barriers to market entry, and collaborators or partners identified as early adopters for the new system. PHASE II: Finalize the design from Phase I and initiate construction of and production from the new DNA synthesis and assembly platform. Establish performance parameters through experimentation to determine: sequence fidelity of oligonucleotides, assemblies and final constructs; cost per base pair of final assemblies; maximum feasible construct length; turn time and production rate of the DNA synthesis platform; and limitations on sequence complexity. Develop, demonstrate, and validate a DNA synthesis and assembly platform that meets the key performance goals and metrics of sequence verified, dsDNA constructs of at least 20,000 bp in length (including A/T- and G/C- rich sequences), at a cost of less than $0.05/bp, and a turn time of less than one week. Deliverables include a prototype device and valid test data, appropriate for a commercial production path. PHASE III DUAL USE APPLICATIONS: The industrial biotechnology and pharmaceutical sectors are deeply reliant on synthetic DNA constructs to produce novel and high value products. A successful DNA synthesis platform that achieves the key metrics stated for Phase II has significant potential to rapidly transition to commercial use, enabling the biologically-based production of new chemicals, enzymes, fuels, diagnostics, therapeutics, and industrial products. A successful DNA synthesis platform will enable the rapid programming of biologically-based manufacturing platforms through synthesis and assembly of DNA"code"for the production of previously unattainable technologies and products. Such technologies may support a number of current DoD challenges in the areas of novel materials production, diagnostics and vaccine development, as well as enabling new manufacturing capabilities and paradigms. For example, the capability to program systems to rapidly and dynamically prevent, seek out, identify, and repair corrosion/materials degradation in situa challenge which costs the DoD $23B/yr and has no near term solution in sight. REFERENCES: 1) M. Baker."Microarrays, megasynthesis,"Nature Methods, 8(6), p. 457-460, 2011. 2) D.G. Gibson, L. Young, R.-Y. Chuang, J.C. Venter, C.A. Hutchison, and H.O. Smith."Enzymatic assembly of DNA molecules up to several hundred kilobases,"Nature Methods, 6(5), p. 343-345, 2009. 3) J. Quan, I. Saaem, N. Tang, S. Ma, N. Negre, H. Gong, K.P. White, and J. Tian."Parallel on-chip gene synthesis and application to optimization of protein expression,"Nature Biotechnology, 29(5), p. 449-452, 2011. 4) C.-C. Lee, T.M. Synder, and S.R. Quake."A microfluidic oligonucleotide synthesizer,"Nucleic Acids Res., 38(8), p. 2514-2521, 2010. 5) M. Matzas, P.F. Sthler, N. Kefer, N. Siebelt, V. Boisguerin, J.T. Leonard, A. Keller, C.F. Sthler, P. Hberle, B. Gharizadeh, F. Babrzadeh, and G.M. Church."High-fidelity gene synthesis by retrieval of sequence-verified DNA identified using high-throughput pyrosequencing,"Nature Biotechnology, 28(12), p. 1291-1294, 2010. 6) S. Kosuri, N. Eroshenko, E.M. LeProust, M. Super, J. Way, J.B. Li, and G.M. Church."Scalable gene synthesis by selective amplification of DNA pools from high-fidelity microchips,"Nature Biotechnology, 28(12), p. 1295-1299, 2010. 7) M. Algire, R. Krishnakumar, and C. Merryman."Megabases for kilodollars,"Nature Biotechnology, 28(12), p. 1272-1273, 2010. 8) P. Carr."DNA construction: homemade or ordered out?"Nature Methods, 7(11), p. 887-889, 2011.
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