A Fast, Scalable, Automated, One-pot Process for Assembly of DNA Oligonucleotides into Large Gene Sequences

The field of synthetic biology is reliant on large quantities of artificial DNA sequences in order to engineer cellular activity and study precisely which genetic elements are effective for producing a desired result in an engineered cell. The goal of this project is to improve the throughput of the “design-build-test” workflows used in synthetic biology by developing new methods for producing high-fidelity genome-length DNA sequences. A set of well-characterized molecular biology methods will be combined to create a novel multiplexable process for the assembly of synthesized DNA oligonucleotides into larger sequences that is more rapid, efficient, and automatable than those currently used in industry or described in recently published studies. The process receives as inputs oligonucleotide pools derived from DNA microarray slides and outputs large, kilobase-scale fragments ligated into vectors. The procedure can then be iterated again to form larger fragments, which can then be further combined in yeast to ultimately form megabase-scale DNA sequences. In Phase I, parameters for each step of this process will be optimized for speed and robustness and for the fidelity of output sequences. Ideally, this effort will result in the discovery of sets of reaction conditions for each step that are compatible with all other steps, which will allow the process to be iterated for multiple cycles to achieve larger output fragments. Such a procedure would be fully automatable and scales similarly with microarray synthesis. The Phase I work will culminate in a proof-of-concept demonstration in which the process is used to construct DNA fragments of up to 1 Mb in length. Anticipated Phase II work will increase the multiplexing ability of the assembly process and reduce its cost, as well as develop a novel process and device for oligonucleotide synthesis that can be automated and integrated upstream of the assembly process. This integrated process would be unique in its capacity to respond to acute demands related to national defense, public health, and biosecurity, and even the first- generation vision of the device would be portable and deployable for point-of-care DNA manufacturing.