Project Summary Methods for rapidly generating synthetic nucleic acid constructs have dramatically changed biological and biomedical research. Improvements in these arenas will continue to impact varied areas of genomics and biomedicine such as synthetic genomics and associated functional screens. Enabling significant advances with new nucleic acid synthesis and synthetic construct capabilities has the potential to lead to remarkable improvements in the understanding, diagnosis, treatment and prevention of disease; advances in agriculture, environmental science and remediation; and our understanding of evolution and ecological systems. Current abilities to work with a variety of synthetic constructs have been enabled by cost reductions in oligonucleotide synthesis along with vastly improved techniques for hierarchal assembly of larger constructs, largely in yeast. Our group has led the way both in assembling the yeast genome (i.e. the international Sc2.0 project) and in a NHGRI-sponsored CEGS that has launched the “Dark Matter Project”, aiming to functionally dissect noncoding DNA and its contribution to human/mammalian transcriptional regulation. We have also developed an in-house design software application environment linked to our LIMS called MenDEL (Mentored Design Environment and LIMS). But we need to do much more to make such projects ever easier to do – ultimately, in any lab. In this project we plan to develop the “Assemblatron”, a workflow/Design platform/Host vector system that systematically optimizes the “Yeast assembly” process, and is capable of producing very large DNA molecules of up to a megabase in size. We will develop a system in which one person can assemble 1-2 Megabases of 20-30 kb DNA pieces in a few days’ work, and 1-2 Mb of ~100 kb pieces in 2-4 weeks. The specific goal is >10X improvement in Big DNA assembly efficiency, manifested as the ability of a single researcher, starting with 3 kb starting materials, to do the following: 1) Assemble 1-2 Mb of DNA in 1-2 weeks and 2) Finalize assembly into 10 to 20 100+ kb pieces in 2-4 weeks. Ultimately this research program will lead to development of an Assemblatron device that automates much or all of the process. We plan to achieve this improvement in efficiency, which is currently limited by a series of bottlenecks that exist throughout the workflow using a combination of dry lab and wet lab methods, outlined below.