1. 1 16-Oct-2008 DARPA Cambridge, MA Connecting Minimal Synthetic Biology with Millibiology Classification of replication/reproduction How close to atomic inputs ? (e.g. autotrophs use CO2, N2, etc.) Non-translational, non-universal construction replication: Schulman & Winfree. ECAL 2005. Self-Replication and Evolution of DNA crystals Sievers D, von Kiedrowski G. Nature. 1994 Self-replication of complementary nucleotide-based oligomers.

2. 2 3 Exponential technologies “law of accelerating returns” Shendure et al. 2004 Nature Reviews of Genetics. Kurzweil 2002; Moore 1965 (24 month doubling til 2020) urea B12 tRNA telegraph Computation & Communication Analytic tRNA Synthetic chemistry

3. 3 NextGen Synthesis: off chip 8K Xeotron Photo-Generated Acid 12K Combimatrix Electrolytic 120K Roche, Febit Photolabile 5'protection 244K Agilent Ink-jet standard reagents Tian et al. 2004 Nature Elledge, Hannon, et al. $500 per 15Mbp Application to shRNA & exon capture libraries

4. 4 Not minimal genomes High speed & accuracy Mycoplasma 500 RNA & protein genes Mitochondria 1500 genes Mike Jewett E.coli in vitro 151 genes Ease of synthesis More radical designs Existing applications -Roche Rapid Translation System (RTS) -Ambion ActivePro In Vitro Translation Kit -Novagen, Promega, Invitrogen, Qiagen Stratagene, Paragon, Amersham

5. 5 113 kbp DNA 151 genes Pure translation: Forster & Church MSB ’05 GenomeRes.’06 Shimizu, Ueda ’01 Not minimal: High speed & accuracy requires a few extra genes (E.coli 20 min. doubling) Reconstituted ribosomes: Jewett & Church

6. 6 Charalel Church Isaacs Sun Wang Carr Jacobson Kong Sterling New genetic code: viral-resistance, novel amino acids no functional GMO DNA exchange TTT F 30362TCT S 11495TAT Y 21999TGT C 7048 TTC22516TCC11720TAC16601TGC8816 TTA L 18932TCA9783TAA STOP 2703TGA STOP 1256 TTG18602TCG12166TAG314TGGW20683 CTT L 15002CCT P 9559CAT H 17613CGT R 28382 CTC15077CCC7485CAC13227CGC29898 CTA5314CCA11471CAA Q 20888CGA4859 CTG71553CCG31515CAG39188CGG7399 ATT I 41309ACT T 12198AAT N 24159AGT S 11970 ATC34178ACC31796AAC29385AGC21862 ATA 5967ACA9670AAA K 45687AGA R 2896 ATGM37915ACG19624AAG14029AGG1692 GTT V 24858GCT A 20762GAT D 43719GGT G 33622 GTC20753GCC34695GAC25918GGC40285 GTA14822GCA27418GAA E 53641GGA10893 GTG35918GCG45741GAG24254GGG15090 PEG-pAcPhe-hGH (Ambrx) high serum stability 314 TAG to TAA changes 1 2 3 4