1. DNA Sequencing (& synthesis) Microbe-weather-maps: single cell sequencing @ $ 0.02/kb 30 bp microbial species then drug resistance (~1000 cells/$) Personal IDs from dust: 30 bp from single cells then immune VDJ Cost of DNA Synthesis & Sequencing have dropped 100 - 1000X in the last few years Polony Sequencing & Personal Genome Project Identifiable traits & common (low risk) pharmacogenomics

2. DNA Instrumentation Sequencing $1K/ 1E8*50bp/2days DNA Synthesis $1K/ 1E6*160bp/day Cell/Genome programming 4/day/well

3. In vivo/vitro Synthetic Biology Initial applications Basic Enabling Technologies More general applications cell-cell signals A-to-D & thresholds Biosensors & 3D fabrication Light input Cell-CPU interfaces Smart materials A synthetic multicellular system for programmed pattern formation Basu, et al. Nature 434:1130

4. DNA origami highly -- predictable 3D nanostructures. Example application to NMR of membrane proteins Douglas, Shih, Rothemund

5. Programmable ligand-controlled riboregulators to monitor metabolites. Bayer & Smolke; Isaacs & Collins 2005 Nature Biotech. ON OFF

6. In vivo Counter Designs XisTF4 XisTF3 Int XisTF5 XisTF6 Int 1 2 3 4 Riboswitch counter   Integrase bit counter Cell-cycle counter  01100110 123123

7. Reconstitution of Circadian Oscillation of Cyanobacterial KaiC Phosphorylation in vitro Nakajima, et al. Science. 2005 Apr 15;308(5720):414-5

8. Engineering a mevalonate pathway in Escherichia coli for production of terpenoids. Martin VJ, et al. Nat. Biotech 2003 Production of the antimalarial drug precursor artemisinic acid in engineered yeast. Ro DK, et al. Nature. 2006 8

9. Smart therapeutics: Environmentally controlled invasion of cancer cells by engineered bacteria. Anderson et al. J Mol Biol. 2006 Optical imaging: bacteria, viruses, and mammalian cells encoding light- emitting proteins reveal the locations of primary tumors & metastases in animals. Yu, et al. Anal. Bioanal. Chem. 2003. accumulate in tumors at ratios in excess of 1000:1 compared with normal tissues. http://www.vionpharm.com/tapet_virulence.htmlhttp://www.vionpharm.com/tapet_virulence.html Regulated Capsule TonB, DapD & new genetic code for safety

10. Synthetic Genomics 113 kbp mini-genomes ribosome-display selection 4.7 Mbp new genetic codes protein drugs 7*7 * 4.7 Mbp mini-ecosystems biosensors, surface selection, high secretors, DNA & metabolic isolation Top Design Utility, safety & scalability CAD-PAM Synthesis (chip & error correction) Combinatorics Evolution Sequence rEcoded rE.coli v2.0

11. Genome synthesis pipeline 50b 15Kb  5Mb 250 Mb Polymerase in vitro Isaacs, Carr, Emig, Gong, Tian, Reppas, Jacobson, Church Recombination in vivo E.coli Error correction Recombination in human cells Bacterial (Artificial) Chromosomes BACs Human(Artificial) Chromosomes HACs

12. Rationale for in vitro synthetic biology Existing powerful molecular biology kits: DNA RNA Protein PCR, T7 RNA pol, in vitro translation. Build structures incompatible with cells (systems interfering with cells or vice versa) Higher yield, accuracy,purity and/or speed.

13. Why new genetic codes? Precise "post-translational" modifications New reactive AAs (not w/ biomolecules) e.g. PEG-pAcPhe-hGH (Ambrx, Schultz) higher serum stability Azide Strained cyclo-octyne + + Prescher, JA & CR Bertozzi (2005) Nature Chem Biol. Chemistry in living systems hydrazide Ketone triazole hydrazone

14. Mirror world: resistant to enzymes, parasites, predators L-amino acids & D-ribose (rNTPs, dNTPs) Transition: EF-Tu, peptidyl transferase, DNA-ligase D-amino acids & L-ribose (rNTPs, dNTPs) Dedkova, et al. (2003) Enhanced D-amino acid incorporation into protein by modified ribosomes. J Am Chem Soc 125, 6616-7

15. Mirror world: resistant to enzymes, parasites, predators Alternative to ambidextrous ribosome & polymerases is chiral chemical synthesis (Taq Polymerase is 834 AA) > 174 AA African Swine Fever Virus DNA Pol X. MLTLIQGKKIVNHLRSRLAFEYNGQLIKILSKNIVAVGSLRREEKMLND VDLLIIVPEKKLLKHVLPNIRIKGLSFSVKVCGERKCVLFIEWEKKTYQ LDLFTALAEEKPYAIFHFTGPVSYLIRIRAALKKKNYKLNQYGLFKNQT LVPLKITTEKELIKELGFTYRIPKKRL Duhee Bang & Stephen Kent (2004) A One-Pot Total Synthesis of Crambin

16. Mini-genone: 113 kbp, 151 genes

18. Forster & Church (2006) Mol. Syst. Biol. rRNAs precursor RNAs, P RNA, mRNAs tRNAs small molecules RNA pol recombinase AA-tRNAs RNase P chaperones P protein Gln methylase translation factors rProteins ribosome modifying enzymes synthetases, MFT DNA DNA pol Synthesizing A Minimal Cell

19. DNA replication

20. 31 Tagged translation proteins Forster, Nalam, Qu, Ying & Blacklow Synthe- tases: Factors:

21. 33 tRNAs with anticodon modifications

22. Systems Biology of Translation and Replication 151 Genes required.

23. Totally unnatural genetic codes 5' mS yU eU UGG UUG CAG AAC... GUU A 3' GAAACCAUG fMTNVE | | | 5' Second base 3' U A C C U mS yU eU A C U G A Forster, et al. (2003) 80% average yield per unnatural coupling. bK = biotinyllysine, mS = Omethylserine eU=2-amino-4-pentenoic acid yU = 2-amino-4-pentynoic acid

24. 3 Exponential technologies (synergistic) Shendure J, Mitra R, Varma C, Church GM, 2004 Nature Reviews of Genetics. Carlson 2003 ; Kurzweil 2002; Moore 1965 urea E.coli B12 tRNA operons telegraph Computation & Communication (bits/sec) Synthesis (daltons) Analysis (bp/$) tRNA

25. .