1. George Church Wed 20-Sep-2006 Science Center Hall E Thanks to: Synthetic, constructive, Adaptive Biology NHGRI Seq Tech 2004: Agencourt, 454, Microchip, 2005: Nanofluidics, Network, VisiGen Affymetrix, Helicos, Solexa-Lynx

2. iGEM International Genetically Engineered Machines : 37 teams

3. Harvard iGEM team 2006 DNA Nanostructures Tiffany Chan Katherine Fifer Valerie Lau Matthew Meisel Cyanobacteria Hetmann Hsieh Jeffrey Lau Zhipeng Sun David Ramos Cell surface targeting Perry Tsai Lewis Hahn Teaching Fellows Chris Doucette Shawn Douglas Nicholas Stroustrup Advisors Pamela Silver George Church Radhika Nagpal Jagesh V. Shah William Shih Alain Viel http://openwetware.org/wiki/IGEM:Harvard/2006

4. The Central Dogma of Molecular Biology Inheritance Messenger Sensors/structures/catalysts Structures Sensors Inherited epigenetic states (non-standard view) DNA RNA Protein Genetic code: UUU encodes Phe = F etc Stop codons: UAG, UAA, UGA

5. iGEM project 1: Cyanobacterial Kai clock 24 hr cycle programmable time (works in vitro with only 3 proteins)

6. 2. DNA nanobox for controlled release of protein-pharmaceuticals

7. iGEM project 3: Cell surface targeting / bridging

8. 3 Exponential technologies 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~m$) Synthesis (amu/project~M$) Analysis (kamu~base/$) tRNA

9. DOE Biofuel Goals Miscanthus v Panicum (switchgrass) 22 v 10 tons/ha Goals: 2kg Hybrid seeds v 2 tons rhizomes self-destruction to aid crop rotation, pretreatment $0.10/L goal (NEB >4, corn-EtOH:1.3 soy-diesel:1.93) Integrated cellulases & fermentation to ethanol, butanol, biodiesel, alkanes $0.02/L via metabolic engineering & lab evolution

10. 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

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

12. Genome & Metabolome Computer Aided Design (CAD) 4.7 Mbp new genetic codes new amino acids 7*7 * 4.7 Mbp mini-ecosystems biosensors, bioenergy, high secretors, DNA & metabolic isolation Top Design Utility, safety & scalability CAD-PAM Synthesis (chip & error correction) Combinatorics Evolution Sequence

13. How? 10 Mbp of oligos / $1000 chip 8K Atactic/Xeotron/Invitrogen Photo-Generated Acid Sheng, Zhou, Gulari, Gao (Houston) 12K Combimatrix Electrolytic 44K Agilent Ink-jet standard reagents 380K Nimblegen Photolabile 5'protection Tian et al. Nature. 432:1050; Carr & Jacobson 2004 NAR; Smith & Modrich 1997 PNAS ~1000X lower oligo costs (= 2 E.coli genomes or 20 Mycoplasmas /chip) Amplify pools of 50mers using flanking universal PCR primers and three paths to 10X error correction Digital Micromirror Array

14. ‘Next Generation’ Technology Development Multi-molecule Our role AB/APG/BEC Seq by Ligation (SbL) 454/Roche Paired ends, emulsion SLXA/IVGN/NEB Multiplexing & polonies CGI Seq by Ligation (SbL) Affymetrix Software Single molecules Helicos Biosci SAB, cleavable fluors Pacific Biosci Advisor KPCB Agilent Nanopores Visigen Biotech AB

15. 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

16. New in vivo genetic code: resistant to all viruses; novel amino acids TTT F 30362TCT S 11495TAT Y 21999TGT C 7048 TTC22516TCC11720TAC16601TGC8816 TTA L 18932TCA9783TAA STOP 2703TGA STOP 1256 TTG18602TCG12166TAG326TGGW20683 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 Freeing 4 tRNAs, 7 codons: UAG, UUR, AGY, AGR e.g. PEG-pAcPhe-hGH (Ambrx, Schultz) high serum stability Isaacs Church Forster Carr Jacobson Jahnz Schultz 1 2 3 4

17. To change the genetic code 1.Free up 1 or more codons by change 30 to 30,000 codons throughout the genome 2.Remove the RF or tRNA dedicated to those codon(s) 3.Add orthogonal tRNA and synthetase protein 4.Add selectable gene dependent on above

18. Schultz tRNAs http://www.sciencemag.org/cgi/reprint/302/5645/584.pdf

19. Evolving orthogonal tRNA charging enzymes http://schultz.scripps.edu/Images_103105/research_fig3.jpg

20. Microbial lab evolution LenskiCitrate utilization ChurchTrp/Tyr exchange PalssonGlycerol utilization Edwards Radiation resistance IngramLactate production StephanopoulosEthanol resistance MarliereThermotolerance J&JDiarylquinoline resistance DuPont1,3-propanediol production

21. Cross-feeding symbiotic systems: aphids & Buchnera obligate mutualism nutritional interactions: amino acids and vitamins established 200-250 million years ago close relative of E. coli with tiny genome (618~641kb) Aphids Internal view of the aphid. (by T. Sasaki) Bacteriocyte (Photo by T. Fukatsu) Buchnera (Photo by M. Morioka) http://buchnera.gsc.riken.go.jp

22. Shigenobu et al. Genome sequence of the endocellular bacterial symbiont of aphids Buchnera sp.APS. Nature 407, 81-86 (2000).

23.  trp/  tyrA pair of genomes shows the best co-growth Reppas, Lin & Church ; Shendure et al. Accurate Multiplex Polony Sequencing of an Evolved Bacterial Genome(2005) Science 309:1728 Second Passage First Passage Synthetic combinatorics & evolution of 7*7* 4.7 Mbp genomes

24. Sequence monitoring of evolution (optimize small molecule synthesis/transport) Sequence trp - Reppas, Lin & Church

25. Consensus error rate Total errors (E.coli) (Human) 1E-4 Bermuda/Hapmap 500 600,000 4E-5 454 @40X 200 240,000 3E-7 Polony-SbL @6X 0 1800 1E-8 Goal for 2006 0 60 Goal of genotyping & resequencing  Discovery of variants E.g. cancer somatic mutations ~1E-6 (or lab evolved cells) Why low error rates? Also, effectively reduce (sub)genome target size by enrichment for exons or common SNPs to reduce cost & # false positives.

26. PositionType GeneLocation ABI Confirm Comments 986,334 T > GompFPromoter-10 Only in evolved strain 985,797 T > GompFGlu > Ala Only in evolved strain 931,960 ▲ 8 bplrpframeshift Only in evolved strain 3,957,960 C > TppiC5' UTR MG1655 heterogeneity -3274 T > CcIGlu > Glu  red heterogeneity -9846 T > CORF61Lys > Gly  red heterogeneity Mutation Discovery in Engineered/Evolved E.coli Shendure, Porreca, et al. (2005) Science 309:1728

27. Glu-117 → Ala (in the pore) Charged residue known to affect pore size and selectivity Promoter mutation at position (-12) Makes -10 box more consensus-like -12 -11 -10 -9 -8 -7 -6 A AAGAT C AAGAT Can increase import & export capability simultaneously ompF - non-specific transport channel

28. 3 independent lines of Trp/Tyr co-culture frozen. OmpF: 42R-> G, L, C, 113 D->V, 117 E->A Promoter: -12A->C, -35 C->A Lrp: 1bp deletion, 9bp deletion, 8bp deletion, IS2 insertion, R->L in DBD. Heterogeneity within each time-point reflecting colony heterogeneity. Co-evolution of mutual biosensors sequenced across time & within each time-point

29. .