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Synthetic, constructive, Adaptive Biology

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

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Synthetic, constructive, Adaptive Biology

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

  2. iGEM International Genetically Engineered Machines : 37 teams

  3. 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 Harvard iGEM team 2006 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. DNA RNA Protein The Central Dogma of Molecular Biology Genetic code: UUU encodes Phe = F etc Stop codons: UAG, UAA, UGA Inheritance Messenger Sensors/structures/catalysts Structures Sensors Inherited epigenetic states (non-standard view)

  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 Computation & Communication (bits/sec~m$) E.coli operons Synthesis (amu/project~M$) tRNA urea B12 Analysis (kamu~base/$) telegraph tRNA Shendure J, Mitra R, Varma C, Church GM, 2004 Nature Reviews of Genetics. Carlson 2003 ; Kurzweil 2002; Moore 1965

  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. OFF ON ON Bayer & Smolke; Isaacs & Collins 2005 Nature Biotech.

  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. (= 2 E.coli genomes or 20 Mycoplasmas /chip) How? 10 Mbp of oligos / $1000 chip Digital Micromirror Array ~1000X lower oligo costs 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 Amplify pools of 50mers using flanking universal PCR primers and three paths to 10X error correction Tian et al. Nature. 432:1050; Carr & Jacobson 2004 NAR; Smith & Modrich 1997 PNAS

  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 Regulated Capsule TonB, DapD & new genetic code for safety 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.html

  16. New in vivo genetic code: resistant to all viruses; novel amino acids Freeing 4 tRNAs, 7 codons: UAG, UUR, AGY, AGR e.g. PEG-pAcPhe-hGH (Ambrx, Schultz) high serum stability 4 1 Isaacs Church Forster Carr Jacobson Jahnz Schultz 3 2

  17. To change the genetic code • Free up 1 or more codons by change 30 to 30,000 codons throughout the genome • Remove the RF or tRNA dedicated to those codon(s) • Add orthogonal tRNA and synthetase protein • 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 Lenski Citrate utilization Church Trp/Tyr exchange Palsson Glycerol utilization Edwards Radiation resistance Ingram Lactate production Stephanopoulos Ethanol resistance Marliere Thermotolerance J&J Diarylquinoline resistance DuPont 1,3-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) Internal view of the aphid. (by T. Sasaki) Bacteriocyte (Photo by T. Fukatsu) Buchnera (Photo by M. Morioka) Aphids 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. Synthetic combinatorics & evolution of 7*7* 4.7 Mbp genomes Second Passage First Passage 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

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

  25. Why low error rates? Goal of genotyping & resequencing  Discovery of variants E.g. cancer somatic mutations ~1E-6 (or lab evolved cells) Consensus error rateTotal 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 Also, effectively reduce (sub)genome target size by enrichment for exons or common SNPs to reduce cost & # false positives.

  26. Mutation Discovery in Engineered/Evolved E.coli Shendure, Porreca, et al. (2005) Science 309:1728

  27. ompF - non-specific transport channel AAAGAT CAAGAT -12 -11 -10 -9 -8 -7 -6 Can increase import & export capability simultaneously • 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

  28. Co-evolution of mutual biosensors sequenced across time & within each time-point 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.

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