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Exponential technologies for reading & writing genomes

Exponential technologies for reading & writing genomes. 8:30-9:15 am 6-Nov-2008 Forum on Science and Biothreats FAZD Lansdowne, VA. Thanks to:. Sequencing tracked Moore’s law (2X / 2 yr) until 2004-8 (10X / yr). $/bp. 40X 98% genome $5K in 2008 ($50 for 1%?). Writing DNA '80 to '08.

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Exponential technologies for reading & writing genomes

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  1. Exponential technologies forreading & writing genomes 8:30-9:15 am 6-Nov-2008 Forum on Science and Biothreats FAZD Lansdowne, VA Thanks to:

  2. Sequencing tracked Moore’s law (2X / 2 yr) until 2004-8 (10X / yr) $/bp 40X 98% genome $5K in 2008 ($50 for 1%?)

  3. Writing DNA '80 to '08 7-logs: $600 to $3E-5 / bp doubling 14 month 2008: $500 / (244K * 60b)

  4. Synthetic Genomics High-Throughput Components • HT Chemical oligo Synthesis • HT DNA assembly • HT in vivo (& in vitro) systems • HT selection • HT sequencing • Integration & applications Why not out-sourced or off-the-shelf?

  5. $500 per 15Mbp Chemical synthesis, enzymatic assembly: on/off chips 8K Xeotron Photo-Generated Acid 12K Combimatrix Electrolytic 120K Roche, Febit Photolabile 5'protection 244K Agilent Ink-jet standard reagents Amplify pools of 50mers using flanking universal PCR primers & 3 paths to 10X error correction Tian et al. 2004 Nature Carr & Jacobson 2004 NAR Smith & Modrich 1997 PNAS

  6. 1 open-architecture hardware, software, wetware e.g. 1981 IBM PC $150K - 2 billion beads/run Polonator Rich Terry

  7. Personal Genome ProjectInherited + Environmental Genomics One in a life-time genome + yearly ( to daily) tests Public Health Bio-weather map : Allergens, Microbes, Viruses VDJ-ome Multi-tissue Epigenome (RNA,mC) TRAITS (Phenome) PERSONAL GENOME 1 to 98% Microbiome

  8. PGP Microbiome-Resistome: 18 Antibiotics Dantas, Sommer, Church unpublished

  9. Multiple Phyla Subsisting on 18 Antibiotics Dantas Sommer Church Science 2008

  10. Antibody (& TCR) VDJ regions VH*DH*NH*JH*Vkl*Jkl 46*23*N * 6 * 67* 5 = > 2M combinations , 750 bp, >1E10 cells Roth DB et al Mol Cell Biol. 1989 9:3049 N (1-13): 14 22 13 15 10 4 5 4 2 2 3 2 1 Lefranc, The Immunoglobulin FactsBook; Janeway, Immunobiology 2001

  11. Maintaining clonal VDJ (H & L) mRNA phase water-in-oil emulsion 4 Encapsulation approaches Dantas, Sommer, Agresti, Rowat Science 309: 1728 Nature Methods 3: 551 Anal. Biochem. 320: 55 NAR 20: 3831 2 Chain co-amplification approaches NAR 20: 3831 Embleton et al. In-cell PCR from mRNA: amplifying and linking heavy and light chain V-genes within single cells. index

  12. Time Series Vaccine Experiment Tracking human dynamic response to vaccination to 11 strains: Hepatitis A+B, Flu A/Brisbane/59/2007 (H1N1)-like, 10/2007 (H3N2)-like, B/Florida/4/2006-like virus Polio, Yellow fever Meningococcus Typhoid, Tetanus Diptheria, Pertussis Collect samples at -14d, 0d, +1d, +3d, +7d, +14d, +21d, +28d

  13. N-region lengths in circulating B-cells 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 Length (aa)

  14. Today: 16 antigens &3 PGP-VDJ(H) combinationsFuture lookup-table: (20K self + 4K pathogen antigens) * (>2M VDJs) Uri Laserson, Francois Vigneault ImMunoGeneTics database http://imgt.cines.fr/

  15. Genome writing example 2000-2006 E.coli Klebsiella Yeast yqhD DAR1 1,3 propanediol dhaB1-3 GPP2 Glycerol-3-P Glycerol 3HPA - NADH coB12 - NADPH Dupont/Genencor: 1,3 Propanediol (7 years & $400M R&D) 135 g/l at 3.5 g/l/h, 51% yield (90% of theoretical) from glucose 27 changes to 4.6 Mbp E.coli ackA aldA aldB arcA crr edd gldA glpK mgsA pta ptsH ptsI yqhC Saccharomyces: DAR1 GPP2 Klebsiella: dhaB1,B2,B3,X; orfX,Y P1.5.gapA P1.6.ppc P1.6.btuR P1.6.yqhD Ptrc.galP Ptrc.glk (13 knock-outs, 8 insertions, 6 regulatory changes) http://www.patentstorm.us/patents/6432686-description.html

  16. 3,000 100 50 90 organic 80 70 60 Distribution (% total) 50 10 1 40 aqueous 30 20 1 2 3 4 10 0 Extracellular Intracellular 3 months Localization Bio-petroleum from grasses or algae Immiscible Products Facilitate Purification • Separate from water without distillation • Decrease toxicity to producer strain • >2 million liters in 2009 Fatty acid derived Leverage current infrastructure & engines

  17. Improving process yield, health, safety: What threatens all biological systems? What do all viruses have in common? or lack?

  18. New genetic code: viral-resistance, novel amino acidsno functional GMO DNA exchange PEG-pAcPhe-hGH (Ambrx) high serum stability 314 TAG to TAA changes 4 Isaacs Charalel Church Sun Wang Carr Jacobson Kong Sterling 1 3 2

  19. Improved Recombination Frequency: 10-4 up to 90% (> 3 log increase!) without selection (#1: ds-circle, #2: linear ds) Allele replacement strategy #3: ss-Oligonucleotide Repair DNA Replication Fork Ellis et al. PNAS 2001 Constantino & Court. PNAS 2003 Obtain >25% recombination efficiency in E. coli strains lacking mismatch repair genes (mutH, mutL, mutS, uvrD, or dam)

  20. Genome Engineering Multiplex Automation (GEMASS)in vivo homologous allele replacement (lagging SS mimics) 3 hr Cycle time. Application: 314 change for multivirus resistance Harris Wang

  21. 3 kb lacZ wt 3 kb lacZ del ~0.5 % Recomb 2log ss-oligo-genome match Deletions Mismatches & Insertions

  22. Design + Evolution Lenski Citrate utilization Palsson Glycerol utilization Edwards Radiation resistance Ingram Lactate production Marliere Thermotolerance J&J Diarylquinoline resistance (TB) DuPont 1,3-propanediol production Tolonen Biofuel resistance (4 to 8%) Lin&Reppas Trp/Tyr (pharma precursors)

  23. Accelerated Evolution via GEMASS: 314 TAG to TAA Mutation Distribution: 11 oligos, 15 cycles Mutation Distribution: 54 oligos, 45 cycles (70X faster than 2006) • Scaling & Automation • Increase Efficiency of Recombination Wang, Isaacs, Carr, Jacobson, Church

  24. Mirror world :resistant to enzymes, parasites, predators Approach#1: De novo Chemical synthesis (below) #2: Redesigned peptidyl transferase + D-AA-tRNAs (next slide) 352 AA Synthetic Dpo4 Sulfolobus DNA polymerase IV 4 peptide bonds left to construct D-aminoacids L-nucleotides (Mirror-biopolymers) L-aminoacids D-nucleotides (current biosphere) Duhee Bang

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

  26. Bio-Engineering Safety: Minimizing Bioerror/Bioterror • Jun-2004: A Synthetic Biohazard Non-proliferation Proposal. • Dec 2004: DOE Synthetic Genomes: Technologies and Impact • http://www.sc.doe.gov/ober/berac/SynBio.pdf • 2005: National Science Advisory Board for Biosecurity (NSABB) • 2007: DNA synthesis and biological security • Nat Biotechnol. 25:627-629. • 2008: Sloan Foundation, MIT, JCVI Study: Options for Governance of Synthetic Genomics • Industry Association Synthetic Biology

  27. Summary

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