ICSB3: DRPM Measures

ICSB3: DRPM Measures

Exponential technologies for reading & writing genomes 8:30-9:15 am 6-Nov-2008 Forum on Science and Biothreats FAZD Lansdowne, VA Thanks to: 1 Sequencing tracked Moores law (2X / 2 yr) until 2004-8 (10X / yr) 10 1 0.1 0.01 $/bp 0.001 0.0001 0.00001 0.000001 0.0000001 1990 1995 2000 2005 2010 40X 98% genome $5K in 2008 ($50 for 1%?) 2

Writing DNA '80 to '08 7-logs: $600 to $3 / bp doubling 14 month 2008: $500 / (244K * 60b) 3 Synthetic Genomics High-Throughput Components 1. 2. 3. 4. 5. 6. 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? 4 Chemical synthesis, enzymatic assembly: on/off chips $500 per 15Mbp 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 5 1 open-architecture hardware, software, wetware e.g. 1981 IBM PC $150K - 2 billion beads/run Polonator Rich Terry 6 Personal Genome Project Inherited + Environmental Genomics One in a life-time genome + yearly ( to daily) tests Public Health Bio-weather map : Allergens, Microbes, Viruses VDJ-ome PERSONAL GENOME 1 to 98% Multitissue Epigenom e

(RNA,mC) TRAITS (Phenome) Microbiome 7 PGP MicrobiomeResistome: 18 Antibiotics Dantas, Sommer, Church unpublished 8 Multiple Phyla Subsisting on 18 Antibiotics Dantas Sommer Church Science 2008 9 Antibody (& TCR) VDJ regions VH*DH*NH*JH*V*J 46*23*N * 6 * 67* 5 = 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 10 Maintaining clonal VDJ (H & L) mRNA phase

water-in-oil emulsion 4 Encapsulation approaches Dantas, Sommer, Science 309: 1728 Nature Methods 3: 551 NAR 20: 3831 Anal. Biochem. 320: 55 Agresti, Rowat 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. 11 index 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 12 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) 13 Today: 16 antigens & 3 PGP-VDJ(H) combinations Future lookup-table: (20K self + 4K pathogen antigens) * (>2M VDJs) Uri Laserson, Francois Vigneault ImMunoGeneTics database 14 http://imgt.cines.fr/ Genome writing example 2000-2006 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 Klebsiella Yeast DAR1 Glycerol-3-P - NADH GPP2 yqhD dhaB1-3 Glycerol E.coli 3HPA coB12 - NADPH 1,3 propanediol 15 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 100 organic Distribution (% total) 90 80 70 3,000 60 50 40 30 20 10 0 aqueous

Extracellular Intracellular 50 Localization Fatty acid derived 1 1 Leverage current infrastructure & engines 10 2 3 4 3 months 16 Improving process yield, health, safety: What threatens all biological systems?

What do all viruses have in common? or lack? 17 New genetic code: viral-resistance, novel amino acids no functional GMO DNA exchange PEG-pAcPhe-hGH (Ambrx) high serum stability 314 TAG to TAA changes TTT TTC F TTA TTG 4 TCT 11495 TAT 22516 TCC 11720 TAC 18932

TCA 9783 TAA TCG 12166 TAG CTT 15002 CCT 9559 CAT CTC 15077 CCC 7485 CAC CTA

5314 CCA 11471 CAA 71553 CCG 31515 CAG 41309 ACT 12198 AAT 34178 ACC 31796 AAC 5967

ACA 9670 AAA 37915 ACG 19624 AAG GTT 24858 GCT 20762 GAT GTC 20753 GCC 34695 GAC

14822 GCA 27418 GAA 35918 GCG 45741 GAG L ATT ATC I ATA ATG GTA GTG M V

P T A Y STOP S 18602 CTG L 30362 STOP H Q N K D E 1

21999 TGT 7048 16601 TGC C 8816 2703 TGA STOP 1256 314 TGG W 20683 17613 CGT

28382 13227 CGC 29898 20888 CGA 39188 CGG 7399 24159 AGT 11970 29385 AGC 45687 AGA 14029

AGG 43719 GGT 33622 25918 GGC 40285 53641 GGA 24254 GGG 4859 R S R G 3 21862 2

2896 1692 Isaacs Charalel Church Sun Wang Carr Jacobson Kong Sterling 10893 15090 18 Allele replacement strategy #3: ss-Oligonucleotide Repair (#1: ds-circle, #2: linear ds) 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) Improved Recombination Frequency: 10-4 up to 90% (> 3 log increase!) without selection 19

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 20 ss-oligo-genome match Recombination Efficiency 0.25 Deletions Mismatches & Insertions 3 kb lacZ wt 0.2 0.15 MM IN 0.1 0.05 3 kb lacZ del 0 0

5 10 15 20 25 30 2log ~0.5 % Recomb # contiguous bp involved 21 Design + Evolution Lenski Palsson Edwards Ingram Marliere J&J DuPont Tolonen Lin&Reppas Citrate utilization

Glycerol utilization Radiation resistance Lactate production Thermotolerance Diarylquinoline resistance (TB) 1,3-propanediol production Biofuel resistance (4 to 8%) Trp/Tyr (pharma precursors) 22 Accelerated Evolution via GEMASS: 314 TAG to TAA Mutation Distribution: 11 oligos, 15 cycles Mutation Distribution: 54 oligos, 45 cycles 25 Frequency 20 15 10 5 0 0 1 2 3 4

5 6 7 # mutations/clone Oligo Pool # cycles Best Clone (98 %tile) Maximum Fraction of mutated sites Time 11 15 7 7/11 3 days 54

45 23 23/54 9 days 10*32 18 8 251/314 2 days Scaling & Automation Increase Efficiency of Recombination (70X faster than 2006) Wang, Isaacs, Carr, Jacobson, Church 23 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 L-aminoacids

D-nucleotides (current biosphere) D-aminoacids L-nucleotides (Mirror-biopolymers) Duhee Bang 24 113 kbp DNA 151 genes Not minimal: High speed & accuracy requires a few extra genes (E.coli 20 min. doubling) Reconstituted ribosomes: Jewett & Church Pure translation: Forster & Church MSB 05 GenomeRes.06 Shimizu, Ueda 01 25 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 26 Summary Reading DNA Writing DNA Resistance Multi-drug resis. via catabolism Multi-enz resis via new chirality Immunity VDJ-ome Multi-virus resis. via new codes Instruments Open access

SOLiD D.005 Polonator G.007 Off-chip-oligos GE-MASS Ethics / safety open access Personal Genome Project Bio-security IASB 27

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