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

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

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

4 Synthetic Genomics High-Throughput Components 1. HT Chemical oligo Synthesis 2. HT DNA assembly 3. HT in vivo (& in vitro) systems 4. HT selection 5. HT sequencing 6.Integration & applications Why not out-sourced or off-the-shelf?

5 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 Tian et al Nature Carr & Jacobson 2004 NAR Smith & Modrich 1997 PNAS $500 per 15Mbp Amplify pools of 50mers using flanking universal PCR primers & 3 paths to 10X error correction

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

7 Personal Genome Project Inherited + Environmental Genomics VDJ-ome TRAITS (Phenome) Multi- tissue Epigenom e (RNA,mC) PERSONAL GENOME 1 to 98% One in a life-time genome + yearly ( to daily) tests Public Health Bio-weather map : Allergens, Microbes, Viruses 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8 PGP Microbiome  Resistome: 18 Antibiotics Dantas, Sommer, Church unpublished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 Roth DB et al Mol Cell Biol :3049 N (1-13): Lefranc, The Immunoglobulin FactsBook; Janeway, Immunobiology 2001 N H J H J  V H *D H *N H *J H *V  *J  N *23*N * 6 * 67* 5 = > 2M combinations, 750 bp, >1E10 cells

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

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 Length (aa)

14 Today: 16 antigens & 3 PGP-VDJ(H) combinations Future lookup-table: (20K self + 4K pathogen antigens) * (>2M VDJs) ImMunoGeneTics database Uri Laserson, Francois Vigneault

15 Genome writing example Dupont/Genencor: 1,3 Propanediol (7 years & $400M R&D) 135 g/l at 3.5 g/l/h, 51% yieldheoretical) from glucose 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) Glycerol DAR1 GPP2 Glycerol-3-P - NADH 3HPA coB 12 - NADPH yqhD dhaB1-3 1,3 propanediol Yeast Klebsiella E.coli

16 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 aqueous organic Extracellular Intracellular Localization Distribution (% total) , months Leverage current infrastructure & engines Fatty acid derived

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

18 PEG-pAcPhe-hGH (Ambrx) high serum stability 314 TAG to TAA changes Isaacs Charalel Church Sun Wang Carr Jacobson Kong Sterling New genetic code: viral-resistance, novel amino acids no functional GMO DNA exchange TTT F 30362TCT S 11495TAT Y 21999TGT C 7048 TTC22516TCC11720TAC16601TGC8816 TTA L 18932TCA9783TAA STOP 2703TGA STOP 1256 TTG18602TCG12166TAG314TGGW20683 CTT L 15002CCT P 9559CAT H 17613CGT R CTC15077CCC7485CAC13227CGC29898 CTA5314CCA11471CAA Q 20888CGA4859 CTG71553CCG31515CAG39188CGG7399 ATT I 41309ACT T 12198AAT N 24159AGT S ATC34178ACC31796AAC29385AGC21862 ATA 5967ACA9670AAA K 45687AGA R 2896 ATGM37915ACG19624AAG14029AGG1692 GTT V 24858GCT A 20762GAT D 43719GGT G GTC20753GCC34695GAC25918GGC40285 GTA14822GCA27418GAA E 53641GGA10893 GTG35918GCG45741GAG24254GGG

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

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

22 Design + Evolution LenskiCitrate utilization PalssonGlycerol utilization Edwards Radiation resistance IngramLactate production MarliereThermotolerance J&JDiarylquinoline resistance (TB) DuPont1,3-propanediol production TolonenBiofuel resistance (4 to 8%) Lin&ReppasTrp/Tyr (pharma precursors)

23 Accelerated Evolution via GEMASS: 314 TAG to TAA Mutation Distribution: 11 oligos, 15 cyclesMutation Distribution: 54 oligos, 45 cycles Oligo Pool # cyclesBest Clone (98 %tile)Maximum Fraction of mutated sites Time /113 days /549 days 10* /3142 days  Scaling & Automation  Increase Efficiency of Recombination Wang, Isaacs, Carr, Jacobson, Church (70X faster than 2006)

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 L-aminoacids D-nucleotides (current biosphere) D-aminoacids L-nucleotides (Mirror-biopolymers) Duhee Bang

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

26 Bio-Engineering Safety: Minimizing Bioerror/Bioterror Jun-2004: A Synthetic Biohazard Non-proliferation Proposal.A Synthetic Biohazard Non-proliferation Proposal Dec 2004: DOE Synthetic Genomes: Technologies and Impact : National Science Advisory Board for Biosecurity (NSABB) 2007: DNA synthesis and biological security Nat Biotechnol. 25: : Sloan Foundation, MIT, JCVI Study: Options for Governance of Synthetic Genomics Industry Association Synthetic Biology

27 Reading DNAWriting DNA ResistanceMulti-drug resis. via catabolism Multi-enz resis via new chirality ImmunityVDJ-omeMulti-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 Summary