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Low-cost, high accuracy, long-DNA synthesis technology

Low-cost, high accuracy, long-DNA synthesis technology. George Church, Joe Jacobsen et al. Harvard & MIT. 16-Feb-2005 10 AM NHGRI. 0. Killer Applications 1. Chip synthesis, fluidics 2. Multiplex assembly 3. Error correction methods 4. Software CAD-PAM 5. Proteome (in vitro) synthesis

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Low-cost, high accuracy, long-DNA synthesis technology

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  1. Low-cost, high accuracy, long-DNA synthesis technology George Church, Joe Jacobsen et al. Harvard & MIT 16-Feb-2005 10 AM NHGRI 0. Killer Applications 1. Chip synthesis, fluidics 2. Multiplex assembly 3. Error correction methods 4. Software CAD-PAM 5. Proteome (in vitro) synthesis 6. Homologous recombination & selection for BACs 7. Integrases 8. Process integration, QA, timeline 9. Safety opportunities

  2. Low-cost, high accuracy, long-DNA synthesis technology George Church, Joe Jacobsen et al. Harvard & MIT 16-Feb-2005 10 AM NHGRI All stages: Error correction, Software, QA, safety 50-100 Chip synthesis, fluidics 100-15k Pol-Assembly-Multiplex, Proteome synthesis 15k-100k Annealing assembly 100k-5M. Microbial recombination 100k-200M Mammalian recomb, integrases

  3. Synthetic Genomes&Proteomes. Why? • Test or engineer cis-DNA/RNA-elements • Access to any protein (complex) including • post-transcriptional modifications • Affinity agents for the above. • Protein design, vaccines, solubility screens • Utility of molecular biology DNA -- RNA -- Protein • in vitro "kits" (e.g. PCR -- T7 -- Roche) • Toward these goals design a chassis: • 115 kbp genome. 150 genes. • Nearly all 3D structures known. • Comprehensive functional data.

  4. (PURE) translation utility Removing tRNA-synthetases, translational release-factors, RNases & proteases Allows: Selection of scFvs[antibodies] specific for HBV DNA polymerase using ribosome display. Lee et al. 2004 J Immunol Methods. 284:147 Programming peptidomimetic syntheses by translating genetic codes designed de novo. Forster et al. 2003 PNAS 100:6353 High level cell-free expression & specific labeling of integral membrane proteins. Klammt et al. 2004 Eur J Biochem 271:568 Cell-free translation reconstituted with purified components. Shimizu et al. 2001 Nat Biotechnol. 19:751-5. Also: membrane incompatible expression & diverse amino-acids (>21)

  5. yU mS eU UUG UGG CAG | | | | | | | | | ... AUG AAC ACC GUU GAA 5' A 3' fM N T V E in vitro genetic codes 5' 3' Second base A U A C U C A C yU mS U G eU 80% average yield per unnatural coupling. eU = 2-amino-4-pentenoic acid yU = 2-amino-4-pentynoic acid mS = O-methylserine gS = O-GlcNAc–serine bK = biotinyl-lysine Forster, et al. (2003) PNAS 100:6353 Zhang et al. (2004) Science. 303:371

  6. Oligos for 150 & 776 synthetic genes(for E.coli minigenome & M.mobile whole genome respectively) Forster & Church

  7. Up to 760K Oligos/Chip18 Mbp for $700 raw (6-18K genes) <1K Oxamer Electrolytic acid/base 8K Atactic/Xeotron/InvitrogenPhoto-Generated Acid Sheng , Zhou, Gulari, Gao (U.Houston) 24K Agilent Ink-jet standard reagents 48K Febit 100K Metrigen 380K NimblegenPhotolabile 5'protection Nuwaysir, Smith, Albert Tian, Gong, Church

  8. Improve DNA Synthesis Cost Synthesis on chips in pools is 5000Xless expensive per oligonucleotide, but amounts are low (1e6 molecules rather than usual 1e12) & bimolecular kinetics slow with square of concentration decrease!) Solution: Amplify the oligos then release them. 10 50 10 => ss-70-mer (chip) => ds-90-mer => ds-50-mer 20-mer PCR primers with restriction sites at the 50mer junctions Tian, Gong, Sheng , Zhou, Gulari, Gao, Church Nature 2004

  9. Improve DNA Synthesis Accuracyvia mismatch selection Other mismatch methods: MutS (&H,L) Tian & Church

  10. Computer Aided Design Polymerase Assembly Multiplexing (CAD-PAM) 50 75 125 225 425 825 … 100*2^(n-1) Moving forward: 1. Tandem, inverted and dispersed repeats (hierarchical assembly, size-selection and/or scaffolding) 2. Reduce mutations (goal <1e-6 errors) to reduce # of intermediates 3. 15kb to 5Mb by homologous recombination (Nick Reppas) 4. Phage integrase site-specific recombination, also for counters. Stemmer et al. 1995. Gene 164:49-53;Mullis 1986 CSHSQB.

  11. All 30S-Ribosomal-protein DNAs(codon re-optimized) 1.7 kb 0.3 kb Atactic <4K chip s19 0.3kb Nimblegen 95K chip Tian, Gong, Sheng , Zhou, Gulari, Gao, Church

  12. Improving synthesis accuracy Method Bp/error Chip assembly (PAM) 160 1 Hybridization-selection 1,400 1 MutS-gel-shift 10,000 2 MutHLS cleavage 30,000 3 (10X better than PCR) 1. Tian, Church, et al. 2004 Nature 432:1050 2. Carr, Jacobson, et al. 2004 NAR 32:e162 3. Smith & Modrich 1997 PNAS 94:6847

  13. Extreme mRNA makeoverfor protein expression in vitro RS-2,4,5,6,9,10,12,13,15,16,17,and 21 detectable initially. RS-1, 3, 7, 8, 11, 14, 18, 19, 20 initially weak or undetectable. Solution: Iteratively resynthesize all mRNAs with less mRNA structure. Western blot based on His-tags Tian & Church

  14. Synthetic - homologous recombination testing of DNA motifs 1.3 2.4 (1.3 in DargR) 1.1 1.3 0.7 2.5 0.2 1.4 1.4 3.5 RNA Ratio (motif- to wild type) for each flanking gene Bulyk, McGuire,Masuda,Church Genome Res. 14:201–208

  15. Safe Synthetic Biology Church, G.M. (2004) A synthetic biohazard non-proliferation proposal. http://arep.med.harvard.edu/SBP/Church_Biohazard04c.doc 1. Monitor oligo synthesis via expansion of Controlled substances, Select Agents, &/or Recombinant DNA 2. Computational tools are available; very small number of reagent, instrument & synthetic gene suppliers at present. 3. System modeling checks for synthetic biology projects 4. Multi-auxotroph, novel genetic code for the host genome, prevents functional transfer of DNA to other cells.

  16. Church, G.M. A synthetic biohazard non-proliferation proposal (2004) • http://arep.med.harvard.edu/SBP/Church_Biohazard04c.doc • Monitor oligo synthesis via expanding the purview of • Controlled substances, Select Agents, &/or Recombinant DNA. • Computational tools for the above (e.g. Craic) • System modeling for all Synthetic Biology Projects • Avoid environmental release uses (at least initially) • Beckwith'69, Asilomar'75, AGS-Rifkin'84-6, Starlink'00, Roundup'04… • http://www.americanscientist.org/template/BookReviewTypeDetail/assetid/16207 • http://www.social-ecology.org/article.php?story=2003120211014237 • Jackson et al. (2001) J Virol. 75:1205-10."immunized genetically resistant mice withthevirusexpressingIL-4 resulted in significant mortality due to fulminant mousepox." Public relations & safety

  17. Safety via blocking exchange Can we make a cell which is resistant to all viruses and incapable of *functional* DNA exchange in or out? One option is genetic code remapping. Micrococcus luteus is naturally missing 6 codons: UUA(L), CUA(L), AUA(I), GUA(Q), CAA(Q), AGA(R). Kowal, AK, & Oliver, JS NAR 1997, 25: 4685

  18. Remaking a genome: rE.coli

  19. rE.coli Project: Free up & switch codons in vivo UAG>A AGG>A

  20. Amplifying DNA from single chromosomes Prochlorococcus & Escherchia Zhang, Martiny, Chisholm, Church, unpub. No template control f29 real-time amplification Affymetrix quantitation of independent amplifications

  21. In vitro libraries via paired tag manipulation Monolayered immobilization in acrylamide SOFTWARE Images → Tag Sequences Tag Sequences → Genome Polony Bead Sequencing Pipeline Bead polonies via emulsion PCR [Dre03] Enrichment of amplified beads FISSEQ or “wobble” sequencing Epifluorescence Scope with Integrated Flow Cell Mitra, Shendure, Porreca, Rosenbaum, Church unpub.

  22. Oligo-testing dNTP-extension Capillary-sequencing 1 2.5 NA bp read/cycle of 4 bases 10 14-200 800 bp reads 3e-3 4e-5 1e-4 non-homopolymer errors 3e-3 1e-1 1e-3 homopolymer errors 1M 1M 1K bp/$

  23. Integrating with appropriate sequencing strategies Shendure J, Mitra R, Varma C, Church GM (May 2004) Advanced Sequencing Technologies: Methods & Goals. Nature Reviews of Genetics 5, 335 -344. NHGRI Seeks Next Generation of Sequencing Technologies (Jan 2004) http://www.genome.gov/12513210

  24. Automated homologous recombination • Positive & Negative Selection in same gene: • URA3 (yeast), ThyA(E.coli), GFP(various) • Electroporation, viral, conjugative delivery • 3 oriT regions: IncPa, F, and R64(IncI) • Valenzuela DM, et al. Nat Biotechnol. 2003 Jun;21(6):652-9. High-throughput engineering of the mouse genome coupled with high-resolution expression analysis. up to 25% targeting with BACs. • Yang Y, Seed B. Site-specific gene targeting in mouse embryonic stem cells with intact bacterial artificial chromosomes. Nat Biotechnol. 2003 21:447-51. • Schneckenburger H, et al. J Biomed Opt. 2002 Jul;7(3):410-6. Laser-assisted optoporation of single cells.

  25. Integrase applications (1) In vivo recombination (increase fidelity & efficiency) Nucleofection of muscle-derived stem cells and myoblasts with phiC31 integrase. Mol Ther. 2004 10:679-87. (2) In vitro plasmid construction (Gateway) (3) In vivo counters allow recording & increased analog I/O through digital reuse of functions. For a 3-bit (8 state counter) 0 0 0 lac-GFP 0 0 1 ara-GFP 0 1 0 trp-GFP 0 1 1 tet-GFP 1 0 0 etc.

  26. Int/Xis contacts Mol Cell. 2003 Jul;12(1):187-98. A conformational switch controls the DNA cleavage activity of lambda integrase. Aihara H, Kwon HJ, Nunes-Duby SE, Landy A, Ellenberger T. Sam MD, Cascio D, Johnson RC, Clubb RT. Crystal structure of the excisionase-DNA complex from bacteriophage lambda. J Mol Biol. 2004 Apr 23;338(2):229-40.

  27. Integrase specificity … diversity Mol Cell. 2003 Jul;12(1):187-98. A conformational switch controls the DNA cleavage activity of lambda integrase. Aihara H, Kwon HJ, Nunes-Duby SE, Landy A, Ellenberger T. Sam MD, Cascio D, Johnson RC, Clubb RT. Crystal structure of the excisionase-DNA complex from bacteriophage lambda. J Mol Biol. 2004 Apr 23;338(2):229-40.

  28. Invitrogen Gateway Vectors Parr RD, Ball JM.(2003) Plasmid 49:179. Nakayama M, Ohara O. (2003) BBRC 312:825

  29. Potential Commercial Biology Partners / Competitors Invitrogen Gateway cloning Poetic Genetics Integrases & Gene Therapy Regeneron Mammalian BAC recombination 1% Scarab Genomics Better E. coli strains 20% of genome Avidia/Diversa Shuffling/selection Ensemble DNA catalysts Amyris Terpenoid pathways Kosan Biosciences Polyketide pathways Big & Small Pharma

  30. ibm.com/chips/services/foundry/partners Analog Bits, Artisan Components, Cadence, eSilicon Corporation, GDA Technologies Inc., insyte, Jennic Limited, Kisel Microelectronics, Magma, MOSIS, QThink, QualCore Logic, Inc., RF Integration, Sierra Monolithics, SOCLE Technology, Synopsys, Tahoe RF Semiconductors, TelASIC, TriCN, Triscend, Virtual Silicon, Mosis.org "50,000 designs… keep prototype costs low by aggregating many designs onto one mask set, sharing overhead" Fabrication Processes: AMIS, IBM, Austriamicrosystems, OMMIC/PML, Peregrine, TSMC, Vitesse You are here Example2: linux.org redhat.com Q2 $46M up 60%

  31. Low-cost, high accuracy, long-DNA synthesis technology George Church, Joe Jacobsen et al. Harvard & MIT 16-Feb-2005 10 AM NHGRI 0. Killer Applications 1. Chip synthesis, fluidics 2. Multiplex assembly 3. Error correction methods 4. Software CAD-PAM 5. Proteome (in vitro) synthesis 6. Homologous recombination & selection for BACs 7. Integrases 8. Process integration, QA, timeline 9. Safety opportunities

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