Low-cost, high accuracy, long-DNA synthesis technology George Church, Joe Jacobsen et al. Harvard & MIT 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 16-Feb AM NHGRI

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

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.

(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 J Immunol Methods. 284:147 (largest libraries) Programming peptidomimetic syntheses by translating genetic codes designed de novo. Forster et al PNAS 100:6353 High level cell-free expression & specific labeling of integral membrane proteins. Klammt et al Eur J Biochem 271:568 Cell-free translation reconstituted with purified components. Shimizu et al Nat Biotechnol. 19: Also: membrane incompatible expression & diverse amino-acids (>21)

in vitro genetic codes 5' mS yU eU UGG UUG CAG AAC... GUU A 3' GAAACCAUG fMTNVE | | | 5' Second base 3' U A C C U mS yU eU A C U G A Forster, et al. (2003) PNAS 100:6353 Zhang et al. (2004) Science. 303:371 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 & Church Oligos for 150 & 776 synthetic genes (for E.coli minigenome & M.mobile whole genome respectively)

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

Improve DNA Synthesis Cost Synthesis on chips in pools is 5000X less 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 => ss-70-mer (chip) 20-mer PCR primers with restriction sites at the 50mer junctions Tian, Gong, Sheng, Zhou, Gulari, Gao, Church Nature 2004 => ds-90-mer => ds-50-mer

Improve DNA Synthesis Accuracy via mismatch selection Tian & Church Other mismatch methods: MutS (&H,L)

Computer Aided Design Polymerase Assembly Multiplexing (CAD-PAM) 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 Gene 164:49-53;Mullis 1986 CSHSQB … 100*2^(n-1)

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

Improving synthesis accuracy Method length bp/error ref Chip assembly (PAM) Hybridization-selection 50 1,400 1 MutS-gel-shift ,000 2 MutHLS cleavage >2kb 30,000 3 (10X better than PCR) 1. Tian, Church, et al Nature 432: Carr, Jacobson, et al NAR 32:e Smith & Modrich 1997 PNAS 94:6847

Extreme mRNA makeover for 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. Tian & Church Western blot based on His-tags

Synthetic - homologous recombination testing of DNA motifs (1.3 in  argR) RNA Ratio (motif- to wild type) for each flanking gene Bulyk, McGuire,Masuda,Church Genome Res. 14:201–208

ELSI: Safe Synthetic Biology Church, G.M. (2004) A synthetic biohazard non-proliferation proposal. /SBP/ Church_Biohazard04c.dochttp://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.

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

4.6 Mbp synthesis: rE.coli # total # to next Average bp/chunk primer pairs Overlap bpComments rE.coli 1- 4,648, bp Red bp bio H , kanR 24 camR H , tetR 24 zeoR offset 50 kb from H1 T , AT dinuc-ends of each 25mer (needed for UDG cleavage) constrained by genome F13, Test of PAM at 28-plex C208, mers S417, Tm ~ 25b QA208, Both strands of C-oligos to assess C & F QB83284%25-- Both strands centered on all potential mismatches 1704 AGG>AGA & 378 UAG>UAA

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

Amplifying DNA from single chromosomes  29 real-time amplification No template control Affymetrix quantitation of independent amplifications Prochlorococcus & Escherchia Zhang, Martiny, Chisholm, Church, unpub.

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, NHGRI Seeks Next Generation of Sequencing Technologies (Jan 2004)

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

Oligo-testing sequencing dNTP-extension sequencing Capillary-sequencing NA bp read/cycle of 4 bases bp reads 3e-3 4e-5 1e-4 non-homopolymer errors 3e-3 1e-1 1e-3 homopolymer errors 1M 1M 1K bp/$

Automated homologous recombination Positive & Negative Selection in same gene: URA3, LYS2 (yeast), thyA,LamB(E.coli), GFP(various) Electroporation, viral, conjugative delivery 3 oriT regions: IncP , F, and R64(IncI) Valenzuela DM, et al. Nat Biotechnol Jun;21(6): 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 : Schneckenburger H, et al. J Biomed Opt Jul;7(3): Laser-assisted optoporation of single cells.

Gene Therapy Needs Targeting tissue Transfer across membrane Pairing & recombination Selection (pos&neg) Varnavski AN, Calcedo R, Bove M, Gao G, Wilson JM. Evaluation of toxicity from high-dose systemic administration of recombinant adenovirus vector in vector-naive and pre-immunized mice. Gene Ther Jan 13; McCormack et al. The LMO2 T-cell oncogene is activated via chromosomal translocations or retroviral insertion during gene therapy but has no mandatory role in normal T-cell development. Mol Cell Biol Dec;23(24): Izumikawa, et al. Auditory hair cell replacement and hearing improvement by Atoh1 gene therapy in deaf mammals. Nature Medicine Feb 2005 (adeno) McGuire et al. A library-selected, Langerhans cell-targeting peptide enhances an immune response. DNA Cell Biol Nov;23(11):

Integrase applications (1) In vivo recombination (increase fidelity & efficiency) Nucleofection of muscle-derived stem cells and myoblasts with phiC31 integrase. Mol Ther : (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) lac-GFP ara-GFP trp-GFP tet-GFP etc.

Sam et al. Crystal structure of the excisionase-DNA complex from bacteriophage lambda. J Mol Biol :229. Aihara... Ellenberger et al. A conformational switch controls the DNA cleavage activity of lambda integrase. Mol Cell : Int/Xis contacts

Integrase specificity … diversity Sam et al. Crystal structure of the excisionase-DNA complex from bacteriophage lambda. J Mol Biol :229. Aihara... Ellenberger et al. A conformational switch controls the DNA cleavage activity of lambda integrase. Mol Cell :187-98

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

Potential Commercial Biology Partners 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

DOE funded efforts Ham Smith, John Glass, Clyde Hutchison, Craig Venter Targets: Phage, Mycoplasma, hydrogen production Methods: Deinococcus in vitro recombination Conventional oligos, error screening by sequencing Seed funding supplement for Church lab for photosynthetic microbes

ibm.com/chips/services/foundry/partners 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 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, You are here Example2: linux.org redhat.com Q2 $46M up 60%

Low-cost, high accuracy, long-DNA synthesis technology George Church, Joe Jacobsen et al. Harvard & MIT 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 16-Feb AM NHGRI