a3316dc9e94c9c18cf8a13f6ba225af6.ppt
- Количество слайдов: 31
Low-cost, high accuracy, long-DNA synthesis technology 16 -Feb-2005 10 AM NHGRI 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
Low-cost, high accuracy, long-DNA synthesis technology 16 -Feb-2005 10 AM NHGRI George Church, Joe Jacobsen et al. Harvard & MIT All stages: Error correction, Software, QA, safety 50 -100 Chip synthesis, fluidics 100 -15 k Pol-Assembly-Multiplex, Proteome synthesis 15 k-100 k Annealing assembly 100 k-5 M. Microbial recombination 100 k-200 M Mammalian recomb, integrases
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 -- T 7 -- Roche) Toward these goals design a chassis: • 115 kbp genome. 150 genes. • Nearly all 3 D structures known. • Comprehensive functional data.
(PURE) translation utility Removing t. RNA-synthetases, translational release-factors, RNases & proteases Allows: Selection of sc. Fvs[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)
in vitro genetic codes y. U m. S 5' e. U 3' Second base A A C C U U UUG UGG CAG 5' . . . ||| ||| AUG AAC ACC GUU GAA A 3' f. M N T V A C y. U E m. S U G e. U 80% average yield e. U = 2 -amino-4 -pentenoic acid per unnatural coupling. y. U = 2 -amino-4 -pentynoic acid m. S = O-methylserine g. S = O-Glc. NAc–serine b. K = biotinyl-lysine Forster, et al. (2003) PNAS 100: 6353 Zhang et al. (2004) Science. 303: 371
Oligos for 150 & 776 synthetic genes (for E. coli minigenome & M. mobile whole genome respectively) Forster & Church
Up to 760 K Oligos/Chip 18 Mbp for $700 raw (6 -18 K genes) <1 K Oxamer Electrolytic acid/base 8 K Atactic/Xeotron/Invitrogen Photo-Generated Acid Sheng , Zhou, Gulari, Gao (U. Houston) 24 K Agilent Ink-jet standard reagents 48 K Febit 100 K Metrigen 380 K Nimblegen Photolabile 5'protection Nuwaysir, Smith, Albert Tian, Gong, Church
Improve DNA Synthesis Cost Synthesis on chips in pools is 5000 X less expensive per oligonucleotide, but amounts are low (1 e 6 molecules rather than usual 1 e 12) & 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 50 mer junctions Tian, Gong, Sheng , Zhou, Gulari, Gao, Church Nature 2004
Improve DNA Synthesis Accuracy via mismatch selection Tian & Church Other mismatch methods: Mut. S (&H, L)
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 <1 e-6 errors) to reduce # of intermediates 3. 15 kb to 5 Mb 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.
All 30 S-Ribosomal-protein DNAs (codon re-optimized) 1. 7 kb 0. 3 kb Atactic <4 K chip 0. 3 kb s 19 Nimblegen 95 K chip Tian, Gong, Sheng , Zhou, Gulari, Gao, Church
Improving synthesis accuracy Method Bp/error Chip assembly (PAM) 160 1 Hybridization-selection 1, 400 1 Mut. S-gel-shift 10, 000 2 Mut. HLS cleavage 30, 000 3 (10 X better than PCR) 1. Tian, Church, et al. 2004 Nature 432: 1050 2. Carr, Jacobson, et al. 2004 NAR 32: e 162 3. Smith & Modrich 1997 PNAS 94: 6847
Extreme m. RNA 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 m. RNAs with less m. RNA structure. Western blot based on His-tags Tian & Church
Synthetic - homologous recombination testing of DNA motifs 2. 4 (1. 3 in Darg. R) 1. 3 1. 1 1. 3 2. 5 0. 7 0. 2 1. 4 3. 5 RNA Ratio (motif- to wild type) for each flanking gene Bulyk, Mc. Guire, Masuda, Church Genome Res. 14: 201– 208
Safe Synthetic Biology Church, G. M. (2004) A synthetic biohazard non-proliferation proposal. http: //arep. med. harvard. edu/SBP/Church_Biohazard 04 c. 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.
Public relations & safety Church, G. M. A synthetic biohazard non-proliferation proposal (2004) http: //arep. med. harvard. edu/SBP/Church_Biohazard 04 c. 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/Book. Review. Type. Detail/assetid/16207 http: //www. social-ecology. org/article. php? story=2003120211014237 Jackson et al. (2001) J Virol. 75: 1205 -10. "immunized genetically resistant mice withthevirusexpressing. IL-4 resulted in significant mortality due to fulminant mousepox. "
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
Remaking a genome: r. E. coli # total # to next Average bp/chunk #prime r pairs Overlap bp Comments r. E. coli_ 0 1 - 4, 648, 88 2 H 1 47 47 100, 000 - 760 23 kan. R 24 cam. R H 2 47 47 100, 000 - 760 23 tet. R 24 zeo. R offset 50 kb from H 1 - -3017 bp bio + 12224 bp Red T 470 10 10, 000 470 25 AT dinuc-ends of each 25 mer (needed for UDG cleavage) are constrained by genome F 13, 045 28 400 470 40 Test of PAM at 28 -plex C 208, 721 16 50 1 25 15+50+15 mers S 417, 442 2 25 1 Tm ~25 b QA 208, 721 1 50 - - Both strands of C-oligos to assess C & F - Both strands centered on all potential mismatches 1704 AGG>AGA & 378 UAG>UAA QB 8328 4% 25 -
r. E. coli Project: Free up & switch codons in vivo UAG>A AGG>A
Amplifying DNA from single chromosomes Prochlorococcus & Escherchia No template control Zhang, Martiny, Chisholm, Church, unpub. f 29 real-time amplification Affymetrix quantitation of independent amplifications
Polony Bead Sequencing Pipeline In vitro libraries via paired tag manipulation Bead polonies via emulsion PCR [Dre 03] Enrichment of amplified beads Monolayered immobilization in acrylamide SOFTWARE Images → Tag Sequences → Genome FISSEQ or “wobble” sequencing Epifluorescence Scope with Integrated Flow Cell Mitra, Shendure, Porreca, Rosenbaum, Church unpub.
Oligo-testing d. NTP-extension Capillary-sequencing 1 10 3 e-3 1 M 2. 5 14 -200 4 e-5 1 e-1 1 M NA 800 1 e-4 1 e-3 1 K bp read/cycle of 4 bases bp reads non-homopolymer errors bp/$
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
Automated homologous recombination • Positive & Negative Selection in same gene: URA 3 (yeast), Thy. A(E. coli), GFP(various) • Electroporation, viral, conjugative delivery 3 ori. T regions: Inc. Pa, F, and R 64(Inc. I) 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.
Integrase applications (1) In vivo recombination (increase fidelity & efficiency) Nucleofection of muscle-derived stem cells and myoblasts with phi. C 31 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.
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.
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.
Invitrogen Gateway Vectors Parr RD, Ball JM. (2003) Plasmid 49: 179. Nakayama M, Ohara O. (2003) BBRC 312: 825
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
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Low-cost, high accuracy, long-DNA synthesis technology 16 -Feb-2005 10 AM NHGRI 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
a3316dc9e94c9c18cf8a13f6ba225af6.ppt