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Fabricating with DNA George Church Wed 22-Aug-2007 9:15 – 9:30 AM 4th Fab Lab Forum & Digital Fabrication Symposium Thanks to: AppliedBiosystems, Helicos, Roche454, Illumina, CGI, IBS, Affymetrix, Enzymatics PGP Volunteers & Donors ! Fab vs. Bio-fab + Plays well with digital computers - Doesn’t get DNA - Needs us to replicate - Needs expensive Fab (e.g. ICs) - Intelligent Design - No habla C++ + DNA is it’s native digital media + We need them + Simple or complex inputs + Evolution Bio – Inorganic interfaces • Metal-oxide-semiconductors (silicateins for Ti & Ga oxides) • Magnetic components (magnetosomes in magnetotactic bacteria) • Optical fibers & lenses (e.g. venus basket sponge) • Bacterial reduction of salts to metals (e.g. Se, Au, Ag) DNA origami -- highly predictable 3D nanostructures Rothemund Nature’06 Douglas, et al. PNAS’07 DNA-nanotube-induced alignment of membrane proteins for NMR structure determination Open-source hardware, software, wetware, ELSware for DNA reading & writing 78-84 87-97 Church, Gilbert Genomic Sequencing (electrotransfer, chemiluminescence, film scanner) Higgins, Richerich, Auger, Smith Multiplex Sequencing Polony A 97-99 B 02-03 Rob Mitra Greg Porecca MJR slide-cycler Jay Shendure GSI Microarray scanner Open-source hardware, etc: Personal Genome Project E07 Faster XYZ D05 = C03 + fluidics F07 : $106K (down from $500K) including computer Rich Terry G07: internal autosampler 10 Mbp of DNA / $300 chip Spatially patterned chemistry 8K Atactic/Xeotron/Invitrogen Photo-Generated Acid 12K Combimatrix Electrolytic 44K Agilent Ink-jet standard reagents 380K Nimblegen/GA Photolabile 5'protection Amplify pools of 50mers using flanking universal PCR primers & 3 paths to 10X error correction Tian et al. Nature. 432:1050 Carr & Jacobson 2004 NAR Smith & Modrich 1997 PNAS Synthetic Biology: augmentation & combinatorics (not minimization) 1. 2. 3. 4. 5. 6. 7. Synthetic DNA: 1Mbp per month (Codon Devices) New polymers in vitro – affinity selection (Vanderbilt) Hydrocarbon & other chemical syntheses in E.coli (LS9) Bacterial & stem cell therapies (SynBERC & MGH) New codes: Viral resistant cells & new aminoacids (MIT) Synthetic Ecosystems – Evolve secretion & signaling Interfaces of Genomics & Society Hierarchical, modular, evolvable Mirror world: resistant to enzymes, parasites, predators Mirror aptamers, ribozymes, etc. require mirror polymerases 834 AA Thermus Polymerase 834 AA 352 AA Dpo4 Sulfolobus DNA polymerase IV 174 AA DNA Polymerase X African Swine Fever Virus. 2004: Bang & Kent: A One-Pot Total Synthesis of Crambin (46-mer) 2006: Torbeev &Kent: A 203-mer Covalent Dimer HIV-1 Protease. Why synthesize (minimal) in vitro self-replication? • Molecular biology kits: PCR, T7 RNA pol, in vitro translation. • Production of devices larger than or toxic to cells. • Directed evolution of drugs & affinity agents. • Mirror-image proteins Duhee Bang (HMS) Tony Forster (Vanderbilt) Pure in vitro translating & replicating system ideal for comprehensive atomic, ODE & stochastic models Forster & Church MSB ‘05 GenomeRes.’06 Shimizu, Ueda et al ‘01 113 kbp DNA 151 genes Genome engineering CAD Recombination in vivo E.coli Polymerase in vitro 70b 15Kb Error Correction MutS 1E-4 Chemical Synthesis 1E-2 5Mb Bacterial (Artificial) Chromosomes BACs Recombination in human cells 250 Mb Human(Artificial) Chromosomes HACs Sequencing 1E-7 Isaacs, Carr, Emig, Gong, Tian, Reppas, Jacobson, Church rE.coli Strategy #3: ss-Oligonucleotide Repair 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, dam) Improved Recombination Frequency: 10-4 0.25 (> 3 log increase!) Multiplex Automated Genome Engineering (MAGE) Wash with water & DNA pool (50) Concentrate O-ring Concentrate, electroporate Wang, Isaacs, Terry membrane Resuspend, bubble, select Multiplex Automated Genome Engineering (MAGE) syringe pump computer communication / data acquisition system electrically actuated valves OD sensor electroporation cuvette w/ membrane filter Wang, Isaacs, Terry Recombination-Cycling UAG to UAA E.coli Essential Genes 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) Fraction of mutated sites Time* 11 15 7 7/11 3 days 54 45 23 23/54 9 days * Continuous cycling Scaling & Automation Increase Efficiency of Recombination Wang, Isaacs, Carr, Jacobson, Church Intelligent Design & Metabolic Evolution Lenski Palsson Edwards Ingram Stephanopoulos Marliere J&J DuPont Church Citrate utilization Glycerol utilization Radiation resistance Lactate production Ethanol resistance Thermotolerance Diarylquinoline resistance (TB) 1,3-propanediol production Trp/Tyr exchange Cross-feeding symbiotic systems: aphids & Buchnera • • • • obligate mutualism nutritional interactions: amino acids & vitamins established 200-250 million years ago close relative of E. coli with tiny genome (618~641kb) MILKFTWV MILKFTWV HR Aphids http://buchnera.gsc.riken.go.jp Pink= enzymes apparently missing in Bucherna Shigenobu et al. Genome sequence of the endocellular bacterial symbiont of aphids Buchnera sp.APS. Nature 407, 81-86 (2000). Synthetic genome pair evolution Second Passage First Passage trp/tyrA pair of genomes shows best cogrowth Reppas, Lin et al. ; Accurate Multiplex Polony Sequencing of an Evolved Bacterial Genome 2005 Science Co-evolution of mutual biosensors/biosynthesis sequenced across time & within each time-point Independent lines of Trp & Tyr co-culture 5 OmpF: (pore: large,hydrophilic > small) 42R-> G,L,C, 113 D->V, 117 E->A 2 Promoter: (cis-regulator) -12A->C, -35 C->A 5 Lrp: (trans-regulator) 1b, 9b, 8b, IS2 insert, R->L in DBD. Heterogeneity within each time-point . At late times Tyr- becomes prototroph! Reppas, Shendure, Porecca -12 -11 -10 -9 -8 -7 -6 .