Fabricating with DNA George Church Wed 22 -Aug-2007

Fabricating with DNA George Church Wed 22 -Aug-2007 9: 15 – 9: 30 AM 4 th Fab Lab Forum & Digital Fabrication Symposium Thanks to: Applied. Biosystems, Helicos, Roche 454, 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 3 D 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 E 07 Faster XYZ D 05 = C 03 + fluidics F 07 : $106 K (down from $500 K) including computer Rich Terry G 07: internal autosampler

10 Mbp of DNA / $300 chip Spatially patterned chemistry 8 K Atactic/Xeotron/Invitrogen Photo-Generated Acid 12 K Combimatrix Electrolytic 44 K Agilent Ink-jet standard reagents 380 K Nimblegen/GA Photolabile 5'protection Amplify pools of 50 mers using flanking universal PCR primers & 3 paths to 10 X 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: 1 Mbp per month (Codon Devices) New polymers in vitro – affinity selection (Vanderbilt) Hydrocarbon & other chemical syntheses in E. coli (LS 9) Bacterial & stem cell therapies (Syn. BERC & 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 Dpo 4 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, T 7 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 Genome. Res. ’ 06 Shimizu, Ueda et al ‘ 01 113 kbp DNA 151 genes

Genome engineering CAD Polymerase in vitro Recombination in vivo E. coli 70 b 15 Kb Error Correction Mut. S 1 E-4 Chemical Synthesis 1 E-2 Recombination in human cells 5 Mb 250 Mb Bacterial (Artificial) Chromosomes BACs Human(Artificial) Chromosomes HACs Sequencing 1 E-7 Isaacs, Carr, Emig, Gong, Tian, Reppas, Jacobson, Church

r. E. 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 (mut. H, mut. L, mut. S, uvr. D, 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 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 Ø Scaling & Automation Ø Increase Efficiency of Recombination Wang, Isaacs, Carr, Jacobson, Church

Intelligent Design & Metabolic Evolution Lenski Palsson Edwards Ingram Stephanopoulos Marliere J&J Du. Pont 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 (618~641 kb) 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/ tyr. A 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. D & Tyr. D co-culture 5 Omp. F: (pore: large, hydrophilic > small) 42 R-> G, L, C, 113 D->V, 117 E->A 2 Promoter: (cis-regulator) -12 A->C, -35 C->A 5 Lrp: (trans-regulator) 1 b. D, 9 b. D, 8 b. D, IS 2 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

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