George Church Wed 22-Aug :15 – 9:30 AM 4 th Fab Lab Forum & Digital Fabrication Symposium Thanks to: Fabricating with DNA AppliedBiosystems, Helicos, Roche454, Illumina, CGI, IBS, Affymetrix, Enzymatics PGP Volunteers & Donors !

Fab vs. Bio-fab + Plays well with digital computers - No habla C++ - Doesn’t get DNA + DNA is it’s native digital media - Needs us to replicate + We need them - Needs expensive Fab (e.g. ICs) + Simple or complex inputs - Intelligent Design + 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 DNA-nanotube-induced alignment of membrane proteins for NMR structure determination Rothemund Nature’06 Douglas, et al. PNAS’07

Open-source hardware, software, wetware, ELSware for DNA reading & writing A Greg Porecca Rob Mitra B Jay Shendure MJR slide-cycler GSI Microarray scanner Church, Gilbert Genomic Sequencing (electrotransfer, chemiluminescence, film scanner) Higgins, Richerich, Auger, Smith Multiplex Sequencing Polony

Open-source hardware, etc: Personal Genome Project D05 = C03 + fluidics Rich Terry E07 Faster XYZ F07 : $106K (down from $500K) including computer G07: internal autosampler

10 Mbp of DNA / $300 chip 8K Atactic/Xeotron/Invitrogen Photo-Generated Acid 12K Combimatrix Electrolytic 44K Agilent Ink-jet standard reagents 380K Nimblegen/GA Photolabile 5'protection Tian et al. Nature. 432:1050 Carr & Jacobson 2004 NAR Smith & Modrich 1997 PNAS Spatially patterned chemistry Amplify pools of 50mers using flanking universal PCR primers & 3 paths to 10X error correction

Synthetic Biology: augmentation & combinatorics (not minimization) 1.Synthetic DNA: 1Mbp per month (Codon Devices) 2.New polymers in vitro – affinity selection (Vanderbilt) 3.Hydrocarbon & other chemical syntheses in E.coli (LS9) 4.Bacterial & stem cell therapies (SynBERC & MGH) 5.New codes: Viral resistant cells & new aminoacids (MIT) 6.Synthetic Ecosystems – Evolve secretion & signaling 7.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.

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 Tony Forster (Vanderbilt) Duhee Bang (HMS) Why synthesize (minimal) in vitro self-replication?

113 kbp DNA 151 genes ideal for comprehensive atomic, ODE & stochastic models Forster & Church MSB ‘05 GenomeRes.’06 Shimizu, Ueda et al ‘01 Pure in vitro translating & replicating system

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

rE.coli Strategy #3: ss-Oligonucleotide Repair Obtain 25% recombination efficiency in E. coli strains lacking mismatch repair genes (mutH, mutL, mutS, uvrD, dam) Ellis et al. PNAS 2001 Constantino & Court. PNAS 2003 DNA Replication Fork Improved Recombination Frequency:  0.25 (> 3 log increase!)

Multiplex Automated Genome Engineering (MAGE) Wash with water & DNA pool (50) Concentrate, electroporate Resuspend, bubble, select O-ring membrane Concentrate Wang, Isaacs, Terry

Multiplex Automated Genome Engineering (MAGE) syringe pump electrically actuated valves electroporation cuvette w/ membrane filter OD sensor data acquisition system computer communication / Wang, Isaacs, Terry

Recombination-Cycling UAG to UAA E.coli Essential Genes Mutation Distribution: 11 oligos, 15 cyclesMutation Distribution: 54 oligos, 45 cycles Oligo Pool # cyclesBest Clone (98 %tile)Fraction of mutated sitesTime* /113 days /549 days * Continuous cycling  Scaling & Automation  Increase Efficiency of Recombination Wang, Isaacs, Carr, Jacobson, Church

Intelligent Design & Metabolic Evolution LenskiCitrate utilization PalssonGlycerol utilization Edwards Radiation resistance IngramLactate production StephanopoulosEthanol resistance MarliereThermotolerance J&JDiarylquinoline resistance (TB) DuPont1,3-propanediol production ChurchTrp/Tyr exchange

Cross-feeding symbiotic systems: aphids & Buchnera obligate mutualism nutritional interactions: amino acids & vitamins established million years ago close relative of E. coli with tiny genome (618~641kb) Aphids MILKFTWV MILKFTWV HR

Shigenobu et al. Genome sequence of the endocellular bacterial symbiont of aphids Buchnera sp.APS. Nature 407, (2000). Pink= enzymes apparently missing in Bucherna

 trp/  tyrA pair of genomes shows best co- growth Reppas, Lin et al. ; Accurate Multiplex Polony Sequencing of an Evolved Bacterial Genome 2005 Science Second Passage First Passage Synthetic genome pair evolution

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. Reppas, Shendure, Porecca At late times Tyr- becomes prototroph!

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