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Fabricating with DNA

Fabricating with DNA. George Church Wed 22-Aug-2007 9:15 – 9:30 AM 4 th Fab Lab Forum & Digital Fabrication Symposium. Thanks to:. AppliedBiosystems, Helicos, Roche454, Illumina, CGI, IBS, Affymetrix, Enzymatics. PGP Volunteers & Donors !. Fab vs. Bio-fab.

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Fabricating with DNA

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  1. 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 !

  2. 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

  3. 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)

  4. 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

  5. Open-source hardware, software, wetware, ELSwarefor DNA reading & writing Church, Gilbert Genomic Sequencing (electrotransfer, chemiluminescence, film scanner) Higgins, Richerich, Auger, Smith Multiplex Sequencing 78-84 87-97 Polony A 97-99 Rob Mitra MJR slide-cycler GSI Microarray scanner B 02-03 Greg Porecca Jay Shendure

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

  7. 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

  8. Synthetic Biology: augmentation & combinatorics (not minimization) • 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

  9. Mirror world:resistant to enzymes, parasites, predators Mirror aptamers, ribozymes, etc. require mirror polymerases 834 AA Thermus Polymerase 834 AA 352 AADpo4 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.

  10. 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 Tony Forster (Vanderbilt) Duhee Bang (HMS)

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

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

  13. Improved Recombination Frequency: 10-4 0.25 (> 3 log increase!) 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)

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

  15. 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

  16. Recombination-Cycling UAG to UAA E.coli Essential Genes Mutation Distribution: 11 oligos, 15 cycles Mutation Distribution: 54 oligos, 45 cycles * Continuous cycling • Scaling & Automation • Increase Efficiency of Recombination Wang, Isaacs, Carr, Jacobson, Church

  17. Intelligent Design & Metabolic Evolution Lenski Citrate utilization Palsson Glycerol utilization Edwards Radiation resistance Ingram Lactate production Stephanopoulos Ethanol resistance Marliere Thermotolerance J&J Diarylquinoline resistance (TB) DuPont 1,3-propanediol production Church Trp/Tyr exchange

  18. 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

  19. 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).

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

  21. -12 -11 -10 -9 -8 -7 -6 Co-evolution of mutual biosensors/biosynthesissequenced across time & within each time-point Independent lines of TrpD & TyrD 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) 1bD, 9bD, 8bD, IS2 insert, R->L in DBD. Heterogeneity within each time-point . At late times Tyr- becomes prototroph! Reppas, Shendure, Porecca

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