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DNA Sequencing (& synthesis)

DNA Sequencing (& synthesis). Microbe-weather-maps: single cell sequencing @ $ 0.02/kb 30 bp microbial species then drug resistance (~1000 cells/$) Personal IDs from dust : 30 bp from single cells then immune VDJ.

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DNA Sequencing (& synthesis)

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  1. DNA Sequencing (& synthesis) • Microbe-weather-maps: single cell sequencing @ $ 0.02/kb • 30 bp microbial species then drug resistance (~1000 cells/$) • Personal IDs from dust: 30 bp from single cells then immune VDJ Cost of DNA Synthesis & Sequencing have dropped 100 -1000X in the last few years Polony Sequencing & Personal Genome Project Identifiable traits & common (low risk) pharmacogenomics

  2. DNA Instrumentation Sequencing $1K/ 1E8*50bp/2days DNA Synthesis $1K/ 1E6*160bp/day Cell/Genome programming 4/day/well

  3. In vivo/vitro Synthetic Biology A synthetic multicellular system for programmed pattern formation Basu, et al. Nature 434:1130

  4. DNA origami highly -- predictable 3D nanostructures. Example application to NMR of membrane proteins Douglas, Shih, Rothemund

  5. Programmable ligand-controlled riboregulators to monitor metabolites. OFF ON ON Bayer & Smolke; Isaacs & Collins 2005 Nature Biotech.

  6. Int Xis TF4 1 Int Xis TF3 2 Int Xis TF5 3 Int Xis TF6 4 In vivo Counter Designs 1 2 3 • Riboswitch • counter  • 0 • 1 • 1 • 0 • Integrase bit counter • Cell-cycle counter 

  7. Reconstitution of Circadian Oscillation of Cyanobacterial KaiC Phosphorylation in vitro Nakajima, et al. Science. 2005 Apr 15;308(5720):414-5

  8. Engineering a mevalonate pathway in Escherichia coli for production of terpenoids.Martin VJ, et al. Nat. Biotech 2003 Production of the antimalarial drug precursor artemisinic acid in engineered yeast. Ro DK, et al. Nature. 2006 8

  9. Smart therapeutics: Environmentally controlled invasion of cancer cells by engineered bacteria. Anderson et al. J Mol Biol. 2006 Regulated Capsule TonB, DapD & new genetic code for safety Optical imaging: bacteria, viruses, and mammalian cells encoding light- emitting proteins reveal the locations of primary tumors & metastases in animals. Yu, et al.Anal. Bioanal. Chem. 2003. accumulate in tumors at ratios in excess of 1000:1 compared with normal tissues. http://www.vionpharm.com/tapet_virulence.html

  10. rEcoded rE.coli v2.0 Synthetic Genomics • 113 kbp mini-genomes ribosome-display selection • 4.7 Mbp new genetic codes protein drugs • 7*7 * 4.7 Mbp mini-ecosystems • biosensors, surface selection, high secretors, • DNA & metabolic isolation • Top Design Utility, safety & scalability • CAD-PAM • Synthesis(chip & error correction) • Combinatorics • Evolution • Sequence

  11. Genome synthesis pipeline Recombination in human cells Recombination in vivo E.coli Polymerase in vitro 50b 15Kb 5Mb 250 Mb Human(Artificial) Chromosomes HACs Error correction Bacterial (Artificial) Chromosomes BACs Isaacs, Carr, Emig, Gong, Tian, Reppas, Jacobson, Church

  12. Rationale for in vitro synthetic biology Existing powerful molecular biology kits: DNA RNA Protein PCR, T7 RNA pol, in vitro translation. Build structures incompatible with cells (systems interfering with cells or vice versa) Higher yield, accuracy,purity and/or speed.

  13. Why new genetic codes? Precise "post-translational" modifications New reactive AAs (not w/ biomolecules) triazole Strained cyclo-octyne Azide + Ketone hydrazone hydrazide + e.g. PEG-pAcPhe-hGH (Ambrx, Schultz) higher serum stability Prescher, JA & CR Bertozzi (2005) Nature Chem Biol. Chemistry in living systems

  14. Mirror world:resistant to enzymes, parasites, predators L-amino acids & D-ribose (rNTPs, dNTPs) Transition: EF-Tu, peptidyl transferase, DNA-ligase D-amino acids & L-ribose (rNTPs, dNTPs) Dedkova, et al. (2003)Enhanced D-amino acid incorporation into protein by modified ribosomes. J Am Chem Soc 125, 6616-7

  15. Mirror world:resistant to enzymes, parasites, predators Alternative to ambidextrous ribosome & polymerases is chiral chemical synthesis (Taq Polymerase is 834 AA) >174 AA African Swine Fever Virus DNA Pol X . MLTLIQGKKIVNHLRSRLAFEYNGQLIKILSKNIVAVGSLRREEKMLNDVDLLIIVPEKKLLKHVLPNIRIKGLSFSVKVCGERKCVLFIEWEKKTYQLDLFTALAEEKPYAIFHFTGPVSYLIRIRAALKKKNYKLNQYGLFKNQTLVPLKITTEKELIKELGFTYRIPKKRL Duhee Bang & Stephen Kent (2004) A One-Pot Total Synthesis of Crambin

  16. Mini-genone: 113 kbp, 151 genes

  17. Mini-genone: 113 kbp, 151 genes

  18. Synthesizing A Minimal Cell small molecules DNA pol recombinase DNA RNA pol precursor RNAs, P RNA, mRNAs RNase P P protein modifying enzymes synthetases, MFT Gln methylase tRNAs rRNAs rProteins AA-tRNAs ribosome chaperones translation factors Forster & Church (2006) Mol. Syst. Biol.

  19. DNA replication

  20. 31 Tagged translation proteins Synthe- tases: Factors: Forster, Nalam, Qu, Ying & Blacklow

  21. 33 tRNAs with anticodon modifications

  22. Systems Biology of Translation and Replication 151 Genes required.

  23. yU mS eU UUG UGG CAG | | | | | | | | | ... AUG AAC ACC GUU GAA 5' A 3' fM N T V E Totally unnatural genetic codes 5' 3' Second base A U A C U C A C yU mS U G eU 80% average yield per unnatural coupling. bK = biotinyllysine , mS = Omethylserine eU=2-amino-4-pentenoic acid yU = 2-amino-4-pentynoic acid Forster, et al. (2003)

  24. 3 Exponential technologies(synergistic) Computation & Communication (bits/sec) E.coli operons Synthesis (daltons) tRNA urea B12 Analysis (bp/$) telegraph tRNA Shendure J, Mitra R, Varma C, Church GM, 2004 Nature Reviews of Genetics. Carlson 2003 ; Kurzweil 2002; Moore 1965

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