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25-Jan-2010 3-3:30 DOE  Session E: Synthetic Biology Genome Engineering, Multi-virus Resistance & Accelerated Evolution for Industrial Chemicals (Harvard). Thanks to: .gov || || .edu || || .org || || .com || || Read = = = = = = = = I/O = = = = = = = Write. LSRF. Azco.

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  1. 25-Jan-2010 3-3:30 DOE  Session E: Synthetic BiologyGenome Engineering, Multi-virus Resistance & Accelerated Evolution for Industrial Chemicals (Harvard) Thanks to: .gov || || .edu || || .org || || .com || || Read = = = = = = = = I/O = = = = = = = Write LSRF Azco RBH

  2. Seq bp/$ $/genome 4 logs in 4 years 2009:Lig:$1.5K (Moore’s law) 1.5x/yr for electronicsvs 10x/yr for DNA Sequencing Pol:$50K 2005:capil:$50M 1995:gel: $3G >20 years ahead of the 1970-2004 exponential

  3. 24Mb/$ 30kb/$ Moore’s law =1.5x/yrvs 10x/yr 1st-generationGene synthesis vs2nd-generationSequencing&DNA synthesis

  4. Ultra-low-cost sequencing Polonator 1. PolonatorSbL/POpen-source$170K device, haplotypes 2. CGI SbL $5K genome Rolony grid & 100Kb haplotypes 3. Roche-454 SbP Long reads (>0.4 kb) 4. Illumina-GA SbP Fluorescent read-length 2*110 bp 5. AB-SOLiD SbL Longest ligation reads 6. Helicos SbP-smHigh parallelism & quantitation 7. Halcyon EM-sm Long reads (>Mb), $100 8. Ion Torrent SbP small device 9. Genizon BioSci SbH In situ sequencing 10. LightSpeed SbL 16X higher density, >10X speed 11. Intelligent Bio SbP Hexagonal grid 12. Pacific Bio SbP-smLong reads (>2.0 kb) 13. Bionanomatrix SbP-smFluorescent mapping 14. OxfordNanopore Pore-protein-sm small device 15. Visigen SbP-smPol <> dNTP FRET 16. ZS Genetics EM-smIodine labels 17. NabsysPore-sm small device 18. GE GlobalSbP-sm 19. IBM Pore Si-sm small device 20. Electronic Biosci Pore-protein-sm Polonator

  5. Figure 4.6.1 Polonator instrument Open-architecture hardware, software, wetware A shared resource e.g.: 1981 IBM PC 1998 Google $160K 2 billion beads per run Polonator Rich Terry

  6. Polonator From Sequencer to Bio-Fab: Selective release of Synthetic sequences &/or cell sorting (FACS) Open-access hardware, software, wetware Digital micromirrors Flow- cell 2 billion beads or cells/run Photo-labile immobilization

  7. Cell Chassis Choice

  8. Genome engineering 2000-2006 E.coli Klebsiella Yeast yqhD DAR1 1,3 propanediol dhaB1-3 GPP2 Glycerol-3-P Glycerol 3HPA - NADH coB12 - NADPH Dupont: 1,3 Propanediol (7 years & $400M R&D) 135 g/l at 3.5 g/l/h 90% of theoretical yield from glucose 27 changes to 4.6 MbpE.coli 6 genes up, 13 down, 8 foreign genes for Sorona polymers

  9. 3,000 100 50 90 organic 80 70 60 Fatty acid derived Distribution (% total) 50 10 1 40 aqueous 30 20 1 2 3 4 10 0 Extracellular Intracellular 3 months Localization Bio-petroleum from microbes flotation -- not distillation Gasoline & diesel for current engines & infrastructure

  10. Algal, cyanobacterial & fungal pathways to triglycerides, alkanes, olefins, terpenes Botryococcus braunii decarbonylase Methylelcosene from Prasiola stipitata http://www.biofuelsdatabase.org/map/alkane-decarbonylation_map.shtml http://www.springerlink.com/content/p6451qx982638856/fulltext.pdf

  11. Helioculture™ Technology

  12. Joule Diesel Productivity Model- photon perspective Unless otherwise stated, all values are /m2/year Extraterrestrial: 43,100 MJ Total Radiation 16,900 MJ PAR Atmosphere, Diurnal Cycle 226 kJ/mol incident PAR photons Phoenix: 3400 MJ PAR ≈ 15,100 mols PAR photons NREL insolation data 85% Photons Transmitted into SC 10,900 mols PAR photons 926 mols CO2 reduced 880 mols CO2 reduced 12,800 mols PAR photonsinto SC 11.75 photons per mol CO2 reduced 5% to cellular maintenance 85% photon utilization 5% of operation time to culture growth 835 mols CO2 reduced to Diesel = 70 mols Diesel produced (C12H26) Diesel: 15K gal/acre/yr 11.8 kg Diesel 170 g/mol

  13. Existing Sensors (select for new ligands) 54 DNA binding proteins: ada araC arcA argPR carP cpxR crp cspA cynR cysB cytR deoR dnaA dgsA fadR farR fhlA flhCD fnr fruR fur galR gcvA glpR hipB iclR ilvY lacI lexA lrp malT marR melR metJ metR modE nagC narL narP ntrC ompR oxyR pdhR phoB purR rhaS rpoE rpoH rpoN rpoS soxS torR trpR tyrR 12 Riboswitches: Adenine B12 FMN Guanine Glucosamine-6-phosphate Glycine di-GMP LysineMolybdenum PreQ1 SAM SAH TPP theophylline 3-methylxanthine http://pubs.acs.org/doi/abs/10.1021/ja048634j

  14. Multiplex Automated Genome Engineering 2 hr Cycle time. For optimization & multivirus resistance Harris Wang

  15. Multiplex Automated Genome Engineering (MAGE) • Allelic Replacement • Strain: MG1655, DmutS, integrated -Red • Highly complex oligo pools for multiplexed multi-loci modifications • >4 billion bp of targeted genetic variation produced per day • Optimized Parameters • Oligo length: 90mer • Oligo 2ndary structure: <12 kcal/mol • Oligo half-life: 5’ phosphothiol bps • Oligo conc.: up to 50 uM • Cycle time: 2 to 2.5 hrs • >30% efficiency per cycle Wang HH, in preparation, 2009

  16. Accelerated Evolution 23K combinations per geneLycopene (hydrocarbon): 20 genes up, 4 down, 2 new 5-fold improvement in 3 days AND improve growth rates Wang H et al Nature 2009

  17. Genome Engineering strategies • Random mutagenesis limited by lethal rate • Genome synthesis • Semi-random • Cyclic semi-random (MAGE) • Directed mutations (MAGE)

  18. Improving process yield, health, safety with 314+ changes What threatens all biological systems? What do all viruses have in common? or lack?

  19. New translation code: novel amino acidsSafety features: no functional DNA exchangemulti-virus resistance PEG-pAcPhe-hGH (Ambrx) high serum stability 314 TAG to TAA changes 4 Isaacs Charalel Church Sun Wang Carr Jacobson Kong Sterling 1 3 2

  20. Multi-virus resistance:stop codons: TAG / total fX-174 5,386 b ss-DNA 0 / 9 M13 6,407 b ss-DNA 1 / 10 MS2 3,569 b ss-RNA 2 / 4 T7 39,937 b ds-DNA 6 / 60 T4 168,903 b ds-DNA 19 / 277 E.coli 4,639,675 b ds-DNA 314 /1,360,152 Farren Isaacs ncbi.nlm.nih.gov/nuccore/9626372 56718463 176120924 9627425 29366675 (7 tRNAs: RITSPGL)

  21. 400 tRNAs /sec20 aminoacids/sec Elongation factors 30S 50S/ Ribosome tRNAs mRNA

  22. Applications of in vitro translation • Ribosome display • Membrane protein drug receptor studies • Personal cancer vaccines. • Labeling one protein not the whole cell. • New chemistries (e.g. mirror chirality) • >11 Commercial Systems: Roche, Ambion, Novagen, Promega, Invitrogen, Qiagen, Stratagene, Paragon, Amersham,Sutro,NEB Tony Forster (Vanderbilt)

  23. 2 key chirality gatekeepers: peptidyl transferase & AA-tRNA synthetases So: 10 bp change in 23S rRNA + a 46 nt ribozyme Mirror world : Goals: Multi-enzyme resistance, metabolic isolation, new chiral chemistries

  24. Mirror world : Construction of Modified Ribosomes for Incorporation of d-Amino Acids into Proteins. Hecht lab Biochemistry 2006 A highly flexible tRNA acylation method for non-natural polypeptide Synthesis. Suga lab Nature Methods 2006

  25. Not minimal:High speed & accuracy requires a few extra genes(E.coli 20 min. doubling) Reconstituted ribosomes:Jewett & Church 113 kbp DNA 151 genes Pure translation: Forster & Church MSB ’05 GenomeRes.’06 Shimizu, Ueda ’01

  26. Ribosomes from synthetic 23S rRNA with coupled translation of active luciferase ng/ml Synthetic 23S rRNA (+) (-) 30S -- 30S50S 30SR50S 5S rRNA TP50 30S IVT 23S rRNA 5S rRNA TP50 30S -- Mike Jewett Traub & Nomura 1968 reconstitution of 30S

  27. 25-Jan-2010 3-3:30 DOE  Session E: Synthetic BiologyGenome Engineering, Multi-virus Resistance & Accelerated Evolution for Industrial Chemicals 1. Polonator: Merge next-gen Seq & Synth 2. LS9-Chevron & JouleBio hydrocarbon production 3. MAGE: combinatoric: Lycopene 4. MAGE: 314 change for multi-virus resistance 5. Synthetic ribosome replication

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