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

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

3 1 st -generation Gene synthesis vs 2nd-generation Sequencing & DNA synthesis Moore’s law =1.5x/yr vs 10x/yr 24Mb/$ 30kb/$

4 Ultra-low-cost sequencing 1. Polonator SbL/P Open-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-sm High parallelism & quantitation 7. Halcyon EM-sm Long reads (>Mb), $ 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-sm Long reads (>2.0 kb) 13. Bionanomatrix SbP-sm Fluorescent mapping 14. OxfordNanopore Pore-protein-sm small device 15. Visigen SbP-sm Pol <> dNTP FRET 16. ZS Genetics EM-sm Iodine labels 17. Nabsys Pore-sm small device 18. GE Global SbP-sm 19. IBM Pore Si-sm small device 20. Electronic BiosciPore-protein-sm Polonator

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

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

7 Cell Chassis Choice E.coliS.cerSynec.M.gen Biomass doubling Chemical tolerance++++- ss-MAGE++??? ds-recombination++++- Industrial use++ +-

8 Genome engineering Dupont: 1,3 Propanediol (7 years & $400M R&D) 135 g/l at 3.5 g/l/h heoretical yield from glucose 90% of theoretical yield from glucose 27 changes to 4.6 Mbp E.coli 6 genes up, 13 down, 8 foreign genes Glycerol DAR1 GPP2 Glycerol-3-P - NADH 3HPA coB 12 - NADPH yqhD dhaB1-3 1,3 propanediol Yeast Klebsiella E.coli for Sorona polymers

9 Bio-petroleum from microbes flotation -- not distillation aqueous organic Extracellular Intracellular Localization Distribution (% total) , months Gasoline & diesel for current engines & infrastructure Fatty acid derived

10 Algal, cyanobacterial & fungal pathways to triglycerides, alkanes, olefins, terpenes Botryococcus braunii decarbonylase Methylelcosene from Prasiola stipitata

Helioculture™ Technology 11

12,800 mols PAR photons into SC Joule Diesel Productivity Model - photon perspective Extraterrestrial:43,100 MJ Total Radiation 16,900 MJ PAR Atmosphere, Diurnal Cycle Phoenix:3400 MJ PAR ≈ 15,100 mols PAR photons Historical Average Total Radiation to the Ground (MJ/m 2 /year) Historical Average PAR Radiation to the Ground (MJ/m 2 /year) Lanai, HI El Paso, TX Phoenix, AZ Las Vegas, NV Albuquerque, NM Leander, TX % Photons Transmitted into SC Diesel: 15K gal/acre/yr NREL insolation data kJ/mol incident PAR photons 85% photon utilization 926 mols CO2 reduced Unless otherwise stated, all values are /m 2 /year 880 mols CO2 reduced10,900 mols PAR photons photons per mol CO 2 reduced 5% to cellular maintenance 835 mols CO2 reduced to Diesel = 70 mols Diesel produced (C 12 H 26 ) 170 g/mol 5% of operation time to culture growth 11.8 kg Diesel

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 Lysine Molybdenum PreQ1 SAM SAH TPP theophylline 3- methylxanthine

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

15 Multiplex Automated Genome Engineering (MAGE) Wang HH, in preparation, 2009 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 Allelic Replacement Strain: MG1655,  mutS, integrated -Red Highly complex oligo pools for multiplexed multi-loci modifications >4 billion bp of targeted genetic variation produced per day

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

17 Genome Engineering strategies 1.Random mutagenesis limited by lethal rate 2.Genome synthesis 3.Semi-random 4.Cyclic semi-random (MAGE) 5.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 PEG-pAcPhe-hGH (Ambrx) high serum stability 314 TAG to TAA changes Isaacs Charalel Church Sun Wang Carr Jacobson Kong Sterling New translation code: novel amino acids Safety features: no functional DNA exchange multi-virus resistance TTT F 30362TCT S 11495TAT Y 21999TGT C 7048 TTC22516TCC11720TAC16601TGC8816 TTA L 18932TCA9783TAA STOP 2703TGA STOP 1256 TTG18602TCG12166TAG314TGGW20683 CTT L 15002CCT P 9559CAT H 17613CGT R CTC15077CCC7485CAC13227CGC29898 CTA5314CCA11471CAA Q 20888CGA4859 CTG71553CCG31515CAG39188CGG7399 ATT I 41309ACT T 12198AAT N 24159AGT S ATC34178ACC31796AAC29385AGC21862 ATA 5967ACA9670AAA K 45687AGA R 2896 ATGM37915ACG19624AAG14029AGG1692 GTT V 24858GCT A 20762GAT D 43719GGT G GTC20753GCC34695GAC25918GGC40285 GTA14822GCA27418GAA E 53641GGA10893 GTG35918GCG45741GAG24254GGG

20 Multi-virus resistance: stop codons: TAG / total  X-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 T4168,903 b ds-DNA 19 / 277 E.coli 4,639,675 b ds-DNA 314 /1,360,152 ncbi.nlm.nih.gov/nuccore/ (7 tRNAs: RITSPGL) Farren Isaacs

tRNAs /sec 20 aminoacids/sec mRNA tRNAs 50S/ Ribosome Elongation factors 30S

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

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

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

26 Ribosomes from synthetic 23S rRNA with coupled translation of active luciferase Traub & Nomura 1968 reconstitution of 30S Mike Jewett Synthetic 23S rRNA ( + ) (-) ng/ml 30S R50S 30S 50S 30S -- 5S rRNA TP50 30S IVT 23S rRNA 5S rRNA TP50 30S --

25-Jan :30 DOE Session E: Synthetic Biology Genome 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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