1. DOE GTL Vertically Integrated BioEnergy Research Center (special thanks to Harvard Inst. for Biologically Inspired Engineering) TimeAgenda item (77 PIs 75 pages 390 minutes today) 8:30-8:45Introduction and enabling technologies (GC) 8:45-9:15Plan for integrated response to RFA (JA) 9:15-9:45Plants and Agriculture 9:45-10:15Saccharification + CBP 10:15-10:30Break 10:30-11:00Fermentation 11:00-11:15Process modeling and improvement 11:15-11:30Alternative biofuels and paradigms 11:30-12:00Pretreatment, extraction, use 12:00-1:00Working lunch: IP, other BRCs 1:00-2:00Open Discussion 2:00-2:30Budgets 2:30-3:00Next steps

2. Brazil’s Bioethanol (& GtL Economic modeling needs) Land use:45,000 km² Sugarcane: 344 million tons (76 tons/ha) Sugar: 23 million tons Ethanol:14 million m³ $0.26/L ( feedstock 70% ) yield increase 3.5%/yr Dry bagasse: 50 million tons Electricity: 1350 MW Bagasse ash 2.5% (vs 40% for coal), nearly no sulfur. Burns at low temperatures, so low nitrogen oxides. Saccharum officinarum

3. The ‘G’ in GtL '77 Gilbert & Sanger papers '77 developed 1 st auto-sequence-reading software '77 1 st full plasmid sequence (recombinant DNA) '84 ‘Genomic Sequencing’ PNAS paper & DOE Alta meeting '87 1 st Genome grant (DOE) '90 co-PI on 1 st NIH Genome Centers, Stanford,GTC,MIT (now Broad) '94 1 st genome sequence H.pylori 1.7 Mbp (commercial) '02 Only DOE Center to address all 5 GtL goals '06 SynBERC grant with LBL, MIT, etc. #1: Protein complexes & Mass Spec #2: Regulatory Networks & RNA #3: Microbial Communities #4: Computational models #5: Synthetic Biology

4. Plants  Zea,Miscanthus,Poplar, Crambe, Algae Pretreat  acid, NH 4 fiber explosion (AFEX), silage Saccharify  Trichoderma, Clostridia, Aspergillus Soluble, cellulosome, consolidated Ferment  Saccharomyces, Escherichia, Zymomonas, Pichia, Klebsiella, communities Extract  distill, gas strip, zeolite, phase Process  crack, blend ethanol, butanol, ethylesters, alkanes, H 2 Uses  transportation, chemicals, power Vertical Integration (horizontal options) AGRICULTURE Enabling Tech: Modeling, Synthesis, Evolution, Sequencing

5. Our GTL network Church- Harvard Silver Shih Curtis-PSU Chisholm -MIT Leschine-UMA Codon Devices Collins-BU LS9 Agrivida Hamilton-INL Ausubel-MGH Liu-BNL SynBERC-iGEM Ingram-UFL Chappell-UKY Laible-ANL Richard-PSU Mendel GreenFuel Celunol Chromatin SunEthanol DuPont Shell Vance-WashU BioEnergy International Endy Polz Pendse-UME Cerrina-UW

6. Enabling Technologies #1: Computational & Systems Modeling

7. Enabling Technologies #2: Lab evolution (building on systems design) #3: Functional metagenomics (interspecies DNA transfer) Radiation resistance (Edwards & Battista) Tyr/Trp production & transport (Lin & Reppas) Citrate utilization (Lenski) Lactate production (Ingram) Temperature/acid tolerance (Marliere) Glycerol utilization (Palsson) Aldehyde resistance (Sommer & Dantas)

8. PositionType GeneLocationFunctionMechanism 986,334 T > GompFPromoter-10 Promoter of Non-specific transport channel Makes promoter more consensus-like 985,797 T > GompFGlu > Ala Non-specific transport channel Makes pore bigger and more hydrophobic 931,960  8 bplrpframeshift General Transcriptional Regulator ? Whole-Genome resequencing of evolved  Trp Shendure, et al. (2005) Science 309:1728 ompF – non specific transport channel Glu-117 → Ala (in the pore) Charged residue known to affect pore size and selectivity Can increase import & export capability simultaneously

9. Tech #4: ‘Next Generation’ Sequencing Multi-molecule Reaction Volume AB/APG Ligase beads 1 fL 454/Roche Pol beads 100,000 fL Solexa Pol term 1 fL CGI Ligase 1 fL Affymetrix Hybr array 100 fL IBS Pol beads 10,000 fL Single molecules Helicos Biosci Pol <1fL Visigen Biotech Pol FRET <1fL Pacific Biosci Pol <1fL Agilent Nanopores <1fL fL =1E-15 liters (femto) Our lab has been involved in 8/10

10. #5: Sequencing genomes from single cells 1) Environmental samples (poor or no lab growth) 2) Candidate chromosome region sequencing 3) Prioritizing or pooling (rare) species based on an initial DNA screen (metagenomics) 4) Multiple chromosomes in a cell or virus 5) RNA 5’, 3’ ends & splicing 6) Cell-cell interactions (predator-prey, symbionts, commensals, parasites) Phi-29 Polymerase Stand-displacement amplification (single chromosome, cell, RNA or particle) Zhang, et al. (2006). Nature Biotech. June ’06

11. #6: 10 Mbp of DNA / $1000 chip 8K Atactic/Xeotron/Invitrogen Photo-Generated Acid 12K Combimatrix/Codon 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 #7: Recombinational Genome Engineering Amplify pools of 50mers using flanking universal PCR primers & 3 paths to 10X error correction Digital Micromirror Array

12. #8: 3D Structural Biology – especially membrane and fibrous structures Hanson & Laible Shih, Douglas, iGEM #9, 10: Proteomics, Metabolomics

13. DOE GTL Vertically Integrated Bioenergy and Novel Technologies (VIBRANT) TimeAgenda item (77 PIs 75 pages 390 minutes today) 8:30-8:45Introduction and enabling technologies (GC) 8:45-9:15Plan for integrated response to RFA (JA) 9:15-9:45Plants and Agriculture 9:45-10:15Saccharification + CBP 10:15-10:30Break 10:30-11:00Fermentation 11:00-11:15Process modeling and improvement 11:15-11:30Alternative biofuels and paradigms 11:30-12:00Pretreatment, extraction, use 12:00-1:00Working lunch: IP, other BRCs 1:00-2:00Open Discussion 2:00-2:30Budgets 2:30-3:00Next steps

14. How do we distinguish ourselves from other GTL proposals? The DOE-GTL-BRC competition (2 – 3 will be funded) 1. LBL-LLNL-Sandia-Stanford-U.IL 2. ORNL-NREL-Dartmouth-Noble-OK 3. Harvard-MIT-UMA-UME-BU-UFL-PSU-UWI-ANL-INL-BNL- 11 Companies 4. San Diego-Iowa-JCVI 5. Purdue-MIT2 6. UWI-MSU 7. LANL BP competition (1 will be funded) 1. Imperial College, UK 2. Cambridge, UK 3. LBL-LLNL-Sandia-Stanford 4. San Diego-Iowa-JCVI 5. MIT-Purdue

15. Snapshots from the 1 st DOE-VIBRANT meeting Boston 21-Nov-2006