DOE%20GTL%20Vertically%20Integrated%20BioEnergy%20Research%20Center%20(special%20thanks%20to%20Harvard%20Inst.%20for%20Biologically%20Inspired%20Engineering)

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DOE GTL Vertically Integrated BioEnergy Research
Center(special thanks to Harvard Inst. for
Biologically Inspired Engineering)
Time Agenda item (77 PIs 75 pages 390 minutes today)
830-845 Introduction and enabling technologies (GC)
845-915 Plan for integrated response to RFA (JA)
915-945 Plants and Agriculture
945-1015 Saccharification CBP
1015-1030 Break
1030-1100 Fermentation
1100-1115 Process modeling and improvement
1115-1130 Alternative biofuels and paradigms
1130-1200 Pretreatment, extraction, use
1200-100 Working lunch IP, other BRCs
100-200 Open Discussion
200-230 Budgets
230-300 Next steps
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Brazils Bioethanol( GtL Economic modeling
needs)

• Land use45,000 km²
• Sugarcane 344 million tons (76 tons/ha)
• Sugar 23 million tons
• Ethanol14 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
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The G in GtL
'77 Gilbert Sanger papers '77 developed 1st
auto-sequence-reading software '77 1st full
plasmid sequence (recombinant DNA) '84 Genomic
Sequencing PNAS paper DOE Alta meeting '87 1st
Genome grant (DOE) '90 co-PI on 1st NIH Genome
Centers, Stanford,GTC,MIT (now Broad) '94 1st
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
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Vertical Integration (horizontal options)
Plants ? Zea,Miscanthus,Poplar, Crambe,
Algae Pretreat ? acid, NH4 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, H2 Uses
? transportation, chemicals, power
AGRICULTURE
Enabling Tech Modeling, Synthesis, Evolution,
Sequencing
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Our GTL network
Ingram-UFL
Celunol
Cerrina-UW
Endy
Codon Devices
Collins-BU
Chisholm-MIT
LS9
Polz
DuPont Shell
Church-Harvard Silver Shih
SynBERC-iGEM
BioEnergy International
Richard-PSU
Agrivida
Ausubel-MGH
Mendel
Hamilton-INL
Liu-BNL
Curtis-PSU
Chromatin
Chappell-UKY
GreenFuel
Laible-ANL
Pendse-UME
SunEthanol
Leschine-UMA
Vance-WashU
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Enabling Technologies 1 Computational
Systems Modeling
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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)
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Whole-Genome resequencing of evolved DTrp
Position Type Gene Location Function Mechanism
986,334 T gt G ompF Promoter-10 Promoter of Non-specific transport channel Makes promoter more consensus-like
985,797 T gt G ompF Glu gt Ala Non-specific transport channel Makes pore bigger and more hydrophobic
931,960 D8 bp lrp frameshift General Transcriptional Regulator ?

• 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

Shendure, et al. (2005) Science 3091728
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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 lt1fL Visigen Biotech
Pol FRET lt1fL Pacific Biosci Pol
lt1fL Agilent Nanopores
lt1fL
fL 1E-15 liters (femto)
Our lab has been involved in 8/10
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5 Sequencing genomes from single cells
(single chromosome, cell , RNA or
particle) Zhang, et al. (2006) . Nature Biotech.
June 06
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
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6 10 Mbp of DNA / 1000 chip
7 Recombinational Genome Engineering
Digital Micromirror Array

• 8K Atactic/Xeotron/Invitrogen
• Photo-Generated Acid
• 12K Combimatrix/Codon 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. 4321050 Carr Jacobson
2004 NAR Smith Modrich 1997 PNAS
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8 3D Structural Biology especially membrane
and fibrous structures
Shih, Douglas, iGEM
Hanson Laible
9, 10 Proteomics, Metabolomics
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DOE GTL Vertically Integrated Bioenergy and Novel
Technologies(VIBRANT)
Time Agenda item (77 PIs 75 pages 390 minutes today)
830-845 Introduction and enabling technologies (GC)
845-915 Plan for integrated response to RFA (JA)
915-945 Plants and Agriculture
945-1015 Saccharification CBP
1015-1030 Break
1030-1100 Fermentation
1100-1115 Process modeling and improvement
1115-1130 Alternative biofuels and paradigms
1130-1200 Pretreatment, extraction, use
1200-100 Working lunch IP, other BRCs
100-200 Open Discussion
200-230 Budgets
230-300 Next steps
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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
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Snapshots from the 1st DOE-VIBRANT meeting Boston
21-Nov-2006