doe gtl vertically integrated bioenergy research center (special thanks to harvard inst. for...
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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)
8:30-8:45 Introduction and enabling technologies (GC)
8:45-9:15 Plan for integrated response to RFA (JA)
9:15-9:45 Plants and Agriculture
9:45-10:15 Saccharification + CBP
10:15-10:30 Break
10:30-11:00 Fermentation
11:00-11:15 Process modeling and improvement
11:15-11:30 Alternative biofuels and paradigms
11:30-12:00 Pretreatment, extraction, use
12:00-1:00 Working lunch: IP, other BRCs
1:00-2:00 Open Discussion
2:00-2:30 Budgets
2:30-3:00 Next steps
Brazil’s Bioethanol(& GtL Economic modeling needs)
Land use:45,000 km²Sugarcane: 344 million tons (76 tons/ha)Sugar: 23 million tonsEthanol:14 million m³ $0.26/L (feedstock 70%)
yield increase 3.5%/yrDry bagasse: 50 million tonsElectricity: 1350 MWBagasse ash 2.5% (vs 40% for coal), nearly no sulfur. Burns at low temperatures, so low nitrogen oxides.
Saccharum officinarum
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
Plants Zea,Miscanthus,Poplar, Crambe, Algae
Pretreat acid, NH4 fiber explosion (AFEX), silage
Saccharify Trichoderma, Clostridia, AspergillusSoluble, cellulosome, consolidated
Ferment Saccharomyces, Escherichia, Zymomonas, Pichia, Klebsiella, communitiesExtract distill, gas strip, zeolite, phase
Process crack, blend ethanol, butanol, ethylesters, alkanes, H2 Uses transportation, chemicals, power
Vertical Integration (horizontal options)
AGRICULTURE
Enabling Tech: Modeling, Synthesis, Evolution, Sequencing
Our GTL network
Church-Harvard Silver Shih
Curtis-PSU
Chisholm-MIT
Leschine-UMA
Codon DevicesCollins-BU
LS9
Agrivida
Hamilton-INL
Ausubel-MGH
Liu-BNL
SynBERC-iGEM
Ingram-UFL
Chappell-UKY
Laible-ANL
Richard-PSU
Mendel
GreenFuel
Celunol
Chromatin
SunEthanol
DuPontShell
Vance-WashU
BioEnergy International
Endy
Polz
Pendse-UME
Cerrina-UW
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)
Position
TypeGen
eLocation Function Mechanism
986,334T >
Gomp
FPromoter-
10
Promoter of Non-specific transport channel
Makes promoter more consensus-like
985,797 T >
Gomp
FGlu > Ala Non-specific
transport channel
Makes pore bigger and more hydrophobic
931,9608
bp
lrp frameshiftGeneral
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
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 fLCGI Ligase 1 fLAffymetrix Hybr array 100 fLIBS Pol beads 10,000 fLSingle molecules Helicos Biosci Pol <1fLVisigen Biotech Pol FRET <1fLPacific Biosci Pol <1fLAgilent Nanopores <1fL
fL =1E-15 liters(femto)
Our lab has been involved in 8/10
#5: Sequencing genomes from single cells
1) Environmental samples (poor or no lab growth)2) Candidate chromosome region sequencing3) Prioritizing or pooling (rare) species based on an initial DNA screen (metagenomics)4) Multiple chromosomes in a cell or virus5) RNA 5’, 3’ ends & splicing6) 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
#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
#8: 3D Structural Biology – especially membrane and fibrous structures
Hanson & Laible Shih, Douglas, iGEM
#9, 10: Proteomics, Metabolomics
DOE GTL Vertically Integrated Bioenergy and Novel Technologies(VIBRANT)
Time Agenda item (77 PIs 75 pages 390 minutes today)
8:30-8:45 Introduction and enabling technologies (GC)
8:45-9:15 Plan for integrated response to RFA (JA)
9:15-9:45 Plants and Agriculture
9:45-10:15 Saccharification + CBP
10:15-10:30 Break
10:30-11:00 Fermentation
11:00-11:15 Process modeling and improvement
11:15-11:30 Alternative biofuels and paradigms
11:30-12:00 Pretreatment, extraction, use
12:00-1:00 Working lunch: IP, other BRCs
1:00-2:00 Open Discussion
2:00-2:30 Budgets
2:30-3:00 Next steps
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 Companies4. San Diego-Iowa-JCVI5. Purdue-MIT26. UWI-MSU7. 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