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DOE Bioenergy Research Center (BRC) - Joint BioEnergy Institute
Monthly Progress Report – October 2014
Fiscal Year: 2015
Year / Period: Year 8 / Month 1/ October 1, 2014 – October 31, 2014
Department of Energy’s Joint BioEnergy Institute is supported by the US Department of Energy, Office of Science, Office of Biological and Environmental Research, through Contract DE-AC02-05CH11231 between Lawrence Berkeley National Laboratory and the US Department of Energy.
JBEI contacts Jay Keasling, Chief Executive Officer, [email protected], 510-495-2620 Nick Everson, Chief Operating Officer, [email protected], 510-486-6268
Executive Summary This Monthly Progress Report provides a summary of JBEI’s activities for the month of October 2014, first month of fiscal year 2015:
Item Count Highlights
Publications in Print 5 "Enhancing terpene yield from sugars via novel routes to 1-deoxy-d-xylulose 5-phosphate"
"A droplet-to-digital (D2D) microfluidic device for single cell assays"
Publications in Press 4 “Improving microbial bio-gasoline production in Escherichia coli using tolerance engineering”
“Expression of a bacterial 3-dehydroshikimate dehydratase reduces lignin content and improves biomass saccharification efficiency”
Media Citations 19 Two stories released: “Boosting Biogasoline Production in Microbes” and “New Lab Startup Afingen Uses Precision Method to Enhance Plants”
Invention Disclosures 4 “Active Component of Cellulase Mixture from a Thermophilic Bacterial Community is a Multenzyme Complex”
Patent Applications 2 “Switchable Ionic Liquids for Biomass Pretreatment and Enzymatic Hydrolysis”
Presentations Given 9 Presentations were given at the “Frontiers in Biorefining Conference Chemicals and Products from Renewable Carbon”, The University of Tennessee, Center for Renewable Carbon, St. Simons Island, GA
On-Site Seminars 12 Prof. Michael Hahn, University of Georgia, Complex Carbohydrate Research Center, BioEnergy Science Center
Tours & Visits 11 Educational visit from PhD students from CEA Grenoble, France.
Industry Interactions 12 New interactions with Bayer, Monsanto and BASF.
Awards 1 JBEI’s CEO Jay Keasling won the The Economist 2014 Innovation Award in the Bioscience Category
FY15 Milestones 70 On track.
Completed: “Type 1 fatty acid synthases from several organisms expressed in E. coli and production of fatty acid-based fuels tested”
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Publications
In Print
Kirby, J., Nishimoto, M., Chow, R. W., Baidoo, E. E., Wang, G., Martin, J., Schackwitz, W., Chan, R., Fortman, J. L., & Keasling, J. D. (2014). "Enhancing terpene yield from sugars via novel routes to 1-deoxy-d-xylulose 5-phosphate". [Journal article]. Appl Environ Microbiol, 17, 02920-02914. doi, 10.1128/AEM.02920-14
Lund, C. H., Bromley, J. R., Stenbaek, A., Rasmussen, R. E., Scheller, H. V., & Sakuragi, Y. (2014). "A reversible Renilla luciferase protein complementation assay for rapid identification of protein-protein interactions reveals the existence of an interaction network involved in xyloglucan biosynthesis in the plant Golgi apparatus". [Journal article]. J Exp Bot, 18. doi, 10.1093/jxb/eru401
Poust, S., Yoon, I., Adams, P. D., Katz, L., Petzold, C. J., & Keasling, J. D. (2014). "Understanding the Role of Histidine in the GHSxG Acyltransferase Active Site Motif: Evidence for Histidine Stabilization of the Malonyl-Enzyme Intermediate". PLoS One, 9(10), e109421. doi, 10.1371/journal.pone.0109421
Shih, S. C., Gach, P. C., Sustarich, J., Simmons, B. A., Adams, P. D., Singh, S., & Singh, A. K. (2014). "A droplet-to-digital (D2D) microfluidic device for single cell assays". [10.1039/C4LC00794H]. Lab Chip. doi, 10.1039/c4lc00794h
Sitepu, I., Shi, S., Simmons, B. A., Singer, S. W., Boundy-Mills, K., & Simmons, C. W. (2014). "Yeast tolerance to the ionic liquid 1-ethyl-3-methylimidazolium acetate". FEMS Yeast Res, n/a-n/a. doi, 10.1111/1567-1364.12224
In Press
Foo, J. L., Jensen, H. M., Dahl, R. H., George, K., Keasling, J. D., Lee, T. S., Leong, S.S.J.L & Mukhopadyay, A. (2014). “Improving microbial bio-gasoline production in Escherichia coli using tolerance engineering”. mBio
Eudes, A.,Noppadon, S., Baidoo, E., George, A.; Liang, Y.; Yang, F.; Singh, S.; Keasling, J. D.; Simmons, B. A.; Loque, D. (2014). “Expression of a bacterial 3-dehydroshikimate dehydratase reduces lignin content and improves biomass saccharification efficiency”. Plant Biotechnology Journal.
Furtado, A. L., Jason; Hoang, N. V.; Healey, A.; Singh, S.; Simmons, B. A.; Henry, R. (2014). “Modifying plants for biofuel and biomaterial production”. Plant Biotechnology Journal.
Woo, H. L., Ballor, N., Hazen, T.C., Fortney, J. L., Simmons, B. A., Davenport, K. W., Goodwin, L., Ivanova, N., Kyrpides, N., Mavromatis, K., Woyke, T., Jansson, J., Kimbrel, J., and DeAngelis, K. (2014). “Complete genome sequence of the lignin-degrading bacterium Klebsiella sp. strain BRL6-2”. Standards in Genomic Sciences
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News Citations
Date Article Source
Oct 1 Sifting Through Metagenomes With MaxBin Design & Trend
Oct 1 TeselaGen Receives $750k NSF SBIR Phase II Award IT Business Net
Oct 1 TeselaGen Receives $750k NSF SBIR Phase II Award ABC2 WBay
Oct 27 Boosting biogasoline production in microbes Science Codex
Oct 27 Boosting biogasoline production in microbes Press-News.org
Oct 27 Boosting Biogasoline Production in Microbes Science Newsline - Biology
Oct 28 Boosting biogasoline production in microbes Phys.org
Oct 28 JBEI identifies microbial genes to boost biogasoline production Biofuels Digest
Oct 28 Boosting biogasoline production in microbes R&D Magazine
Oct 28 JBEI Researchers Identify Microbial Genes That Can Improve Biogasoline Production in E. coli AZoCleantech.com
Oct 28 Lygos Inc. receives two grants for microbial technology projects Biomass Magazine
Oct 29 New Lab Startup Afingen Uses Precision Method to Enhance Plants EIN Presswire
Oct 30 New Startup Afingen Uses Precision Method To Enhance Plants Technology.org
Oct 30 Afingen uses precision method to enhance plants R&D Magazine
Oct 30 JBEI researchers boost biogasoline production in microbes Biomass Magazine
Oct 30 Lab breakthrough can lead to cheaper biofuels, improved crops, and new products from plants Phys.org
Oct 31 Research Finds Key to Cheaper Biofuels, Improved Crops Laboratory Equipment
Oct 31 Engineering Boosts Biogas Production in Microbes Laboratory Equipment
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News Citations
Date Article Source
Oct 31 Lab Breakthrough Paves Way for Improved Crops, Cheaper Biofuels China Topix
Inventions Disclosed Inventor(s)
2015-006 “Thermotoloerant mixture of a Glucoside hydrolase family 1 beta-glucosidase and a glucoside hydrolase family 16 laminarinase for algal biomass deconstruction”
John Cunha, Richard Heins, Kenneth L. Sale, Chessa Scullin, Blake A. Simmons and Seema Singh
2015-007 “Jungle Express – inducible Eil-promoters for gene expression” Thomas L Ruegg
2015-010 “Active Component of Cellulase Mixture from a Thermophilic Bacterial Community is a Multenzyme Complex”
Evelyn Denzel, John Gladden, Jennifer Hiras and Steven W Singer.
2015-012 “The Unexpected Importance of Actinobacterial GH12 in Hydrolysis of Crystalline Cellulose”
Jennifer Hiras, Steven W Singer and Yu-Wei Wu.
Patent Applications Inventor(s)
2013-128-01US62/068,595 “Method and device for manipulation of a droplet” Anup Singh, Jess Sustarich, and Chao Chung Shih
2014-064-01US62/073,802 “Switchable Ionic Liquids for Biomass Pretreatment and Enzymatic Hydrolysis”
Blake Simmons, Kenneth L. Sake, and Michael J. Liszka
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Presentations
Date Division Presenter(s) Title Presentation Location
Oct 2 Fuels Synthesis
Keasling, J. D. Synthetic Biology for Synthetic Chemistry
University of Virginia, Charlottesville, VA
Oct 5-8 Feedstocks Mann, G.W., Joshi, H. J., Petzold, C. J., Heazlewood, J.L.
Mining post translational modifications in Arabidopsis using the ModHunter
Human Proteome Organization World Congress, Madrid, Spain
Oct 8 Deconstruction Thelen, M. P. Microbial-Enabled Bioenergy Sonoma State University Biology Colloquium, Sonoma, CA
Oct 21 Fuels Synthesis
Hillson, N. J. Synthetic Biology Informatics: Scalability and Responsible Innovation
Novo Nordisk Center for Biosustainability, Copenhagen, Denmark
Oct 21-24 Deconstruction Singh, S. Development and Demonstration of an Integrated Biomass Sugar Production Platform based on Ionic Liquids
Frontiers in Biorefining Conference Chemicals and Products from Renewable Carbon, The University of Tennessee, Center for Renewable Carbon, St. Simons Island, GA
Oct 21-24 Deconstruction Parthasarathi, R., Simmons, B. A., and Singh, S.
Biomass Pretreatment Using Designer Ionic Liquids
Frontiers in Biorefining Conference Chemicals and Products from Renewable Carbon, The University of Tennessee, Center for Renewable Carbon, St. Simons Island, GA
Oct 23 Fuels Synthesis
Hillson, N. J. Synthetic Biology Informatics: Scalability and Responsible
University of Edinburgh, Edinburgh, Scotland
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Innovation
Oct 27-29 Feedstocks Heazlewood, J. L. Beyond the Golgi proteome. ARC Centre of Excellence in Plant Cell Walls
Annual Retreat. Yarra Valley, Victoria, Australia
Oct 29 Fuels Synthesis
Hillson, N. J. Berkeley Open BioFoundry (BOB) Update
LBNL BioSciences Retreat, Berkeley, CA
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Onsite Seminars
Date Type Presenter Title Affiliation
Oct 1 JBEI Seminar Series Nathan Hillson Berkeley Open Biofoundry (BOB): Overall Vision and a Day in the Life
Director, Synthetic Biology, Fuels Synthesis Division
Oct 1 Group Seminar Kevin George Characterizing IPP toxicity in E. coli DH1
Fuels Synthesis Division
Oct 8 General Seminar Deconstruction Guest
Prof. Michael Hahn Analysis of Biomass Structure and Processing using Antibody Probes
Professor of Plant Biology, Adjunct Professor of Biochemistry and Molecular Biology, and Plant Pathology; University of Georgia, Complex Carbohydrate Research Center, BioEnergy Science Center
Oct 8 Group Seminar Clara Eng Engineering the β-Ketoreductase Domain of Polyketide Synthases
Fuels Synthesis Division
Oct 15 General Seminar Todd Pray, Program Head Process Development and Scale-up for Feedstock Flexibility and Bio-based Product Diversification
Advanced Biofuels (and Bioproducts) Process Demonstration Unit (ABPDU)
Oct 15 Group Seminar Gabriela Guzman Model-driven discovery of underground metabolic functions in Escherichia coli
Fuels Synthesis Division
Oct 15 Group Seminar Elizabeth Brunk Application of Genome-Scale Metabolic Models in Metabolic Engineering
Fuels Synthesis Division
Oct 22 General Seminar Deconstruction Guest
Dr. Huaming Wang Fungal system biology towards fungus enzymes for food and feed
Principal Investigator; Tianjing Institute of Industrial Biotechnology, Chinese Academy of Science;
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applications Chief Scientist, Qingdao Vland Biotech Group, P.R. China
Oct 22 Group Seminar Jeff Wong Identifying novel genes involved in phorbol biosynthesis in Jatropha curcas
Fuels Synthesis Division
Oct 22 Group Seminar Nicolas Koutsoubelis Precursor pathway engineering in Saccharomyces cerevisiae for PKS expression
Fuels Synthesis Division
Oct 29 Group Seminar Kamrun Zargar Discovery of genes associated with bacterial toluene biosynthesis
Fuels Synthesis Division
Oct 29 Group Seminar Margaret Brown Engineering chemical warfare agent catabolism into E. coli
Fuels Synthesis Division
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Tours and VIP Visits at JBEI
Date Company Attendees
Oct 1 CTO of COFCO Dr. Ping Chen
Oct 3 R&D Planning Dept. Ajinomoto Co., Inc. (Tokyo) Kazuya Onomichi, Nobuhisa Shimba
Oct 6 Calif. Inst. of Technology Professor Harry Atwater
Oct 6 New Fuel and Vehicle Systems Optima (NFVSO) Andrew Sutton, Art Pontau, Brad Ziegler, Jennifer Dunn, John Farrell, John Holladay, Matt McNenly, Paul Miles, Richard Boardman, Robert Wagner, Sam Saxena and Tom Foust
Oct 8 Jülich Research Center Wolfgang Marquardt, Svenja Kruse, Thorsten Voss, Rainer Waser
Oct 10 Die Zeit (German Newspaper) Christiane Grefe
Oct 13 University of Queensland Anton Middelberg
Oct 15 Idaho National Laboratory Visit Vicki Thompson & Darryl Reed
Oct 16 Riffyn Tim Gardner
Oct 22 DOE Chicago real estate team Rick Chapman, Kevin Hartnett, Laura Troche, Roger Doole, Susan Bourgart
Oct 29 CEA Grenoble 15 PhD Students
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Industry Interactions
Date Company / Contact
Oct 1 COFCO / Dr. Ping Chen
Oct 1 Tehnology Holding Co. / Jessica Elwell and Mukund Karanjikar
Area of interest: Methyl Butanols SBIR/STTR Request for Information
Oct /3 Ajinomoto / Dr. Kazuya Onomichi
Oct 14 Reliance Industries Limited / Makarand Phadke and Vidhya Rangaswamy
Oct 14 Total / Angelique Chanal and Shital Tripathi
Oct 15 COFCO / Bo Chen (LBL)
Oct 16 Riffyn / Tim Gardner and Claude Calegari
Oct 17 Monsanto / Ginni Ursin
Area of interest: EIB-3144, golgi nucleotide sugar transporters, Engineering tolerance to drought/submergence by over expression of a grass specific protein: 2014-108
Oct 17 Bayer / Garth Hodges
Area of interest: Improved Crops with Increased Galactan Content: EIB-3144, Engineering Competitive Pathways for Tissue-Specific Lignin Reduction: EIB-3266
Oct 17 BASF / Burkhard Ernst and Lahoucine Achnine
Area of interest: Improved Crops with Increased Galactan Content: EIB-3144, Engineering Competitive Pathways for Tissue-Specific Lignin Reduction: EIB-3267
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Awards
Name Award
Jay Keasling The Economist 2014 Innovation Awards – Bioscience Category
Oct 21 Virdia / Robert Jansen
Oct 22 Agilent / Steve Royce
Area of interest: Proposed and onsite meeting at JBEI
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Milestones
Feedstocks Division
Area Initiatives Milestone/Deliverables Due % Complete
Plant Cell Wall Biosynthesis and Its Regulation
Novel acetyl and feruloyltransferase activities characterized (CWB) Sept. 2015 10
Genetic characterization of mutant candidates from rice stem saccharification screen (GG)
Sept. 2015 8
Correlation between cell wall modification and environmental interactions investigated (GG)
Sept. 2015 8
Characterization of novel nucleotide sugar transporters (SB) Sept. 2015 5
Characterization of novel GT activities (SB) Sept. 2015 5
Lignin and Hemicellulose Modification for Fuels Production
Evaluation of engineered plants with new lignin traits (including analysis with the Deconstruction and Technology Divisions) (CWE)
Sept. 2015 10
Transgenic plants with dominant repressors of hemicellulose biosynthesis characterized (CWB)
Sept. 2015 9
Nucleotide sugar transporters used for cell wall engineering (CWB) Sept. 2015 9
Optimization of translation control technologies to improve tissue-specific expression (CWE)
Sept. 2015 15
Analysis of stacked traits in Arabidopsis (CWE) Sept. 2015 0
Heterologous screening of transferases to promote in planta design of polysaccharides (SB).
Sept. 2015 0
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Milestones
Feedstocks Division (continued)
Area Initiatives Milestone/Deliverables Due % Complete
Engineering of Modifications in Different Plant Species
Cell wall related genes in switchgrass functionally annotated, integrated into phylogenomics databases, and integrated with Kbase (GG)
Sept. 2015 8
Resequencing of Kitaake mutants (with JGI) and a database set up for hi throughput in silico screening (GG).
Sept. 2015 8
Translation of best technologies developed in Arabidopsis to other plant species (CWE)
Sept. 2015 9
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Milestones
Deconstruction Division
Area Initiatives Milestone/Deliverables Due % Complete
Biomass Pretreatment
Predict and develop task specific ILs for selective lignin or cellulose dissolution and low temperature pretreatment
May 2015 10
Complete characterization of lignin streams from various IL pretreatment process configurations
July 2015 8
Design, synthesis and assessment of lignin and hemicellulose derived IL mixtures for mixed and engineered feedstock pretreatment
Aug. 2015 7
Use multi-scale simulations strategies (from first principle quantum mechanics to force field based) to understand interactions between biomass derived ILs and lignocellulosic biomass
Sept. 2015 7
Enzyme Optimization
Develop an assay for screening lignin degrading enzymes on insoluble lignin and begin populating a lignin degrading enzyme database (LigDB).
Feb. 2015 15
Expand our database of ionic liquid tolerant enzymes, particularly to include enzymes from the GH3, GH5, GH6, GH9, GH10, AA9 and AA10 families.
May 2015 10
Improve the overall sugar yields of our existing thermo and ionic liquid tolerant multi-component enzyme mixture by including enzymes for hydrolysis of both cellulose and hemicellulose and by including LPMOs.
July 2015 7
Demonstrate using directed evolution to engineer enhanced stability and activit -glucosidase in up to 20% [C2mim][OAc].
Sept. 2015 7
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Milestones
Deconstruction Division (continued)
Area Initiatives Milestone/Deliverables Due % Complete
Microbial Communities
Complete characterization of active component of JTherm March 2015 15
Establish complementary thermophilic cellulase cocktail focusing on crystalline substrates
July 2015 10
Identify five bacterial strains capable of metabolizing mixtures of aromatics from pretreatment-derived lignin streams
Sept. 2015 7
Fungal Biotechnology
Design new expression systems based on recent “omics” discoveries in regard to promoters and assess heterologous GH expression
Feb. 2015 13
Initiate ChIP-Seq analysis of histone modifications in A. niger aimed at identifying epigenetic determinants of high productivity
April 2015 10
Optimize cultivation conditions for heterologous enzyme production, including temperature and pH, and initiate ‘omic analysis of optimal vs. baseline conditions
July 2015 10
Validate candidate genes for hyperproduction phenotypes through gene deletion and/or overexpression
Sept. 2015 7
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Milestones
Fuels Synthesis Division
Area Initiatives Milestone/Deliverables Due % Complete
Discovery of Novel Hydrocarbon Biochemistries
In vitro, anaerobic testing of toluene biosynthesis candidates Sept. 2015 8
In vitro, anaerobic testing of ladderane biosynthesis candidates Sept. 2015 8
Optimization of Hydrocarbon Biosynthetic Pathways
Improve methyl ketone production in E. coli with NIMS-aided HT screening and metabolic modeling
Sept. 2015 8
E. coli pathway engineering for isopentenol production both in aerobic and anaerobic conditions
Sept. 2015 8
Genes encoding isoprenoid pathway integrated into E. coli chromosome
Sept. 2015 8
Three enzymes in S. cerevisiae glycolytic pathway replaced and impact on fatty acid-derived fuel tested
Sept. 2015 8
Type 1 fatty acid synthases from several organisms expressed in E. coli and production of fatty acid-based fuels tested
March 2015 100
Develop analytical method for LC-MS of MEV and DXP intermediates April 2015 30
Host Engineering S. cerevisiae: Consolidate xylose utilizing production strains with production pathways.
Sept. 2015 0
E. coli membrane editing: With JGI, generate 10-20 single and double mutants and test improvement in expression of beneficial tolerance pump and corresponding tolerance to toxic fuels.
Sept. 2015 30
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Milestones
Fuels Synthesis Division (continued)
Area Initiatives Milestone/Deliverables Due % Complete
Synthetic Biology
Codon optimization tool (GeneDesign) integrated into the DIVA platform.
Sept. 2015 8
Clonal sequence validation (Sanger and/or MiSeq) pipeline implemented and integrated with DIVA/ICE
Sept. 2015 8
Quantitative Metabolic Modeling for Host and Pathway Engineering
Usage of EDD, MvT and 2S-13C MFA to improve production of fatty acids in S. cerevisiae.
Sept. 2015 30
Perform flux analysis of S. cerevisiae sip1 strain June 2015 60
Implement interactive metabolic engineering on MvT Sept. 2015 7
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Milestones
Technology Division
Area Initiatives Milestone/Deliverables Due % Complete
High Throughput Screening
Support the optimization of T and IL tolerant cellulose/hemicellulase cocktail using HT-NIMS
Sept. 2015 12
Apply HT NIMS screen for fatty acids based fuel molecules (e.g. FAMK) in collaboration with the Fuels Division.
Sept. 2015 10
Implement software to automate unit operations (e.g., droplet dispensing, routing, merger, and sorting) on an integrated droplet chip
Sept. 2015 8
Demonstrate microfluidic droplet platform for integration of DNA assembly, transformation of cells, and culturing
July 2015 10
Proteomics Develop rapid untargeted proteomics methods for relative quantification of >800 E. coli and S. cerevisiae proteins
Sept. 2014 10
Develop absolute quantification targeted proteomic assays for 150 S. cerevisiae proteins
Sept. 2015 15
Implement membrane proteomics assays to enable E. coli membrane editing with Fuels Synthesis Division
Sept. 2015 15
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Milestones
Technology Division (continued)
Area Initiatives Milestone/Deliverables Due % Complete
Physical Characterization
SEM and TEM analysis of genetically altered feedstock biomass (including lignin and protein mapping) with Feedstocks and Deconstruction Divisions
Sept. 2015 8
Electron tomographic analysis of plant cell walls of engineered feedstocks, and CAD model generation for mechanical properties simulation.
Sept. 2015 8
Structural Biology Test expression of multiple GT clones in insect cell platform to produce quantities of protein for crystallization trials
Sept. 2015 30
Characterize lignin degrading enzymes in collaboration with GLBRC Sept. 2015 25
Informatics Implemented an updated, refactored version of the EDD to support JBEI’s future data needs
Sept. 2015 20
Integrate EDD with data and workflows for characterizing biological parts, with Fuels Synthesis
Sept. 2015 5
Integrate EDD with data and workflows for characterizing enzymes, with Deconstruction Division
Sept. 2015 5
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Milestones
Operations Management Division
Area Initiatives Milestone/Deliverables Due % Complete
Intellectual Property Management
Generate >10 inventions Sept. 2015 60
Educate researchers about the tech transfer process Sept. 2015 10
Licensing Increase number of patent applications or inventions licensed or optioned and/or licensing agreements executed relative to the average of years 1 - 7.
Sept. 2015 9
Start-up Company Creation
Identify any IP or portfolios of IP that could lead to a start-up company and create an internal commercialization team to develop an initial go-to-market plan around that IP.
Sept. 2015 8
Maintain regular JBEI on-site office hours for the Berkeley Lab entrepreneur adviser.
Sept. 2015 8
Business Development Outreach
Promote each JBEI invention to at least 700 contacts in the biofuels industry.
Sept. 2015 9
Conduct meetings with at least fifteen companies each year, at least one-third of which are new to JBEI
Sept. 2015 10
Industry Advisory Committee
Hold an annual meeting of the IAC to inform JBEI research of industry concerns and technology bottlenecks.
Sept. 2015 5
Industry Partnership Program
Strengthen relationships with current partners and pursue collaborations with new industry partners in areas of complementary research.
Sept. 2015 9
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Milestones
Operations Management Division (continued)
Area Initiatives Milestone/Deliverables Due % Complete
Annual Scientific Advisory Meeting
Hold the annual meeting and report to DOE on the findings of the committee
Sept. 2015 8
Annual JBEI Conference
Annual JBEI Retreat Sept. 2015 11
Human Resources Staffing for year 8 complete Sept. 2015 8