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DOE Bioenergy Research Center (BRC) - Joint BioEnergy Institute
Monthly Progress Report – May 2015
Fiscal Year: 2015
Year / Period: Year 8 / Month 8 / May 1 - 31, 2015
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, jdkeasling@lbl.gov, 510-495-2620 Nick Everson, Chief Operating Officer, njeverson@lbl.gov, 510-486-6268
Executive Summary This Monthly Progress Report provides a summary of JBEI’s activities for the month of May 2015, eight month of fiscal year 2015:
Item Count Highlights
Publications in Print 9 "Acute limonene toxicity in Escherichia coli is caused by limonene-hydroperoxide and alleviated by a point mutation in alkyl hydroperoxidase (AhpC)"
Invention Disclosures 2 “PKS producing adipic acid”
Patent Applications 2 “Engineered Microorganisms Having Resistance to Ionic Liquids”
Media Citations 1 Women Getting Science Ph.D.s Still Face Gender Barriers, KQED
Outreach Interactions 29 Education: 10, Public: 1 and Scientific Community: 18.
On-Site Seminars 10 Professor Greg Challis from the Department of Chemistry, University of Warwick gave a seminar on “Discovery and Biosynthesis of Novel Polyketide Antibiotics”.
Industry Interactions 22 Interactions with Cummins and Genomatica.
FY15 Milestones Milestones completed: - Deconstruction: “Predict and develop task specific ILs for selective lignin or cellulose dissolution and low
temperature pretreatment”
- Deconstruction: “Expand our database of ionic liquid tolerant enzymes, particularly to include enzymes from the GH3, GH5, GH6, GH9, GH10, AA9 and AA10 families”
Milestones delayed: - Deconstruction: “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”
- Fuels Synthesis: “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”
- Technology: “Structural characterization of Fatty Acid Synthase and other large complexes by single particle cryo electron microscopy”
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Publications
In Print
Chubukov, V., Mingardon, F., Schackwitz, W., Baidoo, E. E., Alonso-Gutierrez, J., Hu, Q., Lee, T. S., Keasling, J. D., & Mukhopadhyay, A. (2015). "Acute limonene toxicity in Escherichia coli is caused by limonene-hydroperoxide and alleviated by a point mutation in alkyl hydroperoxidase (AhpC)". Appl Environ Microbiol. doi, 10.1128/aem.01102-15 http://aem.asm.org/content/early/2015/04/27/AEM.01102-15.full.pdf
George, K. W., Thompson, M. G., Kang, A., Baidoo, E., Wang, G., Chan, L. J., Adams, P. D., Petzold, C. J., Keasling, J. D., & Soon Lee, T. (2015). "Metabolic engineering for the high-yield production of isoprenoid-based C alcohols in E. coli". Sci Rep, 5, 11128. doi, 10.1038/srep11128 http://www.ncbi.nlm.nih.gov/pubmed/26052683
Loque, D., Scheller, H. V., & Pauly, M. (2015). "Engineering of plant cell walls for enhanced biofuel production". Curr Opin Plant Biol, 25, 151-161. doi, 10.1016/j.pbi.2015.05.018
Mingardon, F., Clement, C., Hirano, K., Nhan, M., Luning, E. G., Chanal, A., & Mukhopadhyay, A. (2015). "Improving olefin tolerance and production in E. coli using native and evolved AcrB". Biotechnol Bioeng, 112(5), 879-888. doi, 10.1002/bit.25511 http://www.ncbi.nlm.nih.gov/pubmed/25450012
Mortimer, J. C., Faria-Blanc, N., Yu, X., Tryfona, T., Sorieul, M., Ng, Y. Z., Zhang, Z., Stott, K., Anders, N., & Dupree, P. (2015). "An unusual xylan in Arabidopsis primary cell walls is synthesised by GUX3, IRX9L, IRX10L and IRX14". Plant J. doi, 10.1111/tpj.12898 http://onlinelibrary.wiley.com/store/10.1111/tpj.12898/asset/tpj12898.pdf?v=1&t=iapho8v0&s=ba7c1d428ade53affab67c35b6e2ed6dfdec0194
Mukhopadhyay, A. (2015). "Tolerance engineering in bacteria for the production of advanced biofuels and chemicals". Trends Microbiol. doi, 10.1016/j.tim.2015.04.008
Parsons, H. T., & Heazlewood, J. L. (2015). "Beyond the Western front: targeted proteomics and organelle abundance profiling". Front Plant Sci, 6, 301. doi, 10.3389/fpls.2015.00301 http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4419601/pdf/fpls-06-00301.pdf
Scullin, C., Stavila, V., Skarstad, A., Keasling, J. D., Simmons, B. A., & Singh, S. (2015). "Optimization of renewable pinene production from the conversion of macroalgae Saccharina latissima". Bioresour Technol, 184, 415-420. doi, 10.1016/j.biortech.2014.09.105 http://www.ncbi.nlm.nih.gov/pubmed/2531890
Velasquez, S. M., Ricardi, M. M., Poulsen, C. P., Oikawa, A., Dilokpimol, A., Halim, A., Mangano, S., Denita Juarez, S. P., Marzol, E., Salgado Salter, J. D., Dorosz, J. G., Borassi, C., Moller, S. R., Buono, R., Ohsawa, Y., Matsuoka, K., Otegui, M. S., Scheller, H. V., Geshi, N., Petersen, B. L., Iusem, N. D., & Estevez, J. M. (2015). "Complex regulation of prolyl-4-hydroxylases impacts root hair expansion". Mol Plant, 8(5), 734-746. doi, 10.1016/j.molp.2014.11.017 http://www.ncbi.nlm.nih.gov/pubmed/25655826
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Media Citations
Date Article Source
May 18 Women Getting Science Ph.D.s Still Face Gender Barriers KQED
Outreach – Education
Date Presenters Audience or Presentation Title Activity Location
May 6 Peter Matlock and Blake Simmons
University of Hawaii, Hilo Meeting Off-site
May 7 Sarah Richardson Rock Canyon High School - Biotech Club Meeting Off-site
May 8 Ee-Been Goh and Belle Smith
Bioscience Career Institute Tour On-site
May 8 Peter Matlock Lisa Margonelli, writer for The Atlantic Meeting On-site
May 14 Jeff Kimbrel, Aram Kang, Tristan de Rond and Irina Silva
Wageningen University Tour On-site
Invention Disclosures Inventor(s)
2015-106 - “PKS producing adipic acid” Andrew R. Hagen, Leonard Katz, Jay Keasling and Sean Poust
2015-114 - “Droplet-based single molecule detection” Anup Singh
Patent Applications Inventor(s)
2013-120-02, US 14/717,962 “Engineered Microorganisms Having Resistance to Ionic Liquids” Thomas L. Ruegg, Michael Thelen and Douglas Higgins
2013-188-02, PCT/US2015/031444 “Compositions and methods for single-molecule construction of DNA”
Jay Keasling, Lane Weaver, William Holtz and Daniel Arlow.
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May 15 Sarah Richardson Tennyson High School Seminar Off-site
May 18 Irina Silva iCLEM Alumni Meeting Off-site
May 21 Irina Silva, Dominique Loque, Patrick Shih and Virginia Tartaglio
Agroecology Program at Florida International University
Tour On-site
May 26 Sarah Richardson Oakland Unified School District Dinner with a Scientist
Event Off-site
May 28 Sarah Richardson Castro Valley High School - Biotechnology Course Project
Interview Off-site
Outreach – Public
Date Presenters Audience or Presentation Title Activity Location
May 11 Diane Pierotti and Irina Silva Lawrence Hall of Science Meeting Off-site
Outreach – Scientific Community
Date Presenters Audience or Presentation Title Activity Location
May 4 - 6 Steven Singer The Promise and Perils of Automating Metagenomic Binning
Presentation Genome Standards Consortium-17, Joint Genome Institute, Walnut Creek, CA
May 6 Taek Soon Lee Microbial production of advanced biofuels Presentation National University of Singapore, Biochemistry Department Seminar, Singapore
May 6 Dominique Loque Plant Engineering to Synthetic Biology and Back
Presentation UC Berkeley, CA
May 8 Jay Keasling Engineering Microbes to Solve Global Challenges
Presentation Abbott Lecture, Rensselaer Polytechnic Institute, Troy, New York
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May 11 Taek Soon Lee Microbial production of advanced biofuels Presentation Kunkuk University, Bioscience and Biotechnology Department Seminar, Seoul, South Korea
May 12 Dominique Loque Plant Synthetic Biology and Engineering Presentation UC Berkeley, CA
May 13 Sacha Escamez Umeå Plant Science Centre Tour Umeå Plant Science Centre
May 14 Taek Soon Lee Microbial production of advanced biofuels Presentation Seoul National University, Chemistry Department Seminar, Seoul, Korea
May 18 Hector Garcia Martin Quantitative metabolic modeling for biofuel synthesis
Presentation Cell factories and sustainability, Hillerod, Denmark
May 18 Blake Simmons, Jenny Mortimer, Seema Singh, Chris Petzold
Exxon Tour Exxon
May 19 Irina Silva, Nathan Hillson LBL Govt Affairs Tour LBL Govt Affairs
May 20 - 22
Blake Simmons Advanced Biofuel Development at JBEI Presentation GLBRC Annual Retreat, South Bend, IN
May 20 Erika Yoshida Ajinomoto Co., Inc. Tour Ajinomoto Co., Inc.
May 20 Steve Singer PNNL Tour PNNL
May 22 Ryan Phelan, Susan Gardner
UC Berkeley Tour UC Berkeley
May 22 Nathan Hillson, Henrik Scheller, Dominique Loque, Aindrila Mukhopadhyay, Chris Petzold
House Energy and Water Development Appropriations Subcommittee
Tour House Energy and Water Development Appropriations Subcommittee
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May 30 - June 2
Ee-Been Goh Substantial Methyl Ketone Production in E. coli using Rational Metabolic Engineering and Random Mutagenesis Approaches
Presentation 115th ASM General Meeting, New Orleans, LA
May 30 - June 2
Blake Simmons Comparative 'Omics for Understanding and Harnessing Lignocellulose Deconstruction in Microbial Communities
Presentation 115th ASM General Meeting, New Orleans, LA
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Onsite Seminars
Date Type Presenter Title Affiliation
May 5 General Seminar Deconstruction Guest
Junko Yaegashi Application of Genome-wide Strategies for the Mining of Secondary Metabolite Biosynthetic Pathways in Filamentous Fungi
Decon Interview Candidate
May 6 JBEI Seminar/Training
Sarah Richardson Cas9 Working Group Fuels Synthesis Division
May 6 Group Seminar Rachel Li Identification of enzymes involved in the biosynthesis of the diterpenoid Taxol
Fuels Synthesis Division
May 11 General Seminar SynBerc Guest
Professor Greg Challis Discovery and Biosynthesis of Novel Polyketide Antibiotics
Department of Chemistry, University of Warwick
May 13 Group Seminar Sarah Rodriguez Biotransformation of methane into n-butanol by a methanotrophic methane monooxygenase
Fuels Synthesis Division
May 13 Group Seminar Edward Baidoo Developing CE-MS methods for metabolite analyses.
Fuels Synthesis Division
May 13 General Seminar SynBerc Guest
Jessica Walter, Ph.D Efficient Multiplexed Integration of Synergistic Alleles and Metabolic Pathways in Yeasts via CRISPR-Cas
Amyris
May 14 General Seminar
Deconstruction Guest
Amanda Kohler Pathogenesis at an Atomic Scale Decon Interview Candidate
May 18 Group Seminar Ee-Been Goh Recent advancements in the production of methyl ketones in E. coli
Fuels Synthesis Division
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May 18 Group Seminar Maren Wehrs Developing a CRISPR/Cas platform for metabolic engineering of Saccharomyces cerevisiae
Fuels Synthesis Division
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Industry Interactions
Date Company / Contact
May 1 Afingen / Ai Oikawa
May 7 California Technology Council / Matt Gardner
May 7 Evolva
May 7 Arzeda / Alexandre Zanghellini
May 7 ZymoChem / Harshal Chaokhawala
May 7 Illium / Aaron Socha
May 12 MMI / Pat Smith
May 14 Monsanto / John Hamer
May 14 Monsanto / Ginni Ursin
May 14 Renault SA / Federic Maire
May 14 Mass Insight / Tom Gilman
May 14 PolyOne / Chris Murphy
May 18 Arbiom / John Evans
May 18 PolyOne / Roger Avakian
May 19 Ajinomoto / Dr. Yoshio Kawahara
May 26 Total / Florence Mingardon
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May 26 Virdia / Manja Sachet (Wilson Sonsini)
May 27 Cummins, Inc. / John Wall
May 28 Total / Vincent Schachter
May 28 Genomatica / Mark Burk
May 28 Dupont / Luis Cascao-Pereira
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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 72
Genetic characterization of mutant candidates from rice stem saccharification screen (GG)
Sept. 2015 66
Correlation between cell wall modification and environmental interactions investigated (GG)
Sept. 2015 66
Characterization of novel nucleotide sugar transporters (SB) Sept. 2015 70
Characterization of novel GT activities (SB) Sept. 2015 75
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 78
Transgenic plants with dominant repressors of hemicellulose biosynthesis characterized (CWB)
Sept. 2015 65
Nucleotide sugar transporters used for cell wall engineering (CWB) Sept. 2015 71
Optimization of translation control technologies to improve tissue-specific expression (CWE)
Sept. 2015 65
Analysis of stacked traits in Arabidopsis (CWE) Sept. 2015 68
Heterologous screening of transferases to promote in planta design of polysaccharides (SB).
Sept. 2015 60
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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 66
Resequencing of Kitaake mutants (with JGI) and a database set up for hi throughput in silico screening (GG).
Sept. 2015 68
Translation of best technologies developed in Arabidopsis to other plant species (CWE)
Sept. 2015 68
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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 100
Complete characterization of lignin streams from various IL pretreatment process configurations
July 2015 85
Design, synthesis and assessment of lignin and hemicellulose derived IL mixtures for mixed and engineered feedstock pretreatment
Aug. 2015 75
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 75
Enzyme Optimization
Develop an assay for screening lignin degrading enzymes on insoluble lignin and begin populating a lignin degrading enzyme database (LigDB).
Feb. 2015 100
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 100
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 60
Demonstrate using directed evolution to engineer enhanced stability -glucosidase in up to 20% [C2mim][OAc].
Sept. 2015 72
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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 100
Establish complementary thermophilic cellulase cocktail focusing on crystalline substrates
July 2015 95
Identify five bacterial strains capable of metabolizing mixtures of aromatics from pretreatment-derived lignin streams
Sept. 2015 95
Fungal Biotechnology
Design new expression systems based on recent “omics” discoveries in regard to promoters and assess heterologous GH expression
Feb. 2015 100
Initiate ChIP-Seq analysis of histone modifications in A. niger aimed at identifying epigenetic determinants of high productivity
April 2015 100
Optimize cultivation conditions for heterologous enzyme production, including temperature and pH, and initiate ‘omic analysis of optimal vs. baseline conditions
July 2015 83
Validate candidate genes for hyperproduction phenotypes through gene deletion and/or overexpression
Sept. 2015 70
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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 67
In vitro, anaerobic testing of ladderane biosynthesis candidates Sept. 2015 67
Optimization of Hydrocarbon Biosynthetic Pathways
Improve methyl ketone production in E. coli with NIMS-aided HT screening and metabolic modeling
Sept. 2015 67
E. coli pathway engineering for isopentenol production both in aerobic and anaerobic conditions
Sept. 2015 75
Genes encoding isoprenoid pathway integrated into E. coli chromosome
Sept. 2015 67
Three enzymes in S. cerevisiae glycolytic pathway replaced and impact on fatty acid-derived fuel tested
Sept. 2015 100
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 100
Host Engineering Improve overall xylose utilization capability in S. cerevisiae strains and examine performance in industrially relevant yeast strains.
Sept. 2015 65
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 45
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Milestones
Fuels Synthesis Division (continued)
Area Initiatives Milestone/Deliverables Due % Complete
Synthetic Bio Informatics
Codon optimization tool (GeneDesign) integrated into the DIVA platform.
Sept. 2015 67
Clonal sequence validation (Sanger and/or MiSeq) pipeline implemented and integrated with DIVA/ICE
Sept. 2015 67
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 100
Perform flux analysis of S. cerevisiae sip1 strain June 2015 94
Implement interactive metabolic engineering on MvT Sept. 2015 85
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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 75
Apply HT NIMS screen for fatty acids based fuel molecules (e.g. FAMK) in collaboration with the Fuels Division.
Sept. 2015 75
Implement software to automate unit operations (e.g., droplet dispensing, routing, merger, and sorting) on an integrated droplet chip
Sept. 2015 66
Demonstrate microfluidic droplet platform for integration of DNA assembly, transformation of cells, and culturing
July 2015 80
Proteomics Develop rapid untargeted proteomics methods for relative quantification of >800 E. coli and S. cerevisiae proteins
Sept. 2015 85
Develop absolute quantification targeted proteomic assays for 150 S. cerevisiae proteins
Sept. 2015 85
Implement membrane proteomics assays to enable E. coli membrane editing with Fuels Synthesis Division
Sept. 2015 80
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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 75
Electron tomographic analysis of plant cell walls of engineered feedstocks, and CAD model generation for mechanical properties simulation.
Sept. 2015 75
Structural Biology Test expression of multiple GT clones in insect cell platform to produce quantities of protein for crystallization trials
Sept. 2015 75
Characterize lignin degrading enzymes in collaboration with GLBRC Sept. 2015 100
Structural characterization of Fatty Acid Synthase and other large complexes by single particle cryo electron microscopy
Sept. 2015 50
Synthetic Bio Informatics
EDD further developed to support enzyme characterization data and workflows, with Deconstruction Division
Sept. 2015 67
EDD further developed to support genetic expression tool data and workflows, with Fuels Synthesis
Sept. 2015 67
BioCad tools enabled to query ICE and EDD for enzymes and expression tools satisfying specified performance characteristics
Sept. 2015 67
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Milestones
Operations Management Division
Area Initiatives Milestone/Deliverables Due % Complete
Intellectual Property Management
Generate >10 inventions Sept. 2015 95
Educate researchers about the tech transfer process Sept. 2015 79
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 65
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 73
Maintain regular JBEI on-site office hours for the Berkeley Lab entrepreneur adviser.
Sept. 2015 70
Business Development Outreach
Promote each JBEI invention to at least 700 contacts in the biofuels industry.
Sept. 2015 79
Conduct meetings with at least fifteen companies each year, at least one-third of which are new to JBEI
Sept. 2015 68
Industry Advisory Committee
Hold an annual meeting of the IAC to inform JBEI research of industry concerns and technology bottlenecks.
Sept. 2015 90
Industry Partnership Program
Strengthen relationships with current partners and pursue collaborations with new industry partners in areas of complementary research.
Sept. 2015 79
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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 67
Annual JBEI Conference
Annual JBEI Retreat Sept. 2015 89
Human Resources Staffing for year 8 complete Sept. 2015 67
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