Progress 07/08/04 to 10/31/06
Outputs
Progress Report 1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? Why does it matter? Lignocellulosic biomass, an abundant and renewable carbon source, has the potential to supplement starch as a feedstock for the production of fuel ethanol. The lack of ethanol-producing microorganisms that can fully utilize all of the sugars derived from biomass poses a significant technical barrier to the commercial use of this commodity for biofuel production. Saccharomyces cerevisiae is a strain of yeast that is currently used for industrial production of ethanol, but it does not ferment xylose, the predominant pentose sugar derived from biomass. In order to develop lignocellulosic biomass into an economical feedstock for fuel ethanol production, there is a need for improved yeast strains that efficiently utilize mixtures of hexose and pentose sugars. The discovery of new strains
would be facilitated by development of a high-throughput robotic platform that will automate the screening of potentially useful strains. Application of the robotic platform to isolate improved yeast strains will benefit not only commercial partners but farmers as well, by increasing the markets for agricultural based feedstocks. In addition, expansion of the production of fuel ethanol would reduce the nation's dependence on foreign oil and improve the environment by developing alternate energy sources from renewable resources. This research project falls under CRIS 3620-41000-121-00D (Microbial Catalysts to Produce Fuel Ethanol and Value Added Products), whose broad goal is to develop new microorganisms and biocatalysts that can be employed in the fermentative conversion of renewable agricultural materials to fuels and other value-added products. This research is expected to increase the efficiency of conversion of biomass to liquid fuel, and to discover new uses for agricultural
by-products. Therefore, portions of the research fall under National Program 307 (Bioenergy and Energy Alternatives) and under National Program 306 (Quality and Utilization of Agricultural Products). Specifically, the research contributes to Component I (Ethanol) of the NP 307 Action Plan and to Problem Area 2b (New Uses for Agricultural By-products) of the NP 306 Action Plan. 2. List by year the currently approved milestones (indicators of research progress) Year 1 1. Identify liquid handling, colony picking, reading, etc. apparatus to perform individual molecular biology tasks. 2. Develop strategies to perform plasmid isolation and purifications, and other molecular biology techniques in a 96-well format amenable to robotic automation. 3. Construct an integrated system to perform multiple molecular biology manipulations and routines in an automated paradigm. Year 2 1. Develop methods to evaluate the integration and train the workcell to conduct basic molecular biology
routines. 2. Provide genetic constructs to be tested in the optimization of the integrated process. 4a List the single most significant research accomplishment during FY 2006. Construction of a Proteomic Workcell. The Ethanol component of the NP307 Action Plan seeks to reduce the cost of producing ethanol from biomass through technological advances in enzymology and microbiology, and in chemical, biochemical, and process engineering. An automated method to screen large numbers of samples is needed to help identify new microbial strains and enzymes which can be utilized for efficient production of ethanol and other value-added products from agricultural feedstocks. In cooperation with Hudson Control Group, Inc., Springfield NJ, scientists in the Bioproducts and Biocatalysis Research Unit at the USDA-ARS-National Center for Agricultural Utilization Research, Peoria, Illinois have constructed a plasmid-based functional proteomic workcell for high-throughput assembly, optimization, and
modification of gene libraries and microbial strains. Mechanical hardware has been integrated and controlling software developed to provide a robotic platform that picks colonies, cultures bacteria, prepares plasmid DNA, performs in vitro transcription/translation, and assays enzyme activity. This workcell and high-throughput strategy will ultimately be used for identifying strains of ethanologenic yeast capable of fermenting xylose. 5. Describe the major accomplishments to date and their predicted or actual impact. A plasmid-based proteomic robotic workcell that integrates the colony picking, plasmid preparation, protein expression, and activity assay functions has been constructed to automate high-throughput screening of microorganisms. This represents a new technology in the field of laboratory automation. Application of this workcell will be a powerful tool to obtain metabolically balanced robust strains for improved industrial anaerobic ethanol production and to identify the
important genes in the metabolic pathway. 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? Technology is being transferred to Hudson Control Group, Inc. under SCA #58-3620-4-146. The collaboration has resulted in construction of the first workcell of this type. Two provisional patents covering the workcell and a third covering its associated biology have been written and will be placed in conjunction with the completion of the workcell. The third provisional patent is being updated to include the new amino acid scanning technologies for full genome, rapid mutational structure/function analysis and will be licensed to various robot and molecular biology companies. Presently one other complete integrated robotic platform has been engineered and sold
to the pharmaceutical industry. We are anticipating the licensing of the mutational strategy to molecular biology companies within the year. 7. List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below). Presented the advanced molecular biology automation necessary to conduct plasmid-based functional proteomics for optimization of enzyme production for use in biomass to ethanol conversion to DOE, National Renewable Energy Laboratory. Jan 16th 2006 in Golden CO. Presented the possibility of using the robotic workcell to produce large numbers of variant open reading frames for hemicellulases and cellulases as well as other catalysts important for biomass to ethanol production at DOE, Argonne National Laboratory January 25th 2006
Impacts
(N/A)
Publications
- Hughes, S.R., Mertens, J.A., Li, X., Bischoff, K.M. 2005. Plasmid-based functional proteomic robotic workcell process for high-throughput screening of multiplexed libraries of mutagenized clones [abstract]. Laboratory Robotics Information Group. p. 1.
- Hughes, S.R., Riedmuller, S.B., Mertens, J.A., Jordan, D.B., Li, X., Qureshi, N., Cotta, M.A., Farrelly, P.J., Bischoff, K.M. 2005. Functional proteomic workcell for high volume plasmid preparations for repeated in vitro protein expression and high throughput screening to identify mutant enyzmes for use at low pH [abstract]. Optimization High-throughput Cultures for Bioprocessing 2005. 13:3.
- Hughes, S.R., Riedmuller, S., Mertens, J.A., Li, X., Qureshi, N., Farrelly, P., Cotta, M.A. 2006. Automated strategy using a functional proteomic assay to identify and isolate cellulase F mutants with improved activity from multiplexed sets of plasmid [abstract]. PepTalk 2006. p. 10.
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Progress 10/01/04 to 09/30/05
Outputs
1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter? Lignocellulosic biomass, an abundant and renewable carbon source, has the potential to supplement starch as a feedstock for the production of fuel ethanol. The lack of ethanol-producing microorganisms that can fully utilize all of the sugars derived from biomass poses a significant technical barrier to the commercial use of this commodity for biofuel production. Saccharomyces cerevisiae is a strain of yeast that is currently used for industrial production of ethanol, but it does not ferment xylose, the predominant pentose sugar derived from biomass. In order to develop lignocellulosic biomass into an economical feedstock for fuel ethanol production, there is a need for improved yeast strains that efficiently utilize mixtures of hexose and pentose sugars. The discovery of new strains would be
facilitated by development of a high-throughput robotic platform that will automate the screening of potentially useful strains. Application of the robotic platform to isolate improved yeast strains will benefit not only commercial partners but farmers as well, by increasing the markets for agricultural based feedstocks. In addition, expansion of the production of fuel ethanol would reduce the nation's dependence on foreign oil and improve the environment by developing alternate energy sources from renewable resources. This research project falls under CRIS 3620-41000-121-00D (Microbial Catalysts to Produce Fuel Ethanol and Value Added Products), whose broad goal is to develop new microorganisms and biocatalysts that can be employed in the fermentative conversion of renewable agricultural materials to fuels and other value-added products. This research is expected to increase the efficiency of conversion of biomass to liquid fuel and to discover new uses for agricultural
by-products. Therefore, portions of the research fall under National Program 307 (Bioenergy and Energy Alternatives) and under National Program 306 (Quality and Utilization of Agricultural Products). Specifically, the research contributes to Component I (Ethanol) of the NP 307 Action Plan and to Problem Area 2b (New Uses for Agricultural By-products) of the NP 306 Action Plan. 2. List the milestones (indicators of progress) from your Project Plan. 1. Identify liquid handling, colony picking, reading, etc., apparatus to perform individual molecular biology tasks. 2. Develop strategies to perform plasmid isolation and purifications and other molecular biology techniques in a 96-well format amenable to robotic automation. 3. Construct an integrated system to perform multiple molecular biology manipulations and routines in an automated paradigm. 4. Develop methods to evaluate the integration and train the workcell to conduct basic molecular biology routines. 5. Provide genetic
constructs to be tested in the optimization of the integrated process. 3a List the milestones that were scheduled to be addressed in FY 2005. For each milestone, indicate the status: fully met, substantially met, or not met. If not met, why. 1. Identify liquid handling, colony picking, reading, etc., apparatus to perform individual molecular biology tasks. Milestone Fully Met 2. Develop strategies to perform plasmid isolation and purifications and other molecular biology techniques in a 96-well format amenable to robotic automation. Milestone Fully Met 3. Construct an integrated system to perform multiple molecular biology manipulations and routines in an automated paradigm. Milestone Substantially Met 3b List the milestones that you expect to address over the next 3 years (FY 2006, 2007, and 2008). What do you expect to accomplish, year by year, over the next 3 years under each milestone? The termination date for this Specific Cooperative Agreement (SCA) is December 2006. The
following milestones are expected to be addressed during FY 2006: 4. Develop methods to evaluate the integration and train the workcell to conduct basic molecular biology routines. 5. Provide genetic constructs to be tested in the optimization of the integrated process. 4a What was the single most significant accomplishment this past year? CONSTRUCTION OF PROTEOMIC WORKCELL. An automated method to screen large numbers of samples is needed to help identify new microbial strains and enzymes which can be utilized for efficient production of ethanol and other value-added products from agricultural feedstocks. In cooperation with Hudson Control Group, Inc., Springfield, NJ, scientists in the Bioproducts and Biocatalysis Research Unit (BBC) at the USDA-ARS-National Center for Agricultural Utilization Research, Peoria, IL, have designed a plasmid-based functional proteomic workcell for high-throughput assembly, optimization, and modification of gene libraries and microbial strains.
Protocols for plasmid preparation, in vitro protein production, and enzymatic assay were developed for components of the workcell and validated in a 96-well plate format by identifying clones of a cellulase enzyme with optimal activity at low pH from a library of mutagenized genes. This workcell and high-throughput strategy will ultimately be used for identifying strains of ethanologenic yeast capable of fermenting xylose. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. A plasmid-based proteomic robotic workcell that integrates the colony picking, plasmid preparation, protein expression, and activity assay functions has been designed to automate high-throughput screening of microorganisms. This represents a new technology in the field of laboratory automation. Application of this workcell will be a powerful tool to obtain metabolically balanced robust strains for improved industrial anaerobic ethanol production and to
identify the important genes in the metabolic pathway. 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? Technology is being transferred to Hudson Control Group, Inc. under this agreement. The collaboration has resulted in construction of the first workcell of this type. A provisional patent covering the workcell and its associated biology has been written and will be placed in conjunction with the completion of the workcell. 7. List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below). Presented functional proteomic plasmid-based integrated workcell platform concepts and preliminary data to Hudson Control Group, Inc.,
Springfield, NJ (December 2004); Sias, Inc., Wilmington, DE (December 2004); and LifeSensors, Inc., Malvern, PA (February 2005). News article written in Dec 2004 Dunlap, IL, Observer newspaper about S. Hughes new Peoria resident providing USDA, NCAUR Ag lab in Peoria, IL, with new technology.
Impacts
(N/A)
Publications
- Hughes, S.R., Li, X., Cotta, M.A., Mertens, J.A., Bischoff, K.M., Riedmuller, S., Farrelly, P., Patel, M., Brown, L., Carter, L. 2005. Modified liquid handler used to produce high throughput high quality plasmids for integration into plasmid-based functional proteomics workcell [abstract]. CHI's Annual PEP Talk Meeting, January 10-14, 2005, San Diego, California. Poster 151.
- Hughes, S.R., Riedmuller, S.B., Bischoff, K.M., Mertens, J.A., Li, X., Cotta, M.A., Farrelly, P.J. 2005. Development of a liquid handler component for a functional plasmid-based proteomic workcell that generates multiplex samples expresses in yeast [abstract]. Association for Laboratory Automation, LabAutomation 2005, January 30 - February 5, 2005, San Jose, California. Poster WP128.
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