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Engineering Yeast to Drive Cars The Road for the Cellulosome Jeffrey Chen August 21, 2008 University of Texas Lab: Wilfred Chen BRITE 2008 Presentation

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Cars and Gasoline Demand and prices for oil have increased Supply of oil is limited Figures left: (1), right: (2)

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Biofuels Definition: liquid or gaseous fuel that is produced predominately from biomass Advantages: Cleaner, more natural fuel Readily available source, sustainable Alternative security to petroleum Reference: (3)

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Bioethanol Ethanol produced as fuel Wide-accepted use worldwide currently; popular fuel Biomass obtained from any plant source with cellulosic material (originally sugar cane / maize) Figure: (4) Reference: (5)

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Processing of Bioethanol Focuses on the natural fermentation process: conversion of glucose into ethanol Current method (2 nd gen), two parts: 1)Obtain the cellulose source and process it with cellulases 2)Fermentation with yeast to produce ethanol (distillation) Figures left: (10), middle: (11), right: (12) glucose pyruvate ethanol

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Disadvantages of Bioethanol Changes in bioethanol have shifted to cellulosic ethanol production, but still not very efficient Not competitive with gasoline yet Food crisis/famine currently Figures -- left: (6), right: (7) References: (8), (9)

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Improvements for Bioethanol More emphasis on cellulosic ethanol; use of biomass Increasing efficiency of cellulase digestion Consolidating the two processes** Reference: (13)

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Costs: Consolidated Process Figure: (14)

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Increasing Efficiency: The Cellulosome Cellulosome: complex of enzymes that forms outside the cell for digestion Not free-floating! Organized complex Can be specialized** Only found in certain bacteria naturally Reference: mentor Shen-long Tsai

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The Cellulosome Reference: mentor Shen-long Tsai Cellulase Scaffoldin Cohesin Dockerin

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The Cellulosome Reference: mentor Shen-long Tsai Cellulose Reducing Sugars

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The Cellulases Figures top/middle: (15), bottom: (16) Endoglucanase Exoglucanase

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The Cellulases Figures top/middle: (15), bottom: (16) Endoglucanase Exoglucanase -glucosidase

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Engineering Cellulases **Can be specialized new cohesin/cellulase constructs Cloning Idea for New Constructs: -glucosidase: BglA + DocGf / exoglucanase: CelS + DocCc Secretion signal (with protein): MF1 + Gf protein Reference: mentor Shen-long Tsai C F

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Method: Cloning 1.Identify the insert(s) and vector to use a)pET-24a b)protein: BglA c)cohesin: DocGf 2.Plan the order and restriction enzymes needed a)Restriction enzymes: cut a palindrome sequence of DNA b)Avoid restriction sites in inserts (destroys it)

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Method: Cloning Figures left: (17), right: (18)

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Method: Cloning References .TAGACCAAACGGTGAAAGACACGAAAAATACTCTTTGGCTCCAGTTGCTAAGGAATTGCAATCATTGTTGGGTAAGGATGTCACCTTCTTGAACGACTGTGTCGGTCCAGAAGTT GAAGCCGCTGTCAAGGCTTCTGCCCCAGGTTCCGTTATTTTGTTGGAAAACTTGTGTTACCACATCGAAGAAGAAGGTTCCAGAAAGGTCGATGGTCAAAAGGTCAAGGCTTCCAAG GAAGATGTTCAACAGTTCAGACACGAATTGAGCTCTTTGGCTGATGTTTACATCAACGATGCCTTCGGTACCGCTCACAGAGCTCACTCTTCTATGGTCGGTTTCGACTTGCCACAACG TGCTGCCGGTTTCTTGTTGGAAAAGGAATTGAAGTACTTCGGTAAGGCTTTGGAGAACCCAACCAGACCATTCTTGGCCGCGGCCGCAtgagatttccttcaatttttactgcagttttattcgcagca tcctccgcattagctgctccagtcaacactacaacagaagatgaaaccgcacaaattccggctgaagctgtcatcggttacttggatttagaaggggatttcgatgttgctgttttgccattttccaacagcacaaataacgggttattgtttata aatactactattgccagcattgctgctaaagaagaaggggtatctttggataaaagagaggaaggcgaaccaGGATCCGGAACATATAACTATGGAGAAGCATTACAGAAATCAATAATGTTCTATGAATTCC AGCGTTCGGGAGATCTTCCGGCTGATAAACGTGACAACTGGAGAGACGATTCCGGTATGAAAGACGGTTCTGATGTAGGAGTTGATCTTACAGGAGGATGGTACGATGCAGGTGAC CATGTGAAATTTAATCTACCTATGTCATATACATCTGCAATGCTTGCATGGTCCTTATATGAGGATAAGGATGCTTATGATAAGAGCGGTCAGACAAAATATATAATGGACGGTATAA AATGGGCTAATGATTATTTTATTAAATGTAATCCGACACCCGGTGTATATTATTACCAAGTAGGAGACGGCGGAAAGGACCACTCTTGGTGGGGCCCTGCGGAAGTAATGCAGATGG AAAGACCGTCTTTTAAGGTTGACGCTTCTAAGCCCGGTTCTGCAGTATGTGCTTCCACTGCAGCTTCTCTGGCATCTGCAGCAGTAGTCTTTAAATCCAGTGATCCTACTTATGCAGAA AAGTGCATAAGCCATGCAAAGAACCTGTTTGATATGGCTGACAAAGCAAAGAGTGATGCTGGTTATACTGCGGCTTCAGGCTACTACAGCTCAAGCTCATTTTACGATGATCTCTCAT GGGCTGCAGTATGGTTATATCTTGCTACAAATGACAGTACATATTTAGACAAAGCAGAATCCTATGTACCGAATTGGGGTAAAGAACAGCAGACAGATATTATCGCCTACAAGTGGG GACAGTGCTGGGATGATGTTCATTATGGTGCTGAGCTTCTTCTTGCAAAGCTTACAAACAAACAATTGTATAAGGATAGTATAGAAATGAACCTTGACTTCTGGACAACTGGTGTTAA CGGAACACGTGTTTCTTACACGCCAAAGGGTTTGGCGTGGCTATTCCAATGGGGTTCATTAAGACATGCTACAACTCAGGCTTTTTTAGCCGGTGTTTATGCAGAGTGGGAAGGCTGT ACGCCATCCAAAGTATCTGTATATAAGGATTTCCTCAAGAGTCAAATTGATTATGCACTTGGCAGTACCGGAAGAAGTTTTGTTGTCGGATATGGAGTAAATCCTCCTCAACATCCTCA TCACAGAACTGCTCACGGTTCATGGACAGATCAAATGACTTCACCAACATACCACAGGCATACTATTTATGGTGCGTTGGTAGGAGGACCGGATAATGCAGATGGCTATACTGATGA AATAAACAATTATGTCAATAATGAAATAGCCTGCGATTATAATGCCGGATTTACAGGTGCACTTGCAAAAATGTACAAGCATTCTGGCGGAGATCCGATTCCAAACTTCAAGGCTATC GAAAAAATAACCAACGATGAAGTTATTATAAAGGCAGGTTTGAATTCAACTGGCCCTAACTACACTGAAATCAAGGCTGTTGTTTATAACCAGACAGGATGGCCTGCAAGAGTTACG GACAAGATATCATTTAAATATTTTATGGACTTGTCTGAAATTGTAGCAGCAGGAATTGATCCTTTAAGCCTTGTAACAAGTTCAAATTATTCTGAAGGTAAGAATACTAAGGTTTCCGG TGTGTTGCCATGGGATGTTTCAAATAATGTTTACTATGTAAATGTTGATTTGACAGGAGAAAATATCTACCCAGGCGGTCAGTCTGCGTGCAGACGAGAAGTTCAGTTCAGAATTGCC GCACCACAGGGAAGAAGATATTGGAATCCGAAAAATGATTTCTCATATGATGGATTACCAACCACCAGTACTGTAAATACGGTTACCAACATACCTGTTTATGATAACGGCGTAAAA GTATTTGGTAACGAACCCGCAGGTGGATCAGAACCCGGCACAAAGCTCGTTCCTACATGGGGCGATACAAACTGCGACGGCGTTGTAAATGTTGCTGACGTAGTAGTTCTTAACAGA TTCCTCAACGATCCTACATATTCTAACATTACTGATCAGGGTAAGGTTAACGCAGACGTTGTTGATCCTCAGGATAAGTCCGGCGCAGCAGTTGATCCTGCAGGCGTAAAGCTCACAG TAGCTGACTCTGAGGCAATCCTCAAGGCTATCGTTGAACTCATCACACTTCCTCAACTCGAGCACCACCACCACCACCACTGAGGATCCTCTAGAATCATGTAATTAGTTATGTCACGC TTACATTCACGCCCTCCCCCCACATCCGCTCTAACCGAAAAGGAAGGAGTTAGACAACCTGAAGTCTAGGTCCCTATTTATTTTTTTATAGTTATGTTAGTATTAAGAACGTTATTTATA TTTCAAATTTTTCTTTTTTTTCTGTACAGACGCGTGTACGCATGTAACATTATACTGAAAACCTTGCTTGAGAAGGTTTTGGGACGCTCGAAGGCTTTAATTTGCGGCCCTGCATTAATG AATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAA GGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGCCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCAT AGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCT CCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCT..

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Method: Cloning 3.Perform digestion of the insert and vector. 4.Perform ligation if successful. 5.Transformation of the DNA into a host cell.

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Method: Cloning

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6.Mini-preparation of colonies to test construct 7.Transformation into strain BL21.

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Detection: SDS-Page Immunofluorescence Reducing Sugar Measurements

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Reducing Sugar Prediction Time Glucose (OD) BglA + Ec + At Ec + At Ec + BglA control

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The Graph Figure: (14)

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Future Work Run more varied conditions to express the Gf construct Finish constructs; if successful, create new constructs Perform growth curve, reducing sugar, and glucose level experiments/measurements

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References 1. Flickr. $2.299 [picture]. (August 2008) Available at http://flickr.com/photos/69282447@N00/241986026 2. Crazy-jokes. "Gas Pump #2" [picture]. (August 2008) Available at http://blogs.e-rockford.com/applesauce/category/michael-bloomberg/ 3. Demirbas, Ayhan (2006). Progress and recent trends in biofuels. Progress in Energy and Combustion Science, 33:1, 1-18. doi:10.1016/j.pecs.2006.06.001 4. my-biodiesel.org. "Corn Fuel" [picture] (August 2008) Available at http://my-biodiesel.org/web/ 5. Wikipedia (2008). Bioethanol. http://en.wikipedia.org/wiki/Biofuel 6. Experiments using urine and humus dervied from ecological toilets. Experimental maize field under investigation [picture]. (August 2008) Available at http://aquamor.tripod.com/KYOTO.htm 7. Wikipedia. Cornfield in South Africa2 [picture]. (August 2008) Available at http://en.wikipedia.org/wiki/Image:Cornfield_in_South_Africa2.jpg 8. Wikipedia (2008). Ethanol fuel. http://en.wikipedia.org/wiki/Bioethanol 9. Rosegrant, Mark (2008). Biofuels and Grain Prices: Impact and Policy Responses. International Food Policy Research Institute. http://www.ifpri.org/pubs/testimony/rosegrant20080507.pdf 10. Masters Athlete Physiology & Performance. Lactate Threshold [picture]. (August 2008) Available at http://home.hia.no/~stephens/lacthres.htm 11. Wikipedia. "Pyruvic acid 2D skeletal" [picture]. (August 2008) Available at http://en.wikipedia.org/wiki/Image:Pyruvic-acid-2D-skeletal.png 12. Wikipedia. "Ethanol 2D skeletal" [picture]. (August 2008) Available at http://en.wikipedia.org/wiki/Image:Ethanol-2D-skeletal.svg 13. Wikipedia (2008). Cellulosic ethanol. http://en.wikipedia.org/wiki/Cellulosic_ethanol 14. Lynd, Lee et al (2005). Consolidated bioprocessing of cellulosic biomass: an update. Current Opinion in Biotechnology, 16:5, 577-583. doi:10.1016/j.copbio.2005.08.009 15. Colebrook, Michael. Life Chemistry. Cellulose [picture] (August 2008) Available at http://www.greenspirit.org.uk/resources/LifeChemistry.htm 16. Sci-toys. Lactose [picture]. (August 2008) Available at http://sci-toys.com/ingredients/lactose.html 17. Novagen. "pET-24a" [picture]. (August 2008) Available at http://www.merckbiosciences.com/docs/docs/PROT/TB070.pdf 18. Queens University of Charlotte. Recombinant DNA Technology [picture]. (August 2008) Available at http://campus.queens.edu/faculty/jannr/bio103/helpPages/C16DNAtech.htm

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Acknowledgements The BRITE program (NSF), Jun Wang, and UCR Ph.D. candidate Shen-long Tsai Professor Wilfred Chen All of the people in the Chen Lab All of the professors at UCR Others

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Thank you!

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Questions?

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Engineering Yeast to Drive Cars The Road for the Cellulosome Jeffrey Chen August 21, 2008 University of Texas Lab: Wilfred Chen BRITE 2008 Presentation