“Methanococcus: The New Archaea-type” for Synthetic Biology UGA-Georgia Peyton Smith, Rebecca Buchanan, Mengyin Cheng, Kevin Moriles Why Study Archaea? ! ! ! Expand the field of synthetic biology Inexpensive substrates to produce high value chemicals Industrial applicationsanaerobic digestion and biogas Bacteria Archaea Eukarya h#p://www.premiumbeautynews.com/local/cache-‐vigne#es/L400xH300/600_deo_candy-‐2-‐0102f.jpg Why Study Archaea? ! ! ! Expand the field of synthetic biology Inexpensive substrates to produce high value chemicals Industrial applicationsanaerobic digestion and biogas H2 CO2 Glucose E. coli Substrates Methanococcus Substrates h#p://brinkercapital.files.wordpress.com/2013/07/7-‐12-‐13_magno#a_marketoutlook_3.jpg Why Study Archaea? ! ! ! Expand the field of synthetic biology Inexpensive substrates to produce high value chemicals Industrial applicationsanaerobic digestion and biogas Waste/Biomass CO2 2H2O CO2 8H++ 8e- CH4 Lyu, Zhe, 2014 Creative Energy Engineering, 2014 What is Geraniol? ! ! ! ! ! Fragrances Flavorings Biofuel Insect Repellant Tumor Suppressor h#p://www.premiumbeautynews.com/local/cache-‐vigne#es/L400xH300/600_deo_candy-‐2-‐0102f.jpg Why Geraniol from Archaea? Construct for Geraniol Production ORFLESS1: Methanococcal origin of replication PhmvA: Methanococcal promoter RBS: ribosome binding site GS: geraniol synthase Pmcr: Methanococcal promoter Pac: puromycin resistance cassette Tmcr: Methanococcal terminator amp: ampicillin resistance cassette Geraniol Production Transformation Concentration Extraction GC/MS Objectives ! ! ! Objective 1: To Create Protein Expression tools: Ribosome Biding Site Library Objective 2: Modeling & Flux Balance Analysis Objective 3: Improving Extraction of Geraniol Isoprenoid Biosynthesis Pathway ! Investigated using the databases: ! ! ! KEGG Biocyc SEED Geranyl-PP Geranylgeranyl 9 Diphosphate synthase Isopentyl-PP PPi (E,E)-Farnesyl-PP Geranylgeranyl 10 Diphosphate synthase Isopentyl-PP PPi What is a Ribosome Binding Site? The sequence on the mRNA that is complementary to the 3’ end of the 16S rRNA. Jenner, L.B., et al, 2010 mCherry as a Quantitative Reporter pMEV4-mCherry RBS Variants Spacer Native Mutations (19-33) Start Codon Mutations (34-36) RBS Mutations (1-18) Theoretical “positive” (37) Theoretical “negative” (38) The Snake in the Grass h#p://patenJmages.storage.googleapis.com/EP0505023A1/imgb0001.png Oxygen Maturation of mCherry Results and Outlook BioBrick Parts ! Bba_K1383000 ! ! Bba_K1383001 ! ! Positive Bba_K1383002 ! Native Negative Objectives ! ! ! Objective 1: To Create Protein Expression Tools: Ribosome Biding Site Library Objective 2: Modeling & Flux Balance Analysis Objective 3: Improving Extraction of Geraniol Model ! ! BioModels Database: MODEL 130412000 COBRA toolbox in MATLAB Goyal, Nishu, et al., 2014 Addition of Geraniol Reactions Added Metabolites Metabolite Number Metabolite Name 605 grl 606 grl_e Added Geraniol Reactions Simulation Results All values displayed in mmol/gDCW/hr Future of the Model ! ! ! Optimize the production of geraniol through knockout strategies. Include regulatory elements and kinetics upon each reaction. Develop strategies for the overproduction of isoprenoid compounds Objectives ! ! ! Objective 1: To Create Protein Expression Tools: Ribosome Biding Site Library Objective 2: Modeling & Flux Balance Analysis Objective 3: Improving Extraction of Geraniol Improving Extraction of Geraniol ! Tested different solvents ! ! DCM , butanol, diethyl ether, hexanes and acetone Tested different extraction methods ! Separatory funnel vs.Balch tube Outreach Seminars Bioenergy Systems Research Institute Awarded best undergraduate presentation Georgia Environmental Conference Student scholarship award Accomplishments ! ! ! ! ! ! Improved upon a previous submitted part Novel protocol for maturation of mCherry Characterization of 3 RBS variants Submission of 3 BioBrick parts Successful simulation of geraniol production in Methanococcus Human practice: outreach and seminars Mentors ! ! ! ! ! ! Dr. William Whitman Dr. Yajun Yan Dr. Zhe Lyu Rachit Jain Narendran Sekar Naga Sirisha Parimi Sponsors ! ! ! ! ! ! ! ! President’s Venture Fund Office of the Vice President for Research College of Engineering Department of Microbiology Department of Biochemistry and Molecular Biology Department of Genetics Franklin College Student Activity Fee Allocation Committee UGA Alumni Association
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