Modification of Methanococcus maripaludis for Production of Geraniol University of Georgia iGEM Team University of Georgia iGEM Team Foundational Advance Regional Jamboree East−10/13/2012 Presenters: James Bevington, Travis Fetchko, Annika McNally
Who we are… Jay Bevington Annika McNally Travis Fetchko Environmental Biology/German Biochemistry & Engineering Class of 2013 Molecular Biology Class of 2013 Class of 2013
Idea Generation Aromatic compound Isoprenoid Discussing lipid what to do synthesis synthesis http://blog.copdfoundation.org/questions-about-insurance/c-users-sschlegel-pictures-question-mark-man/
Hypothesis Geraniol synthase gene isolated from Ocimum basilicum can be expressed in Methanococcus maripaludis to can be expressed in Methanococcus maripaludis to produce a detectable level of geraniol.
Why Methanococcus maripaludis? • First isolated from salt marshes in South Carolina • Related to methanogenic archaea found in the gut • Opportunity to explore prokaryotic diversity "Methanococcus." Microbe Wiki . N.p., n.d. Web. 19 Sept. 2012. <http://microbewiki.kenyon.edu/ index.php/File:Methanococcus.jpeg>.
Methanococcus maripaludis � Obligate anaerobe � Mesophilic � Autotrophic � Isoprenoid lipids http://genome.jgi-psf.org/metm6/metm6.home.html
Methanogenesis � Unique anaerobic respiration � Novel biochemistry with unusual coenzymes as C1 carriers http://faculty.washington.edu/leighj/mmmethanogenesis.html
Geraniol � Rose like smell � Trans Geraniol/3,7-Dimethylocta-2,6-dien-1-ol � C 10 H 18 O
Uses of Geraniol � Hydrocarbon/alcohol � Potential use as biofuel � Fragrance � Component of rose, lemon and many other scents � Insect repellent � Precursor to terpenes � Important compounds � Commercial applications http://www.aohdiv19.com/rose-sale/
Our Task � Use synthetic biology techniques to modify Methanococcus maripaludis for the production of Geraniol http://complex.upf.es/wet-lab/
Membrane lipid biosynthesis in Methanococcus Intermediate Geraniol synthase Isoprenoid Membrane Lipid Geraniol
Overview Gene Design Construction of shuttle vector •PCR/cloning/screening/plasmid •PCR/cloning/screening/plasmid prep. For transformation Transformation into Methanococcus Product Verification
Gene Design � Identified enzyme of origin � Ocimum basilicum (basil) � Inserted two restriction sites � Codon optimization for M. maripaludis � Plant to Archaea � Synthesized by GenScript From GenScript
Construction of Shuttle Vector � PCR amplify synthetic GS gene, adding the flanking restriction sites to the restriction sites to the primers � Inserted GS gene into pAW42 at NsiI-XbaI sites Walter et al., AEM, 77.7 (2011): 2549-51.
Construction of Shuttle Vector � pAW42 has � Strong promoter � RBS binding site upstream � RBS binding site upstream of the NsiI site � Ampicillin resistance for selection in E. coli � Puromycin resistance for selection in Methanococcus Walter et al., AEM, 77.7 (2011): 2549-51.
Cloning � Transformed shuttle vector into E.coli � Plated on ampicillin plates � Extracted/Purified � Picked resulting colonies for plasmid minipreparations � Picked resulting colonies for plasmid minipreparations � Restrict plasmids with EcoRV and screen by electrophoresis
Verification of Shuttle Vector � Lanes 1-4 are plasmids from four representative colonies. All contain the gene for GS � Lane 5 was negative (abandoned) Gel electrophoresis of plasmid minipreps
Transformation � Polyethylene Glycol transformation into M. maripaludis
Verification of Transformation into M. maripaludis � Colony PCR � Lanes 1-4 are selected colonies � Lanes 1-4 are selected colonies � Lane C is a control � Lane 3 contains the gene
Product Formation � How much geraniol was expected (5% of lipid content) � 20 nmol Geraniol / 5mL of culture � 0.6 µg/mL of Geraniol � Extraction methods � Hexane extraction, then evaporated by N 2 � 60% recovery on control experiments � Verification of Geraniol by GC/MS
Verification of Geraniol Production Standard (1µg/mL) of geraniol in hexane) Extracted Product
Future work � Increase geraniol production � Verification of expression � Improve expression by growing the cells under different growth conditions. � Try GS genes from other sources � Try GS genes from other sources � Add a gene to convert geraniol to other compounds � ATT Geranyl Acetate
Conclusion � Methanococcus maripaludis can make geraniol � Research adds to the foundational knowledge of � Research adds to the foundational knowledge of synthetic biology � Started first successful iGEM team at UGA!
University of Georgia iGEM Team Acknowledgment Other Team Members: Jessica Calkins Rachit Jain Andrew Upchurch Brice Bowerman Brice Bowerman Tuan Nguyen Advisors: Dr. William Whitman Dr. Yajun Yan Dr. Brahm Verma GC/MS Facility: Dr. Dennis Phillips
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