Modification of Methanococcus maripaludis for Production of Geraniol - - PowerPoint PPT Presentation

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Modification of Methanococcus maripaludis for Production of Geraniol University of Georgia iGEM Team University of Georgia iGEM Team Foundational Advance Regional Jamboree East10/13/2012 Presenters: James Bevington, Travis Fetchko, Annika

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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

SLIDE 2

Who we are…

Jay Bevington Environmental Engineering Class of 2013 Annika McNally Biology/German Class of 2013 Travis Fetchko Biochemistry & Molecular Biology Class of 2013

SLIDE 3

Discussing what to do Aromatic compound Isoprenoid lipid synthesis

Idea Generation

http://blog.copdfoundation.org/questions-about-insurance/c-users-sschlegel-pictures-question-mark-man/

synthesis

SLIDE 4

Geraniol synthase gene isolated from Ocimum basilicum can be expressed in Methanococcus maripaludis to

Hypothesis

can be expressed in Methanococcus maripaludis to produce a detectable level of geraniol.

SLIDE 5

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>.

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Obligate anaerobe Mesophilic

Methanococcus maripaludis

Autotrophic Isoprenoid lipids

http://genome.jgi-psf.org/metm6/metm6.home.html

SLIDE 7

Methanogenesis

Unique anaerobic

respiration

Novel biochemistry with

unusual coenzymes as C1 carriers

http://faculty.washington.edu/leighj/mmmethanogenesis.html

SLIDE 8

Geraniol

Rose like smell Trans Geraniol/3,7-Dimethylocta-2,6-dien-1-ol C10H18O

SLIDE 9

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/

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Our Task

Use synthetic biology techniques to modify

Methanococcus maripaludis for the production of Geraniol

http://complex.upf.es/wet-lab/

SLIDE 11

Membrane lipid biosynthesis in Methanococcus

Isoprenoid Membrane Lipid Geraniol Intermediate Geraniol synthase

SLIDE 12

Overview

Gene Design

Construction of shuttle vector

PCR/cloning/screening/plasmid
PCR/cloning/screening/plasmid
prep. For transformation

Transformation into Methanococcus Product Verification

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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

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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.

SLIDE 15

pAW42 has

Strong promoter RBS binding site upstream

Construction of Shuttle Vector

RBS binding site upstream

f 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.

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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

SLIDE 17

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

SLIDE 18

Transformation

Polyethylene Glycol

transformation into M. maripaludis

SLIDE 19

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

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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 N2 60% recovery on control experiments

Verification of Geraniol by GC/MS

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Verification of Geraniol Production

Standard (1µg/mL) of geraniol in hexane) Extracted Product

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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

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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!

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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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