z . c o l i - 2015.igem.org2015.igem.org/files/presentation/csu_fort_collins.pdf · z . c o l i...
TRANSCRIPT
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Z . C O L IUsing frying oil to produce trans-zeatin in
an engineered strain of E. coli
Colorado State University iGEM
Giant Jamboree 2015
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Background
3 billion gallons produced per year in US[1]
Breakdown used frying oil to produce zeatin
1. US EPA. “Learn About Biodiesel”. Web. 8 Jan 2014. Retrieved 20 Aug 2014.
2. Photograph courtesy of Dan Dickinson. Used with permission under creative commons with attribution.
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Our Project
ExtracellularBreakdown
TransportMEP
PathwayZeatin
IntracellularBreakdown
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Why frying oil?
High energy
CheapFatty acids { } Glycerol
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Limiting Steps in Breakdown
Breakdown of Frying Oil Components
Breakdown of Fatty Acids
Transport of Fatty Acids
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Breakdown of DAGs
Lipase from Bacillus stearothermophilus
Turns DAGs into FAs and Glycerol
http://www.emhmagazine.com/uploaded/images/123035292.jpg
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Transport of LCFAs into Cell
Natural Transporter: long-chain fatty acid outer membrane porin
Encoded for by fadL
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Increased Breakdown of FAs
Beta-oxidation pathway
fadD encodes limiting enzyme
Fatty acyl-CoA synthetase
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faddfadD Experiment
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Growth Experiment with Used Frying Oil
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Growth Experiment with Used Frying Oil
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Growth Experiment with Used Frying Oil
Initial 50% Solution 50% Solution after 12 hours of Growth
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Future Improvements
Quantitative vs. Qualitative Data
Cell Growth Measurement
Optimize Feed and Growth Conditions
Metabolic Engineering
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Trans-zeatin
Very expensive
Currently only used in research
Many other applications
https://en.wikipedia.org/wiki/Zeatin
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Plant Growth Experiment
Biological Activity of the tzs Gene of Nopaline Agrobacterium tumefaciens GV3101 in Plant Regeneration and Genetic Transformation (Zhao-Fen Han et al., 2013)
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MEP Pathway
D-Glyceraldehyde 3-phosphate Pyruvate
MEP
HMBDP
http://www.pet.hw.ac.uk/research/fast1/research/res_images/chem_inhibitors/hmdp.gif
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Biosynthesis PathwayZeatin Biosynthesis
Trans-zeatin Synthase
LOGzeatin
https://en.wikipedia.org/wiki/Zeatin
zeatin riboside 5’-phosphate N6-dimethylallyladeninehydroxylation
HMBDPhttp://www.pet.hw.ac.uk/research/fast1/research/res_images/chem_inhibitors/hmdp.gif
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Characterization
Zeatin Construct Growth Experiment
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Extraction methods Solid Phase Extraction Methods
1. Column Wash
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2. Bind Supernatant 3. Wash
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4. Elute
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Zeatin HPLC Results
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Design Considerations
http://instalacionesgrau.com/en/products/bioreactores/
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https://pbs.twimg.com/media/CE4Ad2JWAAA6Rp4.jpg
KillerRed
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Killer Red Literature
Bulina, M., et. al. "A genetically encoded photosensitizer.” Nature Biotechnology, 95-99. 2005. October 2014.
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Kill Switch
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• Functional improvement• Removed trp promoter
• Characterization improvement• Collected data on efficacy
KillerRed Improvement
Control KillerRed
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Kill Switch Constructs
Improved characterization of KillerRed biobrick
Suspected mutation
Working in a jury-rigged darkroom with KillerRed
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interlabInterlab Study
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Research
Industry
Public
Spoke with experts and compiled articles on:
Laws and regulation of GMOs
Commercialization and industry
Intellectual property
Bridging the Gap
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EntrepreneuershipEntrepreneurship
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• Construction and submission of multiple BioBrick Parts (Bba_KI702000-Bba_K1702005)
• Experimental validation of fatty acid breakdown (BBa_K1702001, Bba_K1702002)
• Addressed questions beyond the bench about IP, commercialization, and bridging the gap between academia, industry, and the public
• Used beyond the bench IP research to file provisional patent
• Improved function and characterization of previously existing BioBrick Part, KillerRed (Bba_K1495000)
• Functional prototype of E. coli growing on frying oil
Key Achievements
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Key challenges
• Reworking breakdown construct ideas after fadDexperiment
• Inconclusive results about trans-zeatin production
• Proper handling and experimental design for testing KillerRed
Key Challenges
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Thank You
Dr. Christie Peebles, Dr. Tom Santangelo, and Dr. Ashok Prasad
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Fatty acid growth experimentsLac Promoter
"Lac operon-2010-21-01" by Lac_operon.png: G3proderivative work: Tereseik (talk) - Lac_operon.png. Licensed under CC BY 2.0 via Commons - https://commons.wikimedia.org/wiki/File:Lac_operon-2010-21-01.png#/media/File:Lac_operon-2010-21-01.png
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Growth Experiment with 100% Used Frying Oil
Before Inoculation
After 4 hours of growth
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Fatty acid growth experimentsFatty Acid Growth Experiment
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E. coli Growth on 0.04% Used Frying oil
fadD + fadL
fadD
fadL
lac Promoter
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Why Zeatin?
Benefits:• Increase lateral bud growth• Increases immunity against pathogenic attack on plants[1]
• Anti-aging properties• Green (natural) product• Synthetic production is complex and low yield
Competitors:Benzyl adenine• Synthetic• Not as well researched• Zeatin is 10x more effective[2]
1. Grubkinsky, Dominik et al. ‘Cis- and trans- zeatin differentially modulate plant immunity’. Plant Signal Behav. 2013 8(7); e247982. Bajaj, Y.P.S. Plant Protoplasts and Genetic Engineering IV 2012. Springer Science and Business Media.
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Zeatin Biosynthesis
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MEP Pathway fullMEP Full Pathway
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Tz bioreactor with dataTrans-zeatin Bioreactor Data
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Killer red dataKiller Red Data