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CSU iGEMUsing Frying Oil to Product High Value Products in
an engineered strain of Escherichia coli
2014 Team
Advisers
Dr. Christie Peebles
Dr. Ashok Prasad
Jiayi Sun, Graduate Adviser
Students
Adriana Collings
Anthony Roulier
Chauncy Hinshaw
Josiah Racchini
Krista Henderson
Matt Sabel
Olivia Smith
Renee Plomondon
Savannah Roemer
Our Story
Breaking down used frying
oil to produce a value-added
terpenoid
Spent frying oil can be used
to power cars, but there are
still 3 billion gallons of spent
frying oil produced each
year in the U.S.
Overview of Project
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.
1. Biosensor
2. Breakdown of frying
oil
3. High-value product
4. Kill switch
Project Components
● Detect the presence of fatty acids
● Function as the promoter for
breakdown
● No unnecessary stress without oils
present
Biosensor - Goal
Biosensor - Background
Fatty Acids Present
GFP Produced
Fatty Acids Absent
No GFP production
Zhang, et. al. "Design of a Dynamic Sensor-regulator
System for Production of Chemicals and Fuels Derived from
Fatty Acids." Nature Biotechnology 30.4 (2012): 354-59.
Web.
Biosensor - Approach and Results
● Assemble 4 promoters from 4
sequenced oligonucleotides
○ Chose PAR and PFL1
● Link to GFP for testing
Future Work:
● Use fully synthesized gene
● Integrate into breakdown
Breakdown of Frying Oil - Goal
● Breakdown used frying oil products
● Create metabolic intermediate
Beta-
Oxidation
Mevalonate
Pathway
Fatty Acid Acetyl-CoA
Breakdown of Frying Oil
● Frying oil is made of
triacylglycerol
o heating makes diacylglycerol
fatty acids
● The fatty acids are broken
down by the beta oxidation
cycle
● FadD is the rate limiting
step Dobarganes, M. Carmen. “Formation of New Compounds during
Frying - General Observations.” AOCS Lipid Library. Web. 2 February
2009. June 2014.
Upregulate Beta-Oxidation
● Test by linking to lac
promoter
● Eventually link to
biosensor promoter
● Creates acetyl-CoA for
use in high-value
product manufacturing
Breakdown of Frying Oil
Zhang, Hanxing, et al. “Molecular effect of FadD on the regulation and metabolism of fatty acid in Escherichia coli.” FEMS
Microbiology Letters, 2. Web. 16 May 2006. June 2014.
Kameda, Kensuke, et al. “Purification and Characterization of Acyl Coenzyme A Synthase from Escherichia coli.” The Journal of
Biological Chemistry, 11. Web. 10 June 1981. June 2014.
High-Value Product: Goal
● Acetyl-CoA
→ IPP
→ Terpenoid
Martin, Vincent J., Douglas J. Pitera, Sydnor T. Withers, Jack D.
Newman, and Jay D. Keasling. "Engineering a Mevalonate Pathway in
Escherichia Coli for Production of Terpenoids." Nature Biotechnology
21.7 (2003): 796-802. PubMed.gov. Web. 27 June 2014.
High-Value Product: Background
● 2 pathways
● Mevalonate and
MEP
Martin, Vincent J., Douglas J. Pitera, Sydnor T. Withers, Jack D.
Newman, and Jay D. Keasling. "Engineering a Mevalonate Pathway in
Escherichia Coli for Production of Terpenoids." Nature Biotechnology
21.7 (2003): 796-802. PubMed.gov. Web. 27 June 2014.
● Created plasmids to put mevalonate pathway into E. coli
● Based on constructing two operons from yeast genes (S.
cerevisiae)
High-Value Product: Approach
Martin, Vincent J., Douglas J. Pitera, Sydnor T. Withers, Jack D. Newman, and Jay D. Keasling. "Engineering a Mevalonate Pathway in Escherichia Coli for Production of Terpenoids." Nature Biotechnology 21.7
(2003): 796-802. PubMed.gov. Web. 27 June 2014.
● Toggleable low-leak kill
switch
o Specific conditions
o Low stress
o Highly-efficient
Kill Switch: Goal
● Tryptophan Repressor
(BBa_K588001)
o Control of KillerRed
expression
● KillerRed gene
o Reactive Oxygen
Species
Kill Switch: Background
Bulina, M., et. al. "A genetically encoded photosensitizer.” Nature
Biotechnology, 95-99. 2005. October 2014.
● Trp + GFP Plasmid
● Trp+ KR Plasmid
● Kill curve of KR plasmid
Kill Switch: Approach
Bulina, M., et. al. "A genetically encoded photosensitizer.” Nature
Biotechnology, 95-99. 2005. October 2014.
Low Stress Design
● Allocates resources to
necessary functionso Promotes growth and
survival
o Prevents cell from
producing products
when unneccessary
o Reserves energy for cell
Safety
Workplace safety
programs can
decrease chances of
injury by up to 50%
1. Safety Services Company. “10 Tweetable Facts About Workplace Safety” .
Web. Jul 2012. Retrieved Sep 2014.
2. Photograph courtesy of Michigan School of Natural Resources and
Environment. Used with permission under creative commons with attribution.
Future Directions
● Combination of components into single
continuous pathway
● Optimization of pathway
● Optimization of environmental conditions
● Economic feasibility analysis
Human Practices
● University of Colorado -
Boulder collaboration
● Tested one of their
biobricks before
submission
Human Practices
● Outreach at elementary
school
● Conducted experiments
with Science Club
Thank You
Thank you to our advisers, iGEM, sponsors, and other teams for making this
competition and our project possible.
Acetyl-CoA Production
Produce one acetyl-
CoA per 2 carbons in
the fatty acid
backbone
Image courtesy of PharmaChange.info
Terpenoids
Created from combination of
isoprene units via condensation
reaction
1. Images (left and top right) courtesy of Cyberlipid Center
2. Image (bottom right) courtesy of Simon Cotton
Examples of terpenoids
Plant vs Bacterial Extraction
● Current yields: ~500 mg/L
● Order of magnitude increase in yield may be possible
1. Ajikumar, Parayil Kumaran, et. al. “Terpenoids: Opportunities for Biosynthesis of Natural Product Drugs Using Engineered Microorganisms.”
2. Image (left) courtesy of German Plant Breeders' Association
3. Image (right) courtesy of Scientific American Magazine
Determining Kill Switch
● KillerRed Info:
o Ordered via Evrogen
o 239 aa length, 27kDa
o Maximum Wavelength: 610 nm
● Alternative Choice: Heme Pathway
o Removal of HemH gene causes
protoporphyrin overproduction
o excess protoporphyrin causes ROS buildup
resulting in cell lysis
