CSU iGEM Using Frying Oil to Product High Value Products in an - - PowerPoint PPT Presentation

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CSU iGEM Using Frying Oil to Product High Value Products in an - - PowerPoint PPT Presentation

Oct 17, 2023 350 likes •635 views

CSU iGEM Using 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 Krista Henderson Adriana Collings Matt Sabel

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SLIDE 1

CSU iGEM

Using Frying Oil to Product High Value Products in an engineered strain of Escherichia coli

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SLIDE 2

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

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SLIDE 3

Our Story

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SLIDE 4

Breaking down used frying

  • il 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.

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SLIDE 5
  • 1. Biosensor
  • 2. Breakdown of frying
  • il
  • 3. High-value product
  • 4. Kill switch

Project Components

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SLIDE 6
  • Detect the presence of fatty acids
  • Function as the promoter for

breakdown

  • No unnecessary stress without oils

present

Biosensor - Goal

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SLIDE 7

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.
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SLIDE 8

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
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SLIDE 9

Breakdown of Frying Oil - Goal

  • Breakdown used frying oil products
  • Create metabolic intermediate

Beta- Oxidation Mevalonate Pathway Fatty Acid Acetyl-CoA

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SLIDE 10

Breakdown of Frying Oil

  • Frying oil is made of

triacylglycerol

  • 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.
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SLIDE 11

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.
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SLIDE 12

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.
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SLIDE 13

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.
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SLIDE 14
  • 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.
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SLIDE 15
  • Toggleable low-leak kill

switch

  • Specific conditions
  • Low stress
  • Highly-efficient

Kill Switch: Goal

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SLIDE 16
  • Tryptophan Repressor

(BBa_K588001)

  • Control of KillerRed

expression

  • KillerRed gene
  • Reactive Oxygen

Species

Kill Switch: Background

Bulina, M., et. al. "A genetically encoded photosensitizer.” Nature Biotechnology, 95-99. 2005. October 2014.
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SLIDE 17
  • 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.
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SLIDE 18

Low Stress Design

  • Allocates resources to

necessary functions

  • Promotes growth and

survival

  • Prevents cell from

producing products when unneccessary

  • Reserves energy for cell
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SLIDE 19

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.
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SLIDE 20

Future Directions

  • Combination of components into single

continuous pathway

  • Optimization of pathway
  • Optimization of environmental conditions
  • Economic feasibility analysis
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SLIDE 21

Human Practices

  • University of Colorado -

Boulder collaboration

  • Tested one of their

biobricks before submission

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SLIDE 22

Human Practices

  • Outreach at elementary

school

  • Conducted experiments

with Science Club

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SLIDE 23

Thank You

Thank you to our advisers, iGEM, sponsors, and other teams for making this competition and our project possible.

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SLIDE 24

Acetyl-CoA Production

Produce one acetyl- CoA per 2 carbons in the fatty acid backbone

Image courtesy of PharmaChange.info

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SLIDE 25

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

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SLIDE 26

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
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SLIDE 27

Determining Kill Switch

  • KillerRed Info:
  • Ordered via Evrogen
  • 239 aa length, 27kDa
  • Maximum Wavelength: 610 nm
  • Alternative Choice: Heme Pathway
  • Removal of HemH gene causes

protoporphyrin overproduction

  • excess protoporphyrin causes ROS buildup

resulting in cell lysis