DEGRADATION in E.coli iGEM 2012_ UC Davis 1 Introduction PET - - PowerPoint PPT Presentation

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DEGRADATION in E.coli iGEM 2012_ UC Davis 1 Introduction PET - - PowerPoint PPT Presentation

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Engineering Pathways for Polyethylene Terepthalate DEGRADATION in E.coli iGEM 2012_ UC Davis 1 Introduction PET Degradation Protein Engineering Chassis Conclusion References Problem Problem Solution The Problem: Environmental The

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Engineering Pathways for

Polyethylene Terepthalate

DEGRADATION

in E.coli

iGEM 2012_UC Davis

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  • The Pacific Gyre patch has a mass of 3.5 million tons of

waste material.

  • 90% of oceanic pollution is plastic.

The Problem: Environmental

PET Degradation Problem Introduction Solution Problem Chassis Conclusion Protein Engineering References

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PET Degradation Problem Introduction Solution Problem Chassis Conclusion Protein Engineering References

Polyethylene Terephthalate

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The Problem: Economical

PET Degradation Problem Introduction Solution Problem Chassis Conclusion Protein Engineering References 4

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Solution

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Goals

PET Degradation Problem Introduction Solution Problem Chassis Conclusion Protein Engineering References

1) Engineer E. coli to produce and secrete a protein that efficiently degrades PET. 2) Use rational protein engineering to increase the enzymatic activity of the PET degrading protein. 3) Engineer E. coli to utilize ethylene glycol as a carbon source.

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PET Degradation with LC-Cutinase

LC-Cutinase

  • Breaks down PET to Ethylene Glycol and TPA

PET Degradation Introduction Chassis Conclusion Protein Engineering References Data Cutinase Experiments 7

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LC-Cutinase Design

  • PelB tag
  • Directs the protein to the periplasm
  • His-tag
  • Purification and localization of the protein

PET Degradation Introduction Chassis Conclusion Protein Engineering References Data Cutinase Experiments

/J23101

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Cutinase Experiments

PET Degradation Introduction Chassis Conclusion Protein Engineering References Data Experiments Cutinase

Three goals:

  • 1. Determine if pelB transports the catalyst outside of the cell.
  • 2. Quantify the esterase activity of LC-Cutinase.
  • 3. Find out how well it degrades PET samples.

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PET Degradation Introduction Chassis Conclusion Protein Engineering References Data Experiments Cutinase

Determine if pelB transports the catalyst outside of the cell.

0h 1h 2h 3h Culture Separate Media and whole cell products SDS-PAGE and Western Blots

Cutinase Secretion

Induced Take Samples

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Uninduced

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Cutinase Secretion

PET Degradation Introduction Chassis Conclusion Protein Engineering References Data Experiments Cutinase

His-tagged protein of length consistent with that of LC-Cutinase (~30 kDa) are being secreted into the media.

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Cutinase Secretion

PET Degradation Introduction Chassis Conclusion Protein Engineering References Data Experiments Cutinase

Western blot of media samples taken at different time points after induction. His-tagged proteins of length consistent with that

  • f LC-Cutinase (~30 kDa) are being secreted into the media.

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Cutinase Secretion

PET Degradation Introduction Chassis Conclusion Protein Engineering References Data Experiments Cutinase

Western blot of media samples taken at different time points after induction. His-tagged proteins of length consistent with that

  • f LC-Cutinase (~30 kDa) are being secreted into the media.

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  • Quantify
  • p-nitrophenyl butyrate (pNPB) assays
  • Incubate pNPB buffer solution with cell cultures.

Esterase Activity

PET Degradation Introduction Chassis Conclusion Protein Engineering References Data Experiments Cutinase 14

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Esterase Activity

PET Degradation Introduction Chassis Conclusion Protein Engineering References Data Experiments Cutinase

Initial assays show that cells expressing the LC-Cutinase gene have higher esterase activity than cells that do not express it.

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Constitutive (J23101) Inducible (K206000) Control (J04450)

Cutinase Activity 1

Aborbance at 405nm per cell 0.0 0.2 0.4 0.6 0.8 1.0

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Esterase Activity

PET Degradation Introduction Chassis Conclusion Protein Engineering References Data Experiments Cutinase 16

Constitutive (J23101) Control (J04450)

Cutinase Activity 2

Aborbance at 405nm per cell 0.0 0.5 1.0 1.5

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Esterase Activity

PET Degradation Introduction Chassis Conclusion Protein Engineering References Data Experiments Cutinase

Secreted protein is active

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Constitutive (J23101) Control (J04450)

Cutinase Activity 3

Aborbance at 405nm per cell 0.0 0.5 1.0 1.5

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Future Directions

PET Degradation Introduction Chassis Conclusion Protein Engineering References Data Experiments Cutinase

  • Better characterize the expression of LC-Cutinase.
  • Purify the enzyme and run further esterase activity assays

with standardized concentrations.

  • Incubate purified enzyme with PET samples.

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Protein Engineering

PET Degradation Introduction Chassis Conclusion Protein Engineering References

  • Goal: to make a more active form of the LC-Cutinase protein
  • Replicate Mutants: replicated from previous literature on homologous

Cutinase protein

  • Personally generated mutations: using Foldit

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Silva C, et al. 2011. Engineered Thermobifida fusca cutinase with increased activity on polyester substrates. Biotechnol. J. 6:1230– 1239.

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Mutation Characterization

  • Mutant variants were assayed for increased esterase activity using

pNPB absorbance assays.

PET Degradation Introduction Chassis Conclusion Protein Engineering References

A

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T96A (pBAD) S101A (pBAD) D98T (pBAD) Y127A (pBAD) F125Y (pBAD) WT (pBAD) Control (J04450)

pNPB Cutinase Mutant Assay

A(405nm) per cell 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5

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Pathway Scheme

Introduction PET Degradation Protein Engineering Chassis Conclusion References Hybrid Rational Engineering Directed Evolution 21

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Pathway Scheme

Introduction PET Degradation Protein Engineering Chassis Conclusion References Hybrid Rational Engineering Directed Evolution 21

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Problem:

“No wild-type E. coli can grow on ethylene glycol (EG)”

> University of Barcelona, 1983 < Hypothesis: Overexpressing two enzymes

  • glycolaldehyde reductase
  • glycolaldehyde dehydrogenase

is sufficient for EG utilization

Boronat, Albert, Caballero, Estrella, and Juan Aguilar. “Experimental Evolution of a Metabolic Pathway for Ethylene Glycol Utilization by Escherichia coli.” Journal of Bacteriology, Vol. 153 No. 1, pp. 134-139, January 1983

Introduction PET Degradation Protein Engineering Chassis Conclusion References Hybrid Rational Engineering Directed Evolution 22

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Introduction PET Degradation Protein Engineering Chassis Conclusion References Hybrid Rational Engineering Directed Evolution 23

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Goals

  • 1. Ask for Strain
  • 2. Generate Mutants
  • 3. Test Hypothesis

Directed Evolution Rational Engineering

Introduction PET Degradation Protein Engineering Chassis Conclusion References Hybrid Rational Engineering Directed Evolution 24

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  • 1. Strain (E-15 EG3)
  • Dr. Juan Aguilar Piera
  • Dr. Laura Baldoma
  • Dr. Josefa Badia

University of Barcelona

Paper published in 1983 Freezer Archaelogy Strain Resurrection in 2012

Introduction PET Degradation Protein Engineering Chassis Conclusion References Hybrid Rational Engineering Directed Evolution 25

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Directed Evolution

Introduction PET Degradation Protein Engineering Chassis Conclusion References Hybrid Rational Engineering Directed Evolution 26

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Directed Evolution

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Directed Evolution

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Directed Evolution

Introduction PET Degradation Protein Engineering Chassis Conclusion References Hybrid Rational Engineering Directed Evolution

50000 100000 200000 300000 0.0 0.2 0.4 0.6 0.8 1.0

Rd 25 In Depth

Time (seconds) OD600 Rd 25 Repassage #2 Rd 25 Repassage #23 Rd 25 Repassage #26 Strain E−15 EG3

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  • 2. EMS Mutagenesis

Introduction PET Degradation Protein Engineering Chassis Conclusion References Rational Engineering Hybrid Directed Evolution 30

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  • 2. EMS Mutagenesis

Introduction PET Degradation Protein Engineering Chassis Conclusion References Rational Engineering Hybrid Directed Evolution 31

50000 100000 200000 300000 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7

Strain E−15 EG3 EMS Mutants

Time (seconds) OD600 E−15 EG3 0min #5 E−15 EG3 0min #10 E−15 EG3 0min #31 E−15 EG3 45min #6 E−15 EG3 45min #44 E−15 EG3 45min #47 Strain E−15 EG3

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Directed Evolution Conclusion

Introduction PET Degradation Protein Engineering Chassis Conclusion References Rational Engineering Hybrid Directed Evolution 31

  • Repassaging of E-15 EG3
  • Increased growth rate by 30%
  • Increased growth yield by 20%
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  • 3. Test Hypothesis: Ethylene Glycol Pathway
  • Dehydrogenase
  • In MG1655 chromosomally
  • Works best in aerobic conditions
  • Reductase
  • in MG1655 chromosomally
  • Naturally efficient in anaerobic conditions
  • Engineered for aerobic conditions

Introduction PET Degradation Protein Engineering Chassis Conclusion References Rational Engineering Hybrid Directed Evolution 32

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Simple-polycistronic assembly of reductase and dehydrogenase.

  • Test if two enzymes perform desired function

Note: This construct was created with both the inducible Bba_K206000 and the constitutive Bba_J23101.

Introduction PET Degradation Protein Engineering Chassis Conclusion References Rational Engineering Hybrid Directed Evolution

  • 3. Test Hypothesis: Ethylene Glycol Pathway

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Introduction PET Degradation Protein Engineering Chassis Conclusion References Rational Engineering Hybrid Directed Evolution 34

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  • 3. Test Hypothesis: Ethylene Glycol Pathway

Introduction PET Degradation Protein Engineering Chassis Conclusion References Hybrid Directed Evolution Rational Engineering 37

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Next Steps: Next Generation Sequencing

Introduction PET Degradation Protein Engineering Chassis Conclusion References Hybrid Directed Evolution Rational Engineering 38

  • Created Illumina Library
  • Strain E-15 EG3
  • Confirm hypothesis through

genomic analysis

  • Future
  • Sequence Repassaged Strains
  • Look for patterns

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PET Degradation Introduction Chassis Conclusion Protein Engineering References Human Practice Accomplishments Future

Educational Outreach

  • The growth of Synthetic Biology is extremely important
  • Current educational standards fall far short of necessity
  • Constructed a Lesson Plan aimed at middle-school level
  • Provides foundational base for basic synthetic biology practices
  • Introduces fundamental concepts used in iGEM
  • Curriculum Development
  • Gave Davis Senior High School materials for basic lab practices

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PET Degradation Introduction Chassis Conclusion Protein Engineering References Human Practice Accomplishments Future

Economic Resource

  • Development of Synthetic Biology applications
  • iGEM generates many great solutions, no tangible product
  • Plastic degradation has clear socioeconomic implications
  • Means for idea proliferation
  • Addresses the market, revenue models, risk, intellectual

Property, and other considerations

  • Intent of taking laboratory procedures, producing practical

products

  • Application to Plastic Degradation
  • Applied to our project, foundation of iGEM Entrepreneurship

team

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PET Degradation Introduction Chassis Conclusion Protein Engineering References Human Practice Accomplishments Future

What We Did

  • PET Degradation
  • Cutinase is secreted and demonstrates esterase activity
  • Some cutinase mutants appear to have increased levls of

esterase activity compared to wild-type

  • Chassis Engineering
  • Freezer Archeology, Directed Evolution of E-15 EG3
  • Characterization of Ethylene Glycol construct
  • Parts Registry
  • 20 new submissions

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PET Degradation Introduction Chassis Conclusion Protein Engineering References Human Practice Accomplishments Future

What We Want to Do

  • Conduct more extensive data collection
  • Purify enzyme and conduct standardized pNPB and PET tests
  • Sequence E-15 EG3 and evolved E-15 EG3
  • For comparison, identification of best module.
  • Combine both Cutinase module with Ethylene Glycol

pathway

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Highlighted References

  • S. Sulaiman, S. Yamato, E. Kanaya, J. Kim, Y. Koga, K. Takano, S. Kanaya.

"Isolation of a Novel Cutinase Homolog with Polyethylene Terephthalate- Degrading Activity from Leaf-Branch Compost by Using a Metagenomic Approach." Applied and Environment Microbiology, vol. 78 no. 5, pp. 1556-1562, March 2012.

  • Boronat, Albert, Caballero, Estrella, and Juan Aguilar. Experimental Evolution
  • f a Metabolic Pathway for Ethylene Glycol Utilization by Escherichia coli.

Journal of Bacteriology, Vol. 153 No. 1, pp. 134-139, January 1983.

  • Danchin, Antoine. "Scaling up synthetic biology: Do not forget the chassis."
  • Elsevier. (2011): December. Print.
  • Ö. Faiz et al. Determination and characterization of thermostable esterolytic

activity from a novel thermophilic bacterium Anoxybacillus gonensis

  • Silva C, et al. 2011. Engineered Thermobifida fusca cutinase with increased

activity on polyester substrates. Biotechnol. J. 6:1230–1239.

MODULES INTRODUCTION CONCLUSION REFERENCES PROTEIN CHASSIS

References Acknowledgements 42

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Sponsors – Thank You!

MODULES INTRODUCTION CONCLUSION REFERENCES PROTEIN CHASSIS

References Acknowledgements 43

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iGEM 2012_UC Davis

THANK YOU

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Dehydrogenase and Reductase Endeavors

  • 2. Complex – compartmentalized organization: one gene per

promoter. “fine-tuning” – able to control which enzyme is expressed (less stress on cells).

PET Degradation Introduction Chassis Conclusion Protein Engineering References Directed Evolution Rational Engineering

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