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 Pacific Gyre patch has a mass of 3.5 million tons of waste material. • 90% of oceanic pollution is plastic. http://24.media.tumblr.com/tumblr_m8eahzOJhC1qztczjo1_500.jpg http://2.bp.blogspot.com/_AdHtqOqrL0w/TAvQKCJQRTI/AAAAAAAACts/LlKxCM2UEe8/ 2 s1600/gyre.jpg
Introduction PET Degradation Protein Engineering Chassis Conclusion References Problem Problem Solution Polyethylene Terephthalate 3
Introduction PET Degradation Protein Engineering Chassis Conclusion References Problem Problem Solution The Problem: Economical 4
Introduction PET Degradation Protein Engineering Chassis Conclusion References Problem Problem Solution Solution 5
Introduction PET Degradation Protein Engineering Chassis Conclusion References Problem Problem Solution Goals 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. 6
Introduction PET Degradation Protein Engineering Chassis Conclusion References Cutinase Experiments Data PET Degradation with LC-Cutinase LC-Cutinase • Breaks down PET to Ethylene Glycol and TPA 7
Introduction PET Degradation Protein Engineering Chassis Conclusion References Cutinase Experiments Data LC-Cutinase Design • PelB tag • Directs the protein to the periplasm • His-tag • Purification and localization of the protein /J23101 8
Introduction PET Degradation Protein Engineering Chassis Conclusion References Cutinase Experiments Data Cutinase Experiments 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. 9
Introduction PET Degradation Protein Engineering Chassis Conclusion References Cutinase Experiments Data Cutinase Secretion Determine if pelB transports the catalyst outside of the cell. Uninduced 0h 1h 2h 3h Culture Take Samples Induced Separate Media and SDS-PAGE and whole cell products Western Blots 10
Introduction PET Degradation Protein Engineering Chassis Conclusion References Cutinase Experiments Data Cutinase Secretion His-tagged protein of length consistent with that of LC-Cutinase (~30 kDa) are being secreted into the media. 11
Introduction PET Degradation Protein Engineering Chassis Conclusion References Cutinase Experiments Data Cutinase Secretion Western blot of media samples taken at different time points after induction. His-tagged proteins of length consistent with that of LC-Cutinase (~30 kDa) are being secreted into the media. 12
Introduction PET Degradation Protein Engineering Chassis Conclusion References Cutinase Experiments Data Cutinase Secretion Western blot of media samples taken at different time points after induction. His-tagged proteins of length consistent with that of LC-Cutinase (~30 kDa) are being secreted into the media. 13
Introduction PET Degradation Protein Engineering Chassis Conclusion References Cutinase Experiments Data Esterase Activity • Quantify • p-nitrophenyl butyrate (pNPB) assays • Incubate pNPB buffer solution with cell cultures. 14
Introduction PET Degradation Protein Engineering Chassis Conclusion References Cutinase Experiments Data Esterase Activity Initial assays show that cells expressing the LC-Cutinase gene have higher esterase activity than cells that do not express it. Cutinase Activity 1 1.0 0.8 Aborbance at 405nm per cell 0.6 0.4 0.2 0.0 15 Constitutive (J23101) Inducible (K206000) Control (J04450)
Introduction PET Degradation Protein Engineering Chassis Conclusion References Cutinase Experiments Data Esterase Activity Cutinase Activity 2 1.5 Aborbance at 405nm per cell 1.0 0.5 0.0 16 Constitutive (J23101) Control (J04450)
Introduction PET Degradation Protein Engineering Chassis Conclusion References Cutinase Experiments Data Esterase Activity Secreted protein is active Cutinase Activity 3 1.5 Aborbance at 405nm per cell 1.0 0.5 0.0 Constitutive (J23101) Control (J04450) 17
Introduction PET Degradation Protein Engineering Chassis Conclusion References Cutinase Experiments Data Future Directions • 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. 18
Introduction PET Degradation Protein Engineering Chassis Conclusion References Protein Engineering • 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 Silva C, et al. 2011. Engineered Thermobifida fusca cutinase with increased activity on polyester substrates. Biotechnol. J. 6:1230– 19 1239.
Introduction PET Degradation Protein Engineering Chassis Conclusion References Mutation Characterization • Mutant variants were assayed for increased esterase activity using pNPB absorbance assays. pNPB Cutinase Mutant Assay 3.5 3.0 2.5 A(405nm) per cell 2.0 A 1.5 1.0 0.5 0.0 T96A S101A D98T Y127A F125Y WT Control 20 (pBAD) (pBAD) (pBAD) (pBAD) (pBAD) (pBAD) (J04450)
Chassis Introduction PET Degradation Protein Engineering Conclusion References Directed Evolution Rational Engineering Hybrid Pathway Scheme 21
Chassis Introduction PET Degradation Protein Engineering Conclusion References Directed Evolution Rational Engineering Hybrid Pathway Scheme 21
Chassis Introduction PET Degradation Protein Engineering Conclusion References Directed Evolution Rational Engineering Hybrid 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 22
Chassis Introduction PET Degradation Protein Engineering Conclusion References Directed Evolution Rational Engineering Hybrid 23
Chassis Introduction PET Degradation Protein Engineering Conclusion References Directed Evolution Rational Engineering Hybrid Goals 1. Ask for Strain Directed Evolution 2. Generate Mutants Rational 3. Test Hypothesis Engineering 24
Chassis Introduction PET Degradation Protein Engineering Conclusion References Directed Evolution Rational Engineering Hybrid 1. Strain (E-15 EG3) Paper published in 1983 Freezer Archaelogy Strain Resurrection in 2012 Dr. Juan Aguilar Piera Dr. Laura Baldoma Dr. Josefa Badia University of Barcelona 25
Chassis Introduction PET Degradation Protein Engineering Conclusion References Directed Evolution Rational Engineering Hybrid Directed Evolution 26
Chassis Introduction PET Degradation Protein Engineering Conclusion References Directed Evolution Rational Engineering Hybrid Directed Evolution 27
Chassis Introduction PET Degradation Protein Engineering Conclusion References Directed Evolution Rational Engineering Hybrid Directed Evolution 28
Chassis Introduction PET Degradation Protein Engineering Conclusion References Directed Evolution Rational Engineering Hybrid Directed Evolution Rd 25 In Depth 1.0 Rd 25 Repassage #2 Rd 25 Repassage #23 Rd 25 Repassage #26 Strain E − 15 EG3 0.8 0.6 OD600 0.4 0.2 0.0 0 50000 100000 200000 300000 Time (seconds) 29
Chassis Introduction PET Degradation Protein Engineering Conclusion References Directed Evolution Rational Engineering Hybrid 2. EMS Mutagenesis 30
Chassis Introduction PET Degradation Protein Engineering Conclusion References Directed Evolution Rational Engineering Hybrid 2. EMS Mutagenesis Strain E − 15 EG3 EMS Mutants 0.7 0.6 0.5 0.4 OD600 0.3 E − 15 EG3 0min #5 0.2 E − 15 EG3 0min #10 E − 15 EG3 0min #31 E − 15 EG3 45min #6 0.1 E − 15 EG3 45min #44 E − 15 EG3 45min #47 Strain E − 15 EG3 0.0 0 50000 100000 200000 300000 31 Time (seconds)
Chassis Introduction PET Degradation Protein Engineering Conclusion References Directed Evolution Rational Engineering Hybrid Directed Evolution Conclusion • Repassaging of E-15 EG3 • Increased growth rate by 30% • Increased growth yield by 20% 31
Chassis Introduction PET Degradation Protein Engineering Conclusion References Directed Evolution Rational Engineering Hybrid 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 32
Recommend
More recommend
