Recovered file 2igem
- 1. Ethanol, whey not?iGEM Jamboree 2009UNIPV-Pavia
- 2. OutlineWhy whey?Engineering whey fermentation to ethanolPromoters characterizationEthanol production and conclusions
- 3. Motivation: why whey?Cheese Whey is a residue of cheese curdling in dairy industriesIts high nutrition load contributes to the proliferation of water microorganisms that steal oxygen in their environment
- 4. It causes water asphyxia, when introduced into river systems
- 5. It is considered as a special waste for Italian law (B.O.D.5 2000 times higher than legal limit)
- 6. Many industries extract high value substances in order to decrease its pollutant power and to valorize itCheese whey valorizationSubstances of interest: - whey proteins - purified fatty acids - dry whey
- 7. The residual liquid of these treatments is still a special waste for its high lactose content (~4.5%)
- 8. Complete lactose extraction and purification is not convenient
- 9. New valorization techniques should be developedSolution: fermentation of lactose into ethanolEthanol is an important alternative and renewable source of energy
- 10. It is already used as a fuel in some countries such as Brazil and Australia
- 11. It is produced from feedstock such as sugar cane by fermentation
- 12. Glucose can be fermented into ethanol by many microbiota (such as S. cerevisiae)
- 13. Lactose can be easily converted into glucose by some microorganisms (such as E. coli)Idea: whey can be considered as a free feedstock
- 14. design a new biological system able to convert lactose into ethanol with high efficiencyProblem: no wild type organism is able to perform both functions efficiently
- 15. Project overview Lactose cleaving moduleChassis used: E.coliPoPs input Ethanol producing modulePoPs input
- 16. Lactose cleaving moduleLacZB0034Alfa-D-glucoseD-galactoseLactoseE. coliβ-galactosidase breaks lactose with high efficiencyβ-galactosidase overexpression to increase lactose cleaving capabilityB0010B0010
- 17. Ethanol-producing modulepdcadhBB0030Zymomonas mobilis is an ethanologenic bacterium of the soil.Pyruvate decarboxilase (pdc)Alcohol dehydrogenase II (adhB)Genes were designed with codon usage bias optimization.B0030pyruvatepdcacetaldehydeadhBethanolB0010B0010
- 18. E. coli fermentation pathwaysWild typeEngineeredVSTheoreticalyields: 0.51 (gEtOHg glucose-1)
- 19. 0.54 (gEtOHg lactose-1)Inducible promoters usedaTc inducible devices:tetRPtetB0010B0012B00343OC6HSL sensor: BBa_F2620LuxRlux pRB0010B0012B0034pTetLac promoter (BBa_R0011), BBa_J231xxPLacR23118R23100R23101
- 20. Relative Promoter UnitsIt quantifies a promoter activity:The studied promoter expresses a GFP gene
- 21. Negative control is a non fluorescent culture
- 22. Benchmark: BBa_J23101 which leads GFP expression
- 24. Hard model to study. We approximated it to the steady state: Simplified in: Definition:
- 25. μlMeasurement systemTecan Infinite F200 Microplate readerbacterial incubation in multiwell platesfluorescence and absorbance kineticsExperimental setupoptimized for promoter characterizationstandard growth conditionsLocal evaporationthe “frame effect”GFP vs O.D.600serial dilutions of fluorescent bacteriaO.D.600 vs culture concentrationSerial dilutions of bacteriaBacterial growth in microplatevs falcon tube/flask
- 26. Device characterization steps:aTc sensor driven by J23118 promoter
- 28. β-galactosidase activity resultsBeta-gal generator expressed by Ptet (TOP10)Negative control (TOP10 with BBa_B0032)Positive control (BW20767 strain)X-Gal plates confirmed the cleaving capability of the Registry’s β-galactosidaseDynamic tests will be done to check if our system cleaves lactose more rapidly than the wild type one
- 29. Ethanol tolerance in TOP10 E. coliToxicity threshold of ethanol: between 3.5 and 4.5 %w/vEthanol production resultsStrong expression of the operon: small coloniesWeak expression of the operon: normal coloniesMeanofthreegrowthcurves (96-well microplate) in LB+10% glucose: ourengineeredstrainsreachhigherODsthan the negative control
- 30. ConclusionsA complete ethanol producing operon has been assembled and submitted using BioBrick Standard Parts.The feasibility of conversion from lactose to ethanol, by a synthetic biological device has been demonstrated.The capability of engineered E. coli in ethanol production was tested, still best fermentation conditions haven’t been found yet.A set of inducible devices and promoters has been tested, in order to fine regulate gene expression, like a genetic knob.A lot of work has still to be done for:Optimizing gene expression of our ethanol-producing operonSpeeding up the lactose cleaving process in E. coli.
- 31. Acknowledgements
Editor's Notes
- #11: A large part ofour project hasbeendedicatedto the design and characterizationofconstitutive and induciblepromoters in ordertooptimize the regulationofourthreeenzymesof interest.WestudiedtwodifferentaTcinducibledevicesofourownconstruction: tetRis a repressorforPtet promoter, buttetR can beinhibitedbyaTcmolecule. So, whentetRisconstitutivelyproduced, Ptet promoter can beinducedbyaTc, thushaving a complete inducible system. In our project wetrieddifferentconstitutivepromotersupstreamoftetR.Thenweconsideredanexistinginducible part thatis the homoserinelactonereceiverdevice, in whichluxRisconstitutivelyproduced and can turn Plux promoter on, butluxRneeds a co-inducerthatishomoserinelactone, thushavinganotherinducible system.Finallywestudied some constitutivepromoterslikelachybrid promoter and constitutivepromotersfrom Anderson Collection.
- #12: Why standard measurements?The importance of a relative unit: same measurements in different labs.