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YesOil

YesOil. ENGINEERED YEAST CELLS : A Yeast Sensor for real Extra Virgin Olive Oil. Federico II University. Irene, biologist. Roberta, biologist. Giovanni, engineer. Lucia, mathematician. Velia, biologist. Alda, biologist. Maria Aurelia, biologist. Giulia, biologist.

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  1. YesOil ENGINEERED YEAST CELLS: A Yeast Sensor for real Extra Virgin Olive Oil Federico II University

  2. Irene, biologist Roberta, biologist Giovanni, engineer Lucia, mathematician Velia, biologist Alda, biologist Maria Aurelia, biologist Giulia, biologist

  3. We are from… ITALY NAPLES

  4. Project idea : BIOLOGICAL SENSOR

  5. What’s a sensor? A sensor is a type of transducer that converts a signal from one form to another. In this case we want S.cerevisiae to convert a metabolic signal, that derives from oleic acid concentration, into light!

  6. Background • Oleic acid is the principal component of Olive Oil • It’s the indicator of Oil’s acidity Olive Oil is classified as follows: • [oleic acid] < 2.8 mM EXTRA VIRGIN • [oleic acid] < 7.1 mM VIRGIN • [oleic acid] > 7.1 mM NOT EDIBLE

  7. Oleic acid is yeast alternative carbon source in absence of glucose

  8. OLEIC ACID IS A COMMON POINT! OLEIC ACID OLIVE OIL’S ACIDITY YEAST METABOLISM

  9. Pho80 d Pho85p SB αB [PHO80-PHO85] dt BIOLOGICAL RESPONSE BIOLOGICAL MODEL PROJECTOVERVIEW MATHEMATICAL MODEL SIMULATIONS d Pho4p αA [PHO4p] SA dt rfp Pho8 gfp d [PHO4p] K3 αC [PHO8] [PHO80-PHO85] dt K1K3+K1K2 K3 [PHO8]

  10. BIOLOGICAL CIRCUIT [O.A]<2.8 mM [O.A.]>7.1 mM RFP

  11. WHAT HAPPENS? Virgin Oil Virgin Oil 2.8mM<[O.A]<7.1mM

  12. INPUTS : Oleic acid concentrations oleic acid concentration is greater than 7.1 mM oleic acid concentration is less than 2.8 mM oleic acid concentration is between 2.8 and 7.1mM Virgin Olive oil Extra Virgin Olive Oil Not edible Olive Oil

  13. OUTPUTS [Oleic acid] < 2,8 mM [Oleic acid] > 7,1 mM Virgin Oil Extra Virgin Olive Oil Not edible Olive Oil

  14. Biological Model • What microorganism? • Which promoters? • Which genes?

  15. Choice of microorganism: S.cerevisiae • In yeast genome there are genes activated by Oleic Acid. • Yeast is not a dangerous microorganism for consumers that will use the oil • Yeast can be easily engineered

  16. Choice of promoters: OREs sequences We cloned one ORE sequence and two OREs sequences from the FOX3 gene and we inserted them upstream of a CYC1 promoter. 1ORE 1ORE: One oleate response element (ORE) 2ORE 2ORE: Two sequences of oleate response element (OREs) 1. " Fungi and animals may sharea common ancestor to nuclear receptors " Morag MacLean, Richard J. Fagan, and Didier Picard Chris Phelps, Valentina Gburcik, Elena Suslova, Peter Dudek, Fedor Forafonov, Nathalie Bot,Morag MacLean, Richard J. Fagan, and Didier Picard.PNAS 2006;103;7077-7081

  17. Choice of genes : PHO-patway PHO8p-GFP PHO4p PHO8 PROMOTER PHO80p-RFP PHO4p PHO85p

  18. Promoter efficiency We cloned the luciferase gene downstream of 2ORE O OLEIC ACID FIREFLY LUCIFERASE 2ORE PROMOTER

  19. We tested the perfomance of 2ORE-Cyc1 via luc-assay Virgin Olive oil Extravirgin Olive Oil

  20. Mathematical Model • We modeled our biochemical network as asystem of non linear ODEs assuming that • Component concentrations are continous functions of time; • trascription factor timescales are much larger than protein-protein interactions; • input changes are very rapid; • the system is closed.

  21. Mathematical Model Inputs Degradations Pho4p d SA αA [PHO4p] dt Pho80 rfp SB αB [PHO80-PHO85] d PHO85p dt PHO8 gfp [ Pho4 free ] K1 + [Pho4 free] d αC [PHO8] dt Michaelis-Menten Term

  22. [PHO4p] K3 [ Pho4 free ] [PHO80-PHO85] K2 Pho80 Pho4p PHO85p PHO8 PHO8 CDS Pho4P d [PHO80-PHO85] [PHO4pfree] [PHO4P] K2 K3 dt STEADY STATE ASSUMPTION (since protein-protein interaction is much faster than transcriptional interaction)

  23. Full Mathematical Model Pho4p SA d αA [PHO4p] dt Pho80 rfp SB αB d [PHO80-PHO85] Pho85p dt [PHO4p] Pho8 gfp K3 d αC [PHO8] dt [PHO80-PHO85] [PHO8] K1K3+K1K2 K3

  24. Including Oleic acid into the model • Than we begun analysis; we wanted to have our inputs as functions of Oleic acid concentration. • So we used data from luciferase assay as input data to perform simulations. SA SB

  25. Simulations: good parameters choice

  26. Simulations: bad parameters choice We performed simulations for different values of parameters. K1=0.5 uncorrected behaviour !!! the repression of PHO8 is too weak when [Oleic acid] >7.1 mM.

  27. Our sistem is Robust !!! ..also with other values...

  28. We tested and implemented one promoter (2ORE) able to identify Extravergine Olive Oil We cloned it upstream of the resistence natMX4 in a yeast vector (pAG25) We integrated “GFP-KanMX6” cassette in yeast strain W303 Conclusions • Response to low conc. of oleic acid • The cassette 2ORE-natMX4 is now ready to be integrated • This is going to be our output for extravergin olive oil

  29. Integration of the second promoter “1ORE” Deletion of the endogenous Pho80 promoter from yeast strain W303 via insertion of the hphMX4 resistance Cloning of RFP downstream of 1 ORE- pho80 CDS Future work

  30. Our BioBrick parts • In the Igem Registry 2007 there were nine Yeast parts available but none of them working... Now with our work the total number of yeast parts is fourteen and our five parts are all “ready to use!!”

  31. Thank y u s much!! Instructors: Maria Pia Cosma Diego di Bernardo Mario di Bernardo Thanks to...

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