YesOilYesOil

ENGINEERED YEAST CELLSENGINEERED YEAST CELLS::A Yeast Sensor for real Extra Virgin A Yeast Sensor for real Extra Virgin

Olive OilOlive Oil

Federico II University

Giovanni,engineer

Lucia, mathematician

Maria Aurelia, biologist

Irene, biologist

Alda, biologist

Velia,biologist

Roberta, biologist

Giulia, biologist

We are from…

ITALYITALY

NAPLES

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!

Olive Oil is classified as follows:

[oleic acid] < 2.8 mM EXTRA VIRGIN2.8 mM EXTRA VIRGIN

[oleic acid] < 7.1 mM VIRGIN7.1 mM VIRGIN

[oleic acid] > 7.1 mM NOT EDIBLE 7.1 mM NOT EDIBLE

Background•Oleic acid is the principal component of Olive Oil •It’s the indicator of Oil’s acidity

Oleic acid is yeast

alternative carbon

source in absence

of glucose

OLEIC ACID IS A COMMON POINT!

OLEIC ACID

OLIVE OIL’S

ACIDITY

YEAST METABOLISM

BIOLOGICALBIOLOGICAL MODELMODEL

BIOLOGICAL BIOLOGICAL RESPONSERESPONSE

MATHEMATICAL MATHEMATICAL MODELMODEL

d

dt Pho4p

SA αA [PHO4p]

Pho80d

dtSB αB [PHO80-PHO85]

Pho85p

[PHO80-PHO85]K3

d

dt

Pho8 gfp K3 [PHO4p]

[PHO8]

αC [PHO8]K1K3+K1K2

rfp

SIMULATIONSSIMULATIONS

PROJECTOVERVIEW

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

RFP

WHAT HAPPENS?

2.8mM<[O.A]<7.1mM 2.8mM<[O.A]<7.1mM

Virgin OilVirgin Oil

INPUTS : Oleic acid concentrations

oleic acid concentration is less than less than

2.8 mM 2.8 mM

oleic acid concentration

is greater than greater than 7.1 mM 7.1 mM

Extra Virgin Olive OilExtra Virgin Olive OilNot edible Olive OilNot edible Olive Oil

oleic acid concentration is between between

2.8 and 7.1mM2.8 and 7.1mM

Virgin Olive oilVirgin Olive oil

OUTPUTS

[Oleic acid] < 2,8 mM [Oleic acid] > 7,1 mM

Virgin OilExtra Virgin Olive Oil Not edible Olive Oil

Biological ModelBiological Model

What microorganism?

Which promoters?

Which genes?

Choice of microorganism: Choice of microorganism: S.cerevisiaeS.cerevisiae

1. In yeast genome there are genes activated by Oleic Acid.

2. Yeast is not a dangerous microorganism for consumers that will use the oil

3. Yeast can be easily engineered

Choice of promoters: OREs sequencesWe 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) One oleate response element (ORE)

1ORE

2ORE

2ORE:2ORE:Two sequences of oleate response Two sequences of oleate response

element (OREs) element (OREs)

1. " Fungi and animals may share a 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

Choice of genes :

PHO-patway

PHO4pPHO4p

PHO8 PROMOTERPHO8 PROMOTER

PHO8p-GFPPHO8p-GFP

PHO80p-RFPPHO80p-RFP

PHO85pPHO85p

PHO4pPHO4p

OOLEIC ACID

2ORE PROMOTER FIREFLY LUCIFERASE

Promoter efficiencyWe cloned the luciferase gene downstream of 2ORE

We tested the perfomance of We tested the perfomance of 2ORE-Cyc1 2ORE-Cyc1 via via luc-assayluc-assay

0

200000

400000

600000

800000

1000000

1200000

1400000

Lu

min

escen

ce/m

icro

g

0NT 5mMNT 0 0,2 0,4 0,6 0,8 1 2 3 4 5

oleic acid mM

Luciferase Assay

Extravirg

in Oliv

e Oil

Extravirg

in Oliv

e Oil

Virgin O

live oil

Virgin O

live oil

Mathematical Model• We modeled our biochemical network as a

system 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.

d

dt

PHO85p

PHO8gfp [ Pho4 free ]

K1 + [Pho4 free]αC

[PHO8]

Pho80

d

dt

d

dt

Pho4p

rfp

SA αA[PHO4p]

SB αB[PHO80-PHO85]

Mathematical ModelInputs

Michaelis-Menten Term

Degradations

d

dt

Pho4P

K2 [PHO80-PHO85] [PHO4pfree] K3 [PHO4P]

STEADY STATE ASSUMPTION (since protein-protein interaction is much faster than transcriptional interaction)

[ Pho4 free ]K3 [PHO4p]

K2 [PHO80-PHO85]

Pho4p

PHO8 PHO8 CDS

PHO85p

Pho80

ddt

Pho4pSA αA [PHO4p]

Pho80

ddt

rfp

SB αB [PHO80-PHO85]Pho85p

[PHO80-PHO85]K3

d

dt

Pho8 gfp K3 [PHO4p]

[PHO8]

αC [PHO8]

K1K3+K1K2

Full Mathematical Model

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

0

200000

400000

600000

800000

1000000

1200000

1400000

Lu

min

esce

nce

/mic

rog

0NT 5mMNT 0 0,2 0,4 0,6 0,8 1 2 3 4 5

oleic acid mM

Luciferase Assay

Simulations: good parameters choiceSimulations: good parameters choice

Simulations: bad parameters choiceSimulations: 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.

Our sist

em is R

obust !!!

..also with other values...

Conclusions• 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

• 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

Future work

• 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

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!!”

Registration number Part's Name

BBa_1735007 Pho80CDS

BBa_1735008 1ore-Cyc1 promoter

BBa_1735009 2ore-Cyc1 promoter

BBa_1735010 Fox3 promoter

BBa_1735011 1ore-pho80 cds

Thanks to...

                                                                                                                                                                   

Instructors:Maria Pia CosmaDiego di BernardoMario di Bernardo