Naples

From 2007.igem.org

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('''Materials & Methods''')
 
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== '''About Us''' ==  
== '''About Us''' ==  
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[[more pictures]]
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[[images from Naples]]
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== '''Tigem'''==
== '''Tigem'''==
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[[Image:Tigem.jpg|left|460px]] The Telethon Institute of Genetics and Medicine (TIGEM)[http://www.tigem.it]  
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was created by the Italian Telethon Foundation in 1994. TIGEM's purpose is understanding pathogenic mechanisms of genetic diseases. The final aim is developing preventive and therapeutic strategies. The research centre currently hosts 17 research groups, and a total of more than 120 people, including students, postdoctoral fellows, technicians, and administrators. It offers training programs in human medical genetics and Synthetic Biology in collaboration with the University of Naples Federico II.
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              [[Image:Tigem.jpg|center]]  
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The Telethon Institute of Genetics and Medicine (TIGEM)[http://www.tigem.it]  
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was created by the Italian Telethon Foundation in 1994. TIGEM mission is the understanding of the pathogenic mechanisms of genetic diseases with the aim of developing preventive and therapeutic strategies.TIGEM currently hosts 17 research groups, and a total of more than 120 persons, including students, postdoctoral fellows, staff scientists, technicians, and administrators and offers training programs in medical human genetics and Synthetic Biology in cooperation with the University of Naples Federico II.
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== '''University of Naples "Federico II"'''==   
== '''University of Naples "Federico II"'''==   
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[[Image:Uni.jpg|left|110px]]
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 +
University of '''Naples''' "Federico II"[http://www.international.unina.it/] was founded by the King of "Sacro Romano Impero" Federico II of Svevia. It is considered one of the oldest University of Europe. It consists of 13 departments divided in three areas: Sciences and Technologies, Humanistic and Social, Medicine.
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[[Image:Uni.jpg|center]]
 
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University of Naples "Federico II"[http://www.international.unina.it/] was established by the King of "Sacro Romano Impero" Federico II of Svevia. It's considered the most ancient public school of the world. It consists of 13 departments divided in three areas: Sciences and technologies, humanistic and social, medicine.
 
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=='''Our Project - [[YeSOil]]: A Yeast Sensor for real Extra Virgin Olive oil'''==               
 +
[[image:logo2.jpg|center|160px]]
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The aim of our project is to engineer a synthetic biological network in yeast. This system will help in evaluating the quality of olive oil, one of the wordly famous product of Italy [http://en.wikipedia.org/wiki/Italy]. Detection of oil quality is now possible only through expensive and bulky machines. In order to render this process easy and cheap we will modify Saccharomyces cerevisiae cells so that they will act as sensors and indicators of different oleate concentrations.
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=='''Our Project - YeSOil: A Yeast Sensor for real Extra Virgin Olive oil'''==                [[image:yesoil.jpg|left|150px]]
 
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The aim of our project is to engineer a synthetic biological network in yeast  able to detect the quality of olive of oil, one of the most famouse product of Italy [http://en.wikipedia.org/wiki/Italy], now possible only through expensive and not-portable machines. In order to achieve this, we will modify Saccharomyces cerevisiae cells so that they will be able to change colour at different oleate concentrations.
 
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==='''System model''' === 
 
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After some ''brainstorming'' we had this idea:
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==='''System Model''' === 
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In the picture the genes are represented with their interactions. The transcription factor for PHO4p is activated when there is a low oleic acid concentration, i.e. ''extra virgin'', while PHO80 gene is activated when the oleic acid concentration is high, i.e. not edible oil. When PHO4p is activated it transcripts PHO8 which is integrated with GFP, indicating that the oil is ''extra virgin''. When PHO80 is activated by the ''not edible oil promoter'' it creates a complex with PHO85: PHO80-PHO85. PHO80PHO85 phosforilates PHO4p and so it inhibits the trascription of PHO8. RFP is integrated with PHO80, indicating when the oil is not edible. When the level of oleic acid concentration is between extra virgin and not edible, the output will be a mix of green and red fluorescence.
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After some brainstorming we had this idea!!!
 +
 +
[[image:Circuito3.jpg|centre|thumb|580px|YeSOil circuit]]
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The input to the system will be the level of oleic acid that will drive expression from appropriate promoters responsive to oleic acid cloned upstream of Pho80Pho85 and Pho4.
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The whole circuit is based on the reaction of the transcription factor for PHO4p which is activated when there is a low oleic acid concentration,
 +
i.e. ''extra virgin olive oil'', while PHO80 gene is activated when the oleic acid concentration is high, i.e. ''not edible oil''.
 +
When PHO4p is activated PHO8, which is integrated with GFP, is expressed: cells turn green indicating that the oil is ''extra virgin''. When PHO80 is transcribed by the ''not edible oil promoter'' it creates a complex with PHO85: PHO80-PHO85. PHO80PHO85 phosforilates PHO4p inhibiting the trascription of PHO8. As PHO80 is integrated with RFP, when it is expressed cells turn red, indicating that the oil is not edible. When the level of oleic acid concentration is between extra virgin and not edible, the output will be a mix of green and red fluorescence: yellow-orange.
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                    [[image:Circuito3.jpg|left|thumb|580px|YeSOil circuit]]
+
The input of the system will be the level of oleic acid that will drive expression from appropriate promoters responsive to oleic acid cloned upstream of Pho80Pho85 and Pho4.
 +
                 
          
          
-
We recall that, for 100 gr of the oil, it will be:
+
We recall that, for 100 gr of the oil, oil will be classified as:
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*''extra virgin'', if the oleic acid conentration will be less than 0.8 gr
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*''extra virgin'', if the oleic acid conentration is less than 0.8 gr
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*''virgin'', if oleic the acid concentration will be less than 2 gr
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*''virgin'', if oleic the acid concentration is less than 2 gr
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*''not edible'', if the oleic acid concentration will be greater than 3-4 gr
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*''not edible'', if the oleic acid concentration is greater than 3-4 gr
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*the oil is ''virgin'' if the oleic acid concentration is less than 7.1 mM
*the oil is ''virgin'' if the oleic acid concentration is less than 7.1 mM
*the oil is ''not edible'' if the oleic acid concentration is greater than 7.1 mM
*the oil is ''not edible'' if the oleic acid concentration is greater than 7.1 mM
-
Oleate is the principal olive oil element and acidity indicator.
 
-
The olive oil is defined "extra vergine" if it has an acidity lower than 0.8 %,"vergine" with an acidity lower than 2% and not edible if has an acidity higher than 3%.[http://en.wikipedia.org/wiki/Olive_oil]
 
-
Oleate induces the transcription of genes involved in peroxisome biogenesis and stimulates the
 
-
proliferation of these organelles in Saccharomyces cerevisiae. Fatty acid-mediated induction is based on a dramatic increase in transcription of several genes encoding peroxisomal functions due to the presence of an oleate response element (ORE) in their promoters.This upstream activating sequence is minimally defined by an inverted repeat of CGG triplets separated by a 15-18-nucleotide spacer. It constitutes the binding target for the transcription factors Oaf1p and Pip2p.
 
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[[Image:tfOre.jpg|center|350px]]
 
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----
 
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==='''Mathematical Model'''===
 
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Over the past decades progress in measurement of rates and interactions of molecular and cellular processes has initiated a revolution in understanding of dynamical phenomena in cells. Generally speaking a ''dynamical phenomenon'' is a process that changes over time. Living cells are inherently dynamic! Indeed, to sustain the characteristic features of life (growth, cell division...) they need to extract and transform energy from their surroundings. This implies that cells function thermodinamically as open ''systems''. So, we have encountered a new keyword: system. The most general definition for system is the following: ''a set of functional elements joint together to perform a specific task''. Cells are astoundingly complex systems: they contain networks of thousands of biochemical interactions.
 
-
System-level understanding, the approach of systems biology, requires a ''shift'' in the notion of ''what to look for''. An understanding of genes and proteins is very important, but now the focus is on understanding system' structure and dynamics. Biologists use ''cartoons'' to capture the complexity of the networks, but because a system is not just an assembly of genes and proteins, its properties cannot be fully understood by drawing these diagrams. They, of course, represent a first step in our modeling, but can be compared to ''static roadmap'', whereas what we really seek to know are the traffic patterns, why they emerge, how to control them. So, we will use a typical approach from systems and control theory.
 
 +
[[Image:tfOre.jpg|right|420px]]
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[[Image:assunzioni.png|left|100px]]
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Oleate is the principal olive oil element and acidity indicator.
 +
The olive oil is defined "extra vergine" if it has an acidity lower than 0.8 %,
 +
"vergine" with an acidity lower than 2% and not edible if has an acidity higher than 3%.[http://en.wikipedia.org/wiki/Olive_oil]
 +
Oleate induces the transcription of genes involved in peroxisome biogenesis and stimulates the
 +
proliferation of these organelles in Saccharomyces cerevisiae.
 +
Fatty acid-mediated induction is based on a dramatic increase in transcription of several genes encoding peroxisomal functions due to the presence of an oleate response element (ORE) in their promoters.This upstream activating sequence is minimally defined by an inverted repeat of CGG triplets separated by a 15-18-nucleotide spacer.
 +
It constitutes the binding target for the transcription factors Oaf1p and Pip2p.
-
[[Basic assumptions]]
 
 +
----
 +
==='''Mathematical Model'''===
-
 
+
Over the past decades progress in measurement of rates and interactions of molecular and cellular processes has initiated a revolution in understanding of dynamical phenomena in cells. Generally speaking a ''dynamical phenomenon'' is a process that changes over time. Living cells are inherently dynamic! Indeed, to sustain the characteristic features of life (growth, cell division...) they need to extract and transform energy from their surroundings. This implies that cells function thermodinamically as open ''systems''. So, we have encountered a new keyword: system. The most general definition for system is the following: ''a set of functional elements joint together to perform a specific task''. Cells are astoundingly complex systems: they contain networks of thousands of biochemical interactions.
-
[[Image:modello matematico.jpg|left|100px]]
+
System-level understanding, the approach of systems biology, requires a ''shift'' in the notion of ''what to look for''. An understanding of genes and proteins is very important, but now the focus is on understanding system' structure and dynamics. Biologists use ''cartoons'' to capture the complexity of the networks, but because a system is not just an assembly of genes and proteins, its properties cannot be fully understood by drawing these diagrams. They, of course, represent a first step in our modeling, but can be compared to ''static roadmap'', whereas what we really seek to know are the traffic patterns, why they emerge, how to control them. So, we will use a typical approach from systems and control theory.
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[[Mathematical model]]
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[[Image:ing.gif|left|100px]]
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[[System analysis and simulations]]
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[[Image:assunzioni.png|110px]]  [[Basic assumptions]]  [[Image:modello matematico.jpg|100px]]  [[Mathematical model]]  [[Image:ing.gif|100px]]    [[System analysis and simulations]]
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We have adopted a strategy of parallel cloning.
We have adopted a strategy of parallel cloning.
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A reporter gene is cloned in parallel into a vector containing one and two tandem copies of the oleate response elements from the FOX3 gene.
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A reporter gene is cloned in parallel into a vector containing one and two tandem copies of the oleate response elements from the FOX3 promoter.
'''Background'''
'''Background'''
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*[[Restriction Enzyme]]  
*[[Restriction Enzyme]]  
*[[Primers Design]]
*[[Primers Design]]
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 +
 +
 +
'''Yeast integration'''
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*[[Integration strategies]]
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*[[Primers Design For Yeast Integration]]
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'''Cloning Process'''
'''Cloning Process'''
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*[[Mini and Midi prep]]
*[[Mini and Midi prep]]
*[[Transformation]]
*[[Transformation]]
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*[[Yeast Integration]]
 
-
== '''Experimental Results''' ==
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 +
----
 +
 
 +
=== '''Experimental Results'''===
*[[Luciferase assay]]
*[[Luciferase assay]]
*[[Cloning in BioBrick vectors]]
*[[Cloning in BioBrick vectors]]
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*[[Cloning in yeast vector]]
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Latest revision as of 18:49, 25 October 2007

NAPOLI ariprova.jpg


Contents

About Us

TUTTI3.jpgCapi2.jpg

Students:

Instructors

  • Diego di Bernardo
  • Maria Pia Cosma
  • Mario di Bernardo

Advisor


more pictures

images from Naples


Tigem

Tigem.jpg
The Telethon Institute of Genetics and Medicine (TIGEM)[http://www.tigem.it]

was created by the Italian Telethon Foundation in 1994. TIGEM's purpose is understanding pathogenic mechanisms of genetic diseases. The final aim is developing preventive and therapeutic strategies. The research centre currently hosts 17 research groups, and a total of more than 120 people, including students, postdoctoral fellows, technicians, and administrators. It offers training programs in human medical genetics and Synthetic Biology in collaboration with the University of Naples Federico II.



University of Naples "Federico II"

Uni.jpg


University of Naples "Federico II"[http://www.international.unina.it/] was founded by the King of "Sacro Romano Impero" Federico II of Svevia. It is considered one of the oldest University of Europe. It consists of 13 departments divided in three areas: Sciences and Technologies, Humanistic and Social, Medicine.




Our Project - YeSOil: A Yeast Sensor for real Extra Virgin Olive oil

Logo2.jpg

The aim of our project is to engineer a synthetic biological network in yeast. This system will help in evaluating the quality of olive oil, one of the wordly famous product of Italy [http://en.wikipedia.org/wiki/Italy]. Detection of oil quality is now possible only through expensive and bulky machines. In order to render this process easy and cheap we will modify Saccharomyces cerevisiae cells so that they will act as sensors and indicators of different oleate concentrations.



System Model

After some brainstorming we had this idea!!!

YeSOil circuit

The whole circuit is based on the reaction of the transcription factor for PHO4p which is activated when there is a low oleic acid concentration, i.e. extra virgin olive oil, while PHO80 gene is activated when the oleic acid concentration is high, i.e. not edible oil. When PHO4p is activated PHO8, which is integrated with GFP, is expressed: cells turn green indicating that the oil is extra virgin. When PHO80 is transcribed by the not edible oil promoter it creates a complex with PHO85: PHO80-PHO85. PHO80PHO85 phosforilates PHO4p inhibiting the trascription of PHO8. As PHO80 is integrated with RFP, when it is expressed cells turn red, indicating that the oil is not edible. When the level of oleic acid concentration is between extra virgin and not edible, the output will be a mix of green and red fluorescence: yellow-orange.

The input of the system will be the level of oleic acid that will drive expression from appropriate promoters responsive to oleic acid cloned upstream of Pho80Pho85 and Pho4.




We recall that, for 100 gr of the oil, oil will be classified as:

  • extra virgin, if the oleic acid conentration is less than 0.8 gr
  • virgin, if oleic the acid concentration is less than 2 gr
  • not edible, if the oleic acid concentration is greater than 3-4 gr


Now, we need to convert gr in mol and we found that:

  • the oil is extra virgin if the oleic acid concentration is less than 2.8 mM
  • the oil is virgin if the oleic acid concentration is less than 7.1 mM
  • the oil is not edible if the oleic acid concentration is greater than 7.1 mM



TfOre.jpg


Oleate is the principal olive oil element and acidity indicator. The olive oil is defined "extra vergine" if it has an acidity lower than 0.8 %, "vergine" with an acidity lower than 2% and not edible if has an acidity higher than 3%.[http://en.wikipedia.org/wiki/Olive_oil] Oleate induces the transcription of genes involved in peroxisome biogenesis and stimulates the proliferation of these organelles in Saccharomyces cerevisiae. Fatty acid-mediated induction is based on a dramatic increase in transcription of several genes encoding peroxisomal functions due to the presence of an oleate response element (ORE) in their promoters.This upstream activating sequence is minimally defined by an inverted repeat of CGG triplets separated by a 15-18-nucleotide spacer. It constitutes the binding target for the transcription factors Oaf1p and Pip2p.





Mathematical Model

Over the past decades progress in measurement of rates and interactions of molecular and cellular processes has initiated a revolution in understanding of dynamical phenomena in cells. Generally speaking a dynamical phenomenon is a process that changes over time. Living cells are inherently dynamic! Indeed, to sustain the characteristic features of life (growth, cell division...) they need to extract and transform energy from their surroundings. This implies that cells function thermodinamically as open systems. So, we have encountered a new keyword: system. The most general definition for system is the following: a set of functional elements joint together to perform a specific task. Cells are astoundingly complex systems: they contain networks of thousands of biochemical interactions. System-level understanding, the approach of systems biology, requires a shift in the notion of what to look for. An understanding of genes and proteins is very important, but now the focus is on understanding system' structure and dynamics. Biologists use cartoons to capture the complexity of the networks, but because a system is not just an assembly of genes and proteins, its properties cannot be fully understood by drawing these diagrams. They, of course, represent a first step in our modeling, but can be compared to static roadmap, whereas what we really seek to know are the traffic patterns, why they emerge, how to control them. So, we will use a typical approach from systems and control theory.


Assunzioni.png Basic assumptions Modello matematico.jpg Mathematical model Ing.gif System analysis and simulations



Yeast Strain

Yeast strain used is W303.

All DNA manipulations and subcloning were done in Escherichia coli


Materials & Methods

We have adopted a strategy of parallel cloning.

A reporter gene is cloned in parallel into a vector containing one and two tandem copies of the oleate response elements from the FOX3 promoter.

Background

References

Cloning Strategies in E.coli


Cloning Strategies in S.cerevisiae


Yeast integration


Cloning Process



Experimental Results



Thanks to...

Synbiocomm.jpg Bandiera comunita europea.jpg

We are partly funded by the European Union SYNBIOCOMM project