Naples
From 2007.igem.org
(→'''Yeast Strain''') |
(→'''Materials & Methods''') |
||
| (77 intermediate revisions not shown) | |||
| Line 1: | Line 1: | ||
| - | [[Image:NAPOLI_ariprova.jpg]] | + | [[Image:NAPOLI_ariprova.jpg|center|420px]] |
| + | |||
| + | |||
== '''About Us''' == | == '''About Us''' == | ||
| Line 26: | Line 28: | ||
| + | [[more pictures]] | ||
| + | |||
| + | [[images from Naples]] | ||
---- | ---- | ||
| - | == '''Tigem''' | + | == '''Tigem'''== |
| - | The Telethon Institute of Genetics and Medicine (TIGEM)[http://www.tigem.it] | + | |
| - | was created by the Italian Telethon Foundation in 1994. TIGEM | + | [[Image:Tigem.jpg|left|460px]] 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"''' | + | == '''University of Naples "Federico II"'''== |
| - | + | [[Image:Uni.jpg|left|110px]] | |
| - | + | ||
| - | |||
| - | |||
| - | |||
| - | + | 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'''== |
| + | [[image:logo2.jpg|center|160px]] | ||
| + | |||
| + | 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!!! |
| + | |||
| + | [[image:Circuito3.jpg|centre|thumb|580px|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 | ||
| - | |||
| + | [[Image:tfOre.jpg|right|420px]] | ||
| + | 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. | ||
| + | [[Image:assunzioni.png|110px]] [[Basic assumptions]] [[Image:modello matematico.jpg|100px]] [[Mathematical model]] [[Image:ing.gif|100px]] [[System analysis and simulations]] | ||
| Line 124: | Line 124: | ||
---- | ---- | ||
| - | == '''Yeast Strain''' == | + | ==='''Yeast Strain''' === |
Yeast strain used is [[W303]]. | Yeast strain used is [[W303]]. | ||
All DNA manipulations and subcloning were done in [[Escherichia coli]] | All DNA manipulations and subcloning were done in [[Escherichia coli]] | ||
| - | |||
---- | ---- | ||
| - | == '''Materials & Methods''' == | + | === '''Materials & Methods''' === |
We have adopted a strategy of parallel cloning. | 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 | + | 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''' | ||
| Line 151: | Line 150: | ||
*[[Restriction Enzyme]] | *[[Restriction Enzyme]] | ||
*[[Primers Design]] | *[[Primers Design]] | ||
| + | |||
| + | |||
| + | |||
| + | '''Yeast integration''' | ||
| + | *[[Integration strategies]] | ||
| + | *[[Primers Design For Yeast Integration]] | ||
| + | |||
| + | |||
'''Cloning Process''' | '''Cloning Process''' | ||
| Line 164: | Line 171: | ||
*[[Transformation]] | *[[Transformation]] | ||
| - | == '''Experimental Results''' == | + | |
| + | ---- | ||
| + | |||
| + | === '''Experimental Results'''=== | ||
*[[Luciferase assay]] | *[[Luciferase assay]] | ||
*[[Cloning in BioBrick vectors]] | *[[Cloning in BioBrick vectors]] | ||
| - | + | ||
| + | |||
| + | |||
| + | ---- | ||
== '''Thanks to...''' == | == '''Thanks to...''' == | ||
Latest revision as of 18:49, 25 October 2007
Contents |
About Us
Students:
- Giovanni Russo
- Lucia Marucci
- Velia Siciliano
- Irene Cantone
- Roberta Bergamasco[1]
- Maria Aurelia Ricci
- Mafalda Graziano
Instructors
- Diego di Bernardo
- Maria Pia Cosma
- Mario di Bernardo
Advisor
Tigem
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"
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
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!!!
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
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.
Basic assumptions 
Mathematical model 
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
Cloning Strategies in E.coli
Cloning Strategies in S.cerevisiae
Yeast integration
Cloning Process
- Yeast DNA extraction
- PCR
- Agarose Gel Electrophoresis
- PCR Purification
- Digestion
- Extraction from Gel
- Ligation
- E.coli Transformation
- Mini and Midi prep
- Transformation
Experimental Results
Thanks to...
We are partly funded by the European Union SYNBIOCOMM project
