Naples

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Revision as of 08:34, 17 October 2007

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

• Giulia Cuccato

Tigem

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.

University of Naples "Federico II"

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.

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.

System Model

After some brainstorming we had the idea represented in the picture.

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.

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.

YeSOil circuit

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

• extra virgin, if the oleic acid conentration will be less than 0.8 gr
• virgin, if oleic the acid concentration will be less than 2 gr
• not edible, if the oleic acid concentration will be 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 gene.

Background

References

Cloning Strategies in E.coli

• Biobrick Vector choice
• Biobrick Restriction Enzyme
• Biobrick Primers Design

Cloning Strategies in S.cerevisiae

• Vector choice
• Restriction Enzyme
• Primers Design

Cloning Process

• Yeast DNA extraction
• PCR
• Agarose Gel Electrophoresis
• PCR Purification
• Digestion
• Extraction from Gel
• Ligation
• E.coli Transformation
• Mini and Midi prep
• Transformation
• Yeast Integration

Experimental Results

• Luciferase assay
• Cloning in BioBrick vectors
• Cloning in yeast vector

Thanks to...

We are partly funded by the European Union SYNBIOCOMM project