Team:UNIPV-Pavia

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<tr><td align="left" valign="top" width="15%">{{UNIPV-Pavia/menu}}</td>
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<html><a href="https://2010.igem.org/Team:UNIPV-Pavia/Project">
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<img src="https://static.igem.org/mediawiki/2010/3/3d/UNIPV_Pavia10_PROJ_LOGO.png" width="200px" height="100px" title="The Project" alt="The Project"/></a>
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<img src="https://static.igem.org/mediawiki/2010/e/e1/UNUPV_Pavia_Team.jpg" width="140px" height="100px" title="The Team" alt="The Team"/></a>
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<td style="padding:20px"><html><a href="https://2010.igem.org/Team:UNIPV-Pavia/Notebook">
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<img src="https://static.igem.org/mediawiki/2010/0/03/UNIPV_Pavia_agenda2.jpg" width="120px" height="120px" title="Notebook" alt="Notebook"/></a>
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<p align="justify">
<p align="justify">
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Despite the long and successful history of E. coli as a "protein
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Despite the long and successful history of ''E. coli'' as a "protein
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factory", there are still many limitations, in both quantity and
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factory", there are still many limitations in the production process of recombinant proteins.
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quality, affecting the production process of recombinant proteins.
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Efficient expression of the recombinant gene can be achieved by
Efficient expression of the recombinant gene can be achieved by
improving several steps of the production cycle, in order to obtain a
improving several steps of the production cycle, in order to obtain a
much better yield/cost ratio, especially at industrial scale. We
much better yield/cost ratio, especially at industrial scale. We
explored different approaches to these manufacturing steps, coming up
explored different approaches to these manufacturing steps, coming up
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with several possible improvements:
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with several possible improvements.
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<br><br><br>
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<html><font size="3"><i><b>Self-inducible promoters</b></i></font></html>
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<br><br>
<br><br>
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<html><font size="3"><i><b>Self-inducible promoters</b></i></font></html>
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<br>
Expression of the recombinant gene has to be induced at a desired
Expression of the recombinant gene has to be induced at a desired
culture density, in order to ease the burden on the organisms,
culture density, in order to ease the burden on the organisms,
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chemical compound) is added to the culture at the desired growth
chemical compound) is added to the culture at the desired growth
phase, thus triggering protein synthesis. A library of self-inducible
phase, thus triggering protein synthesis. A library of self-inducible
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promoters can be realized and characterized, allowing a degree of
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promoters can be realized and characterized to trigger the protein production without the cost associated to
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control over the time of production without the cost associated to
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other inducible systems.
other inducible systems.
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<br><br><br>
 
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<html><font size="3"><i><b>Integrative standard vectors for E. coli and yeast</b></i></font></html>
 
<br><br>
<br><br>
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<html><font size="3"><i><b>Integrative standard vectors for E. coli and yeast</b></i></font></html>
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<br>
Integration of the recombinant gene or standard part in the genome
Integration of the recombinant gene or standard part in the genome
eliminates the need for antibiotics in cultures for selection,
eliminates the need for antibiotics in cultures for selection,
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explored and tested a method that allows us to integrate a part into
explored and tested a method that allows us to integrate a part into
the genome, with the possibility of building a library of integration
the genome, with the possibility of building a library of integration
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sites for both E. coli and yeast (S. cerevisiae).
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sites for both ''E. coli'' and yeast (S. cerevisiae).
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<br><br><br>
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<html><font size="3"><i><b>Self-cleaving affinity tags to easily purify proteins</b></i></font></html>
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<br><br>
<br><br>
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<html><font size="3"><i><b>Self-cleaving affinity tags to easily purify proteins</b></i></font></html>
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<br>
Purification of the target protein is usually achieved with affinity
Purification of the target protein is usually achieved with affinity
resins or columns, often amounting to a very large fraction of
resins or columns, often amounting to a very large fraction of
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pH/temperature shock, that triggers the self-cleavage of inteins and
pH/temperature shock, that triggers the self-cleavage of inteins and
the release of purified product.
the release of purified product.
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<br><br><br>
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<br><br>
These solutions are modular, easily combinable and provide useful
These solutions are modular, easily combinable and provide useful
BioBricks for other applications.
BioBricks for other applications.
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Latest revision as of 08:06, 25 October 2010


FOCUS ON... The Project The Team Notebook Gallery



Despite the long and successful history of E. coli as a "protein factory", there are still many limitations in the production process of recombinant proteins. Efficient expression of the recombinant gene can be achieved by improving several steps of the production cycle, in order to obtain a much better yield/cost ratio, especially at industrial scale. We explored different approaches to these manufacturing steps, coming up with several possible improvements.

Self-inducible promoters
Expression of the recombinant gene has to be induced at a desired culture density, in order to ease the burden on the organisms, allowing the cultures to grow undisturbed before initiating production. This is usually achieved by controlling protein expression with inducible promoters: an inducer molecule (usually an expensive chemical compound) is added to the culture at the desired growth phase, thus triggering protein synthesis. A library of self-inducible promoters can be realized and characterized to trigger the protein production without the cost associated to other inducible systems.

Integrative standard vectors for E. coli and yeast
Integration of the recombinant gene or standard part in the genome eliminates the need for antibiotics in cultures for selection, lowering relative costs, and leading to a more stable system; we explored and tested a method that allows us to integrate a part into the genome, with the possibility of building a library of integration sites for both E. coli and yeast (S. cerevisiae).

Self-cleaving affinity tags to easily purify proteins
Purification of the target protein is usually achieved with affinity resins or columns, often amounting to a very large fraction of production costs; while many different approaches to purification have been explored in literature, we wanted to combine two promising techniques: PolyhydroxyAlkanoates production in the cytoplasm and an affinity tag system based on PHA-binding proteins (phasins) and self-cleaving protein segments (inteins). PHA granules covered by tagged proteins can be separated from the lysate by simple mechanical means, once again reducing costs and simplifying the process. Then the target protein can be easily separated by PHA granules through a pH/temperature shock, that triggers the self-cleavage of inteins and the release of purified product.

These solutions are modular, easily combinable and provide useful BioBricks for other applications.