Team:EPF-Lausanne

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== The "SWITCH" Project ==
== The "SWITCH" Project ==
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===In a nutshell===
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[[File:File-Team-EPF-Lausanne_main_BD.png|left|frameless|550px]]
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Our project is all about implementing a light activated genetic switch in mammalian cells. The production of complex proteins is becoming an increasingly commonplace task, particularly in the pharmaceutical industry. Mammalian cells are an ideal environment to make these proteins, but producing them at the right time and optimal rate requires an expression system controlled by a specific signal. Until now, these systems have mostly been limited to chemical signals which have many drawbacks. By using a light-activated expression system, we hope to enable mammalian cells to respond to a light signal, rather than a chemical signal, by initiating gene expression for a protein of interest.
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===A new expression system: LovTAP fusion protein===
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[[File:Team-EPF-Lausanne_activation_alt.png|center|thumb|650px| LovTAP-VP16 gets activated by blue light (1.), then binds to the DNA (2.) and activates the transcription (3.)]]
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Our main goal this summer was to implement a previously untested expression system in mammalian cells. The LovTAP-VP16 fusion protein we used is based on a previously tested fusion protein, [http://partsregistry.org/wiki/index.php/Part:BBa_K191006 LovTAP] by the [https://2009.igem.org/Team:EPF-Lausanne 2009 EPF Lausanne team], which binds to a specific sequence of DNA once it is activated with blue light. By attaching an activating domain to the protein that is known to work in mammalian cells, we hoped to enable LovTAP-VP16 to bind DNA after exposure to light and regulate expression of a gene next to its binding site in a mammalian cell line.
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===Another approach: Melanopsin===
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[[File:Team-EPF-Lausanne-melanopsinpathway.JPG|thumb|left|250px|
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[[Team:EPF-Lausanne/References#Fussenegger_2011|Fusseneger 2011 et al.]] ]]
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We also worked with another light-activated expression system that has already been proven to work. Martin Fusseneger et al. have designed a pathway that makes extensive use of gene expression pathways already present in the cell. By adding a light-activated receptor on the cell membrane, they promote the release of calcium into the cytoplasm upon exposure to light. This activates the native cellular response to an increased calcium concentration. Promoter proteins sensitive to calcium will react to this change and start expressing genes with specific promoter sites. By introducing a new gene with a calcium-sensitive promoter next to it, we can use light to induce its expression.
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===The Problem===
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===CHO cells & Co===
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Producing complex therapeutic proteins requires biosynthesis in mammalian cells. The proteins produced can sometimes have some level of toxicity for the cells, limiting the productivity if they are produced constantly and thus accumulating. Additionally, in a bioreactor setup, the synthesis of a particular protein is usually activated upon addition of a particular molecule rendering it hard to control production without interfering in various pathways.
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[[File:Team-EPF-Lausanne transfection graphic.jpg |thumb|right|frameless |300px]]
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CHO (Chinese Hamster Ovary) cells are the workhorses of industrial protein production. The light activated expression systems we worked on would be extremely convenient for production of proteins in an industrial context in these cells. For our project, we transfected these cells with each expression system and conducted a variety of tests. Along with the CHO cell line, we also chose to test these light activated systems using HEK (Human Embryonic Kidney) cells since the melanopsin system had already been shown to work in this cell type. The melanopsin system would provide data to compare to our LovTAP-VP16 system.
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The pharmaceutical industry needs an easier way to induce the production of a specific compound in mammalian cell.
 
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This is where our "SWITCH" project steps in.
 
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===The First Switch===
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In each different cell line, we tested a transfection containing a light-sensitive protein and a target gene or readout. We chose to express easily detectable fluorescent proteins so that we could compare the efficiency of both systems in turning on a particular gene. The fluorescent protein gene could eventually be replaced by a gene coding for a medically-relevant protein of interest to the pharmaceutical industry.
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We want to transfect a light switch along with the gene of interest. This means that the product will only be synthesized by the cells in the presence of blue light, allowing them to grow happily in the dark.
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The switch is based on an already existing chimeric protein, LovTAP. This protein was originally intended to act as a light-induced repressor in bacteria. The EPFL 2009 iGEM team proposed to fuse it with VP16, a viral activator, in order to convert it into a light-induced activator in mammalian cells.
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This year we will try to realize this idea by transfecting CHO (Chinese hamster ovary) cells with two plasmids: one coding for the LovTAP-VP16 fusion protein and another with a read-out ( red fluorescence ) protein preceded by a binding site for LovTAP-VP16. If everything goes as expected, LovTAP-VP16 will only bind the plasmid and activate the production of read-out when exposed to light.
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We will thus be able to induce protein transcription without using any chemical reaction.
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===The Other Switch===
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===Not Only Pharma===
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In addition, we will be realizing the light switch described by Fussenegger et al. In this light switch, light-sensitive melanopsin triggers a cascade involving calcium ion channels that eventually leads to the transcription of the gene of interest. Fussenegger's team did this using HEK (human embryo kidney) cells, and we will also try to make it work on CHO cells.
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[[File:Team-EPF-Lausanne-very motivated cell dressed as hamster.png|150px|right]]
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The use of our switch is not exclusively limited to the pharmaceutical industry. The LovTAP-VP16 switch can also be used in experimental biology to observe temporal gene expression suppression. Our project also has a multitude of other valuable applications, yet achieving them requires comprehension and consent of general public. That's why, in addition to our experimental work, we've presented our project and introduced the public to the field of synthetic biology, and attempted to engage in a two-sided dialog by polling different populations to get their opinion about synthetic biology.
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Check the rest of our wiki to see our project more in detail!
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{{:Team:EPF-Lausanne/Template/Footer}}

Latest revision as of 04:07, 27 September 2012


Contents

The "SWITCH" Project

In a nutshell

File-Team-EPF-Lausanne main BD.png

Our project is all about implementing a light activated genetic switch in mammalian cells. The production of complex proteins is becoming an increasingly commonplace task, particularly in the pharmaceutical industry. Mammalian cells are an ideal environment to make these proteins, but producing them at the right time and optimal rate requires an expression system controlled by a specific signal. Until now, these systems have mostly been limited to chemical signals which have many drawbacks. By using a light-activated expression system, we hope to enable mammalian cells to respond to a light signal, rather than a chemical signal, by initiating gene expression for a protein of interest.

A new expression system: LovTAP fusion protein

LovTAP-VP16 gets activated by blue light (1.), then binds to the DNA (2.) and activates the transcription (3.)


Our main goal this summer was to implement a previously untested expression system in mammalian cells. The LovTAP-VP16 fusion protein we used is based on a previously tested fusion protein, [http://partsregistry.org/wiki/index.php/Part:BBa_K191006 LovTAP] by the 2009 EPF Lausanne team, which binds to a specific sequence of DNA once it is activated with blue light. By attaching an activating domain to the protein that is known to work in mammalian cells, we hoped to enable LovTAP-VP16 to bind DNA after exposure to light and regulate expression of a gene next to its binding site in a mammalian cell line.

Another approach: Melanopsin

Fusseneger 2011 et al.

We also worked with another light-activated expression system that has already been proven to work. Martin Fusseneger et al. have designed a pathway that makes extensive use of gene expression pathways already present in the cell. By adding a light-activated receptor on the cell membrane, they promote the release of calcium into the cytoplasm upon exposure to light. This activates the native cellular response to an increased calcium concentration. Promoter proteins sensitive to calcium will react to this change and start expressing genes with specific promoter sites. By introducing a new gene with a calcium-sensitive promoter next to it, we can use light to induce its expression.

CHO cells & Co

Team-EPF-Lausanne transfection graphic.jpg

CHO (Chinese Hamster Ovary) cells are the workhorses of industrial protein production. The light activated expression systems we worked on would be extremely convenient for production of proteins in an industrial context in these cells. For our project, we transfected these cells with each expression system and conducted a variety of tests. Along with the CHO cell line, we also chose to test these light activated systems using HEK (Human Embryonic Kidney) cells since the melanopsin system had already been shown to work in this cell type. The melanopsin system would provide data to compare to our LovTAP-VP16 system.



In each different cell line, we tested a transfection containing a light-sensitive protein and a target gene or readout. We chose to express easily detectable fluorescent proteins so that we could compare the efficiency of both systems in turning on a particular gene. The fluorescent protein gene could eventually be replaced by a gene coding for a medically-relevant protein of interest to the pharmaceutical industry.

Not Only Pharma

Team-EPF-Lausanne-very motivated cell dressed as hamster.png

The use of our switch is not exclusively limited to the pharmaceutical industry. The LovTAP-VP16 switch can also be used in experimental biology to observe temporal gene expression suppression. Our project also has a multitude of other valuable applications, yet achieving them requires comprehension and consent of general public. That's why, in addition to our experimental work, we've presented our project and introduced the public to the field of synthetic biology, and attempted to engage in a two-sided dialog by polling different populations to get their opinion about synthetic biology.

Check the rest of our wiki to see our project more in detail!

Wiki Links

Important pages

Pages

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