Team:EPF-Lausanne/Project

From 2012.igem.org

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===The Problem===
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Producing complex therapeutic proteins requires biosynthesis in mammalian cells. This method of expressing proteins presents many obstacles. Our project focuses on one: getting the cell to express the desired protein when want it to. Currently, bioreactors in industry rely heavily on small signaling molecules to get the cells to respond. The time it takes for these molecules to diffuse through the cell culture medium and the subsequent removal they require when the product is purified are huge problems. To allow a finer control over gene expression our project studied the implementation of two light-induced genetic (or optogenetic) switches. Both allow a tight regulation of gene expression and eliminate the problem of removing signaling molecules during the final purification of the protein that was synthesized.
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===The problem===
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===The Simple Switch===
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Producing complex therapeutic proteins requires biosynthesis using mammalian cells to obtain the desired product. This product sometimes has some level of toxicity for the cells, limiting the productivity.
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The first switch we studied is an untested fusion protein designed to act as a light-induced transcriptional activator in mamallian cells. The LovTAP-VP16 protein consists of a Lov2 domain (from ) a Trp repressor (from ) and a VP16 transactional activating domain (from ). A first version of this fusion protein was developed at the university of Chicago and later characterized and used as a bacterial repressor by the EPFL 2009 iGEM. The protein binds to a TRP promoter after undergoing a conformational change when exposed to light. By fusing the Lov-TAP protein to a viral promoter, the idea was to turn the protein into a light activated DNA binding protein. When the TRP promoter is bound to the LovTAP-VP16 protein the viral promoter on the C terminal recruits RNA polymerase II to the site and favors the transcription of the reporter gene next to the TRP promoter.  
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===Our solution===
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This pathway is simple and light activation of LovTAP-VP16 results in direct activation of transcription. There are also major obstacles this approach presents though. The protein needs to be localized in the nucleus, bind well to DNA and long enough to activate transcription. For more information, you can have a look at the [https://2009.igem.org/Team:EPF-Lausanne/LOVTAP EPFL 2009 iGEM team wiki] or at the [http://addrefhere simulation] provided on the modeling page. --> link
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We want to cotransfect a genetic light switch along with the gene coding for a protein of interest whose expression is controlled by exposure to light. The protein of interest will only be synthesized by cells in the presence of blue light, allowing them to grow happily in the dark until the switch is activated. 
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The switch consists of 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 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 blue light, thereby activating expression of the read-out gene.
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===Plan B===
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In addition, we will be recreating the light switch described by Fussenegger et al (1). 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 with CHO cells.
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(1) Ye H, Baba M, Peng R-W, Fussensgger M. A synthetic optogenetic transcription device enhances blood-glucose homeostasis in mice. Science 332, (2011).
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== Project Details==
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===LovTAP===
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LovTAP is a chimeric protein, made from the light-sensitive domain (LOV) and the TrpR DNA-binding domain (TAP).
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LOV stands for Light, Oxygen, Voltage, to which LOV-domain is sensitive. TrpR is acterial transcription factor trp repressor and can bind it's site on DNA as a dimer. In the cell, two LovTAP proteins dimerize, so that the two LovTAP domains are bound together and TrpR domains face the same direction. When the light shines on the LovTAP domain, it changes conformation, so that [put an image to show how it works].
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For more information, you can have a look at the [https://2009.igem.org/Team:EPF-Lausanne/LOVTAP EPFL 2009 iGEM team wiki] or at the [http://addrefhere simulation] provided on the modeling page. --> link
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You can find the annotated protein sequence of LovTAP [http://addref here ]
You can find the annotated protein sequence of LovTAP [http://addref here ]
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===Melanopsin===
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For this system we will be using a DsRed readout to characterize the speed and efficiency of transcription by quantifying fluorescence.
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=== The Experiments ===
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===The Complex Switch===
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In addition to LovTAP switch, we will be realizing another, more complex, melanopsin-based light switch developed by Fussenegger et al. In this switch, a light-sensitive membrane bound protein, melanopsin, triggers a cascade.  The melanopsin opens calcium channels in the cell which activates the NFAT pathway. By inserting a readout gene next to an NFAT promoter, we can promote the expression by triggering the release of calcium with light.  This optogenetic switch takes advantage of an already existing mammallian pathway and limits the potential flaws related to promoting transcription once the pathway is activated. However, calcium is a broad effector and can have unintended consequences. Different cell types also react differently to calcium influx and this approach might not be generalizable. Fussenegger's team did this using HEK (human embryo kidney) cells, and we will also try to ge the pathway to work in CHO cells.
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The main readout we will be using for this pathway is GFP.

Revision as of 09:18, 25 September 2012


The Problem

Producing complex therapeutic proteins requires biosynthesis in mammalian cells. This method of expressing proteins presents many obstacles. Our project focuses on one: getting the cell to express the desired protein when want it to. Currently, bioreactors in industry rely heavily on small signaling molecules to get the cells to respond. The time it takes for these molecules to diffuse through the cell culture medium and the subsequent removal they require when the product is purified are huge problems. To allow a finer control over gene expression our project studied the implementation of two light-induced genetic (or optogenetic) switches. Both allow a tight regulation of gene expression and eliminate the problem of removing signaling molecules during the final purification of the protein that was synthesized.


The Simple Switch

The first switch we studied is an untested fusion protein designed to act as a light-induced transcriptional activator in mamallian cells. The LovTAP-VP16 protein consists of a Lov2 domain (from ) a Trp repressor (from ) and a VP16 transactional activating domain (from ). A first version of this fusion protein was developed at the university of Chicago and later characterized and used as a bacterial repressor by the EPFL 2009 iGEM. The protein binds to a TRP promoter after undergoing a conformational change when exposed to light. By fusing the Lov-TAP protein to a viral promoter, the idea was to turn the protein into a light activated DNA binding protein. When the TRP promoter is bound to the LovTAP-VP16 protein the viral promoter on the C terminal recruits RNA polymerase II to the site and favors the transcription of the reporter gene next to the TRP promoter.

This pathway is simple and light activation of LovTAP-VP16 results in direct activation of transcription. There are also major obstacles this approach presents though. The protein needs to be localized in the nucleus, bind well to DNA and long enough to activate transcription. For more information, you can have a look at the EPFL 2009 iGEM team wiki or at the [http://addrefhere simulation] provided on the modeling page. --> link

You can find the annotated protein sequence of LovTAP [http://addref here ]


For this system we will be using a DsRed readout to characterize the speed and efficiency of transcription by quantifying fluorescence.

The Complex Switch

In addition to LovTAP switch, we will be realizing another, more complex, melanopsin-based light switch developed by Fussenegger et al. In this switch, a light-sensitive membrane bound protein, melanopsin, triggers a cascade. The melanopsin opens calcium channels in the cell which activates the NFAT pathway. By inserting a readout gene next to an NFAT promoter, we can promote the expression by triggering the release of calcium with light. This optogenetic switch takes advantage of an already existing mammallian pathway and limits the potential flaws related to promoting transcription once the pathway is activated. However, calcium is a broad effector and can have unintended consequences. Different cell types also react differently to calcium influx and this approach might not be generalizable. Fussenegger's team did this using HEK (human embryo kidney) cells, and we will also try to ge the pathway to work in CHO cells.

The main readout we will be using for this pathway is GFP.



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