Team:Valencia/WetLab/YeastTeam

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== '''WetLab Overview''' ==
== '''WetLab Overview''' ==
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<span style="color:black; align:justify; font-size:11pt; font-family: Verdana">At WetLab we are working with '''yeasts''' in order to create each and everyone of the cell-pixels of our bioscreen. Since we want to make yeasts shine, we are using an aequorin-transformed yeast strains.
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The project our team focused on was about '''electrically controlling genetically engineered cells to induce a desired behaviour'''. In particular, our project has achieved '''the ambitious goal of making Light Emitting Cells (LECs)'''.
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<span style="color:black; align:justify; font-size:11pt; font-family: Verdana">'''Aequorin''' is a luminiscent protein that originally was isolated from luminescent jellyfish ''Aequorea''. It can also find in other species of ''Aequorea'' and in many other marine organisms. For more information about de discovery of aequorin, read [https://2009.igem.org/Team:Valencia/A_short_story '''A short story of Aequorin'''], written by [https://2009.igem.org/Team:Valencia/A_short_story/Osamu Osamu Shimomura]. This is an interesting and entertaining text. You should read it. No doubt!
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In order to do so, we have worked with a bioluminiscent protein called aequorin. '''Aequorin''' is present in jellyfish and it is responsible of the emission of light by these marine invertebrates. In order for aequorin to produce light, this protein has to be coupled to '''a prostetic group: coelenterazine'''. Coelenterazine is oxidized when '''calcium ions''' bind to the aequorin-coelenterazine complex and, as a result, light is emitted. '''This is not a fluorescent reaction''', as aequorin does not need to be excited with light, it is bioluminescence.
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<html><center><img src="https://static.igem.org/mediawiki/2009/9/96/Aquorin_reaction.gif" WIDTH="464" HEIGHT="268"></center></html>
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<span style="color:black; align:justify; font-size:11pt; font-family: Verdana">To make light, Aequorin uses '''coelenterazine''' as its cofactor. Aequorin also needs the binding of Ca<SUP>2+</SUP> to produce light. You can see the complete reaction in the next picture:
 
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We did not want to work with jellyfishes in the lab, we used budding yeast, ''Saccharomyces cerevisiae'', for our experiments, as it is easier to implement in a biological screen. A genetically modified yeast strain expressing aequorin was used. '''If grown with the prosthetic group, coelenterazine, this strain has been reported to produce light''' through a calcium channel-based system in response to a chemical stimulation (namely KOH).
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We started to study those works, but screens are controled electrically, so '''our aim was to control electrically our LECs'''. Therefore, we induced a '''membrane depolarization by supplying transient electricity to our yeasts''', thus opening voltage-dependent calcium channels to produce a calcium entry to cytosol and, as a result, '''get light emission from aequorin'''.
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[[Image:Aquorin reaction.gif|500px]]
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In summary, we have '''designed and constructed a yeast-based system for the production of light through a jellyfish protein sensitive to calcium'''. Since calcium enters the cytoplasm through voltage-dependent channels, '''it is possible to have light emission under electrical control'''. We have shown that [https://2009.igem.org/Team:Valencia/WetLab/YeastTeam/Results this is possible]; over and above '''we have proved that having a biological display made of aequorin-expressing yeasts as biological pixels is [https://2009.igem.org/Team:Valencia/Hardware/iLCD also possible]'''.
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Some interesting bibliography used to develop this project and that can be very interesting can be found [https://2009.igem.org/wiki/index.php?title=Team:Valencia/experimentalbiblio here]
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<b>[https://2009.igem.org/Image:Aquorin_val.JPG Aequorin]</b> + <b>[https://2009.igem.org/Image:Coelenterazine.png Coelenterazine]</b> + Ca<SUP>2+</SUP> -->  [[Image:Aquorin in action.jpg|350px]]
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<span style="color:black; align:justify; font-size:11pt; font-family: Verdana">We will use a '''chemical input''' like KOH (alkali shock) to open '''Ca<SUP>2+</SUP> channels''' in the yeasts' membrane (Viladevall L, et al. J Biol Chem. (2004) 279 43614–43624), then Ca<SUP>2+</SUP> will enter into the cells and we will get '''light''' as an '''output'''. We have a [https://2009.igem.org/Team:Valencia/WetLab/YeastTeam/Protocols protocol] and diferent [https://2009.igem.org/Team:Valencia/WetLab/YeastTeam/Protocols#Different experimental designs] to try to reproduce the Arinyo's experiment.
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<span style="color:black; align:justify; font-size:11pt; font-family: Verdana">If we succed in this, we will try to reproduce the same response with an '''electrical stimulus''', using some hardware built by ourselves. We will use an electrical input to open the Ca<SUP>2+</SUP> channels. We hope that we see light by this metod.
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Latest revision as of 03:18, 22 October 2009



WetLab Overview


The project our team focused on was about electrically controlling genetically engineered cells to induce a desired behaviour. In particular, our project has achieved the ambitious goal of making Light Emitting Cells (LECs).

In order to do so, we have worked with a bioluminiscent protein called aequorin. Aequorin is present in jellyfish and it is responsible of the emission of light by these marine invertebrates. In order for aequorin to produce light, this protein has to be coupled to a prostetic group: coelenterazine. Coelenterazine is oxidized when calcium ions bind to the aequorin-coelenterazine complex and, as a result, light is emitted. This is not a fluorescent reaction, as aequorin does not need to be excited with light, it is bioluminescence.


We did not want to work with jellyfishes in the lab, we used budding yeast, Saccharomyces cerevisiae, for our experiments, as it is easier to implement in a biological screen. A genetically modified yeast strain expressing aequorin was used. If grown with the prosthetic group, coelenterazine, this strain has been reported to produce light through a calcium channel-based system in response to a chemical stimulation (namely KOH).

We started to study those works, but screens are controled electrically, so our aim was to control electrically our LECs. Therefore, we induced a membrane depolarization by supplying transient electricity to our yeasts, thus opening voltage-dependent calcium channels to produce a calcium entry to cytosol and, as a result, get light emission from aequorin.

In summary, we have designed and constructed a yeast-based system for the production of light through a jellyfish protein sensitive to calcium. Since calcium enters the cytoplasm through voltage-dependent channels, it is possible to have light emission under electrical control. We have shown that this is possible; over and above we have proved that having a biological display made of aequorin-expressing yeasts as biological pixels is also possible.

Some interesting bibliography used to develop this project and that can be very interesting can be found here




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