Team:Valencia/WetLab/YeastTeam

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== '''Yeast Team''' ==
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An important part of our project is to work with '''yeasts''' in order to create each and everyone of the cell-pixels of our bioscreen. We will use some aequorin-transformed yeast strains.
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'''Aequorin''' is a luminiscent protein isolated from luminescent jellyfish and many other marine organisms that uses '''coelenterazine''' as its cofactor to produce light whenever Ca<SUP>2+</SUP> is bound to it. Using a '''chemical input''' (KOH, alkali shock) we will open '''Ca<SUP>2+</SUP> channels''' in the yeasts' membrane (Viladevall L, et al. J Biol Chem. (2004) 279 43614–43624), Ca<SUP>2+</SUP> will enter into the cells and we will get '''light''' as an '''output'''.
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Later we will try to reproduce the same response with an '''electrical stimulus''', using some hardware built by ourselves.
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== '''WetLab Overview''' ==
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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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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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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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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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[https://2009.igem.org/Team:Valencia/TeamVal https://static.igem.org/mediawiki/2009/2/20/PolaroidTeam.png]
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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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