Team:Valencia/WetLab/YeastTeam/Results

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<br>
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='''Experimental results'''=
<br>
<br>
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Our ultimate goal was to make a bio-screen made with cellular pixels (LECs). But, before to be able to build this iLCD, we had to study the behaviour of one single LEC. Therefore, we focused on the electrical excitation of our transformed yeasts.
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[under construction]
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This is the behaviour that stands as the cornerstone of our project. '''With a serie of voltage inputs, we accomplished a series of light emissions'''. No exciting light was needed, only aequorin, coelenterazine (the prosthetic group) and Ca<sup>2+</sup>.
-
The last goal of our project is to make a bio-screen made with cell pixels as we have described. But, before to be able to build this iLCD, we had to characterize the cell light emission if we wanted to control it better.
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<table>
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We thought about two possible ways to make cells produce light: First, the producion of light with a chemical imput and, second, making the cells glow with electricity
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    <tr>
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        <td>[[Image:Valencia_Grafica_continu_2_pics.jpg|500 px|left]]</td>
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        <td>[[Image:Bursts.gif|100 px|center]]</td>
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    </tr>
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</table>
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<br>
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Arrows indicate when voltage was applied: 16V during 5 seconds applied at 300 seconds (5 minutes) and 600 seconds (10 minutes). Movie on the right shows how the response would be seen in a pixel.
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===Chemical input.===
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*'''WT''' indicates our LEC: our aequorin-engineered yeast, with coelenterazine and wild-type calcium channels
 +
*'''WT -coe''' indicates the same yeast, without the addition of coelenterazine.
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In order to make our yeasts produce light, we firstly reproduce experiments made by Viladevall et al, After a lot of different tries, we finally could characterize the luminiscence curve in a discontinuos luminometer.  
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As we can observe, '''only our LEC is excited when electric current is applied'''. Withour coelenterazine there is no response to that stimulus.
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[[Image:Comparació koh.jpg|500px]]
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==Conclusions of the study==
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<br>
 +
We have investigated a set of '''different voltages and times in order to precisely know how our system respons to electric current. Knowing this, we could control it properly.  
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As we can see in the graphic, a peak of light is emited about 450 seconds before adding 60 microliters of KOH to 170 microliters of medium with WT transformed yeasts. Although we were almost sure that the mechanism that triggered that flash of light was the expected, we found properly make the same experiment with different kind of controls and make sure we were not observing any artiffact:
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We will show and explain here the '''major results''', for a thorough description of the behaviour of the aequorin light emission system please refer to the [https://2009.igem.org/Team:Valencia/Parts/Characterization '''Characterization page'''] or to the [http://partsregistry.org/wiki/index.php?title=Part:BBa_K222000 '''Registry'''].
 +
<br>
 +
===Controls===
 +
<br>
 +
In every single experiment in a molecular biology laboratory, one has to bear in mind the use of negative controls for every logical step of the hypothesis.
 +
Therefore, we have taken advantage of the different experimental designs that were available: we had several calcium channel knock outs (''mid1'' and ''ch1''), as well as functional inhibitors of the calcium channels (KCl) and divalent ion quelant (EDTA).
-
* Mid1: one mutant for a Calcium channel. Light is not observed because Ca2+ can’t enter into the cell and bind to the aequorin-coelenterazine complex.
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All these designs were used in order to reject the idea of looking to an artifact.
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* Cch1: another mutant for Calcium channel, so the absence of light can be explainned in the same way.
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[[Image:Comparació_disc.jpg|700 px||left]]
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* EDTA: Aulthough every compound necessary for the reaction is present (including Ca2+ channels) light is not emited because EDTA is a divalent ion quelant, so Ca2+ is quenched and not useful for the emission.
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This graph shows the behaviour of a set of designs after the supply of a 4V shock.
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* KCl: another negative control. The Absence of the -OH group prevents the oppening of calcium channels and makes yeasts produce no light.
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* '''WT''' is our wild type luminiscent cell, with coelenterazine and fully working calcium channels
 +
* '''mid1''' is a knock out for a calcium channel. Light is not observed because Ca2+ can’t enter into the cell and bind to the aequorin-coelenterazine complex.
 +
* '''cch1''' is another knock out mutant for a calcium channel, so the absence of light can be explainned in the same way.
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But we wanted to characterize in detail this kind of response.
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* '''EDTA''' is a divalent ion quelant, so Ca2+ is quenched and not useful for the light emission, although every compound necessary for the reaction is present.
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To complete the work with the chemical input, we though KOH amounts could influence in the quantity of emited light, so we repited the experiment with different concentrations of KOH.
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[[Image:Comparació koh.jpg|500px]]
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* '''SD +coe''' is growth media with coelenterazine, just to be sure that without cells we had no light.
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As we can see, the volume of added KOH (from 15 microliters to 120) is related to the luminiscent peak. Although there is not directly proporcional, luminiscence intensity is increased when we increase the quantity of KOH we put in the sample (always 170 microliters of medium with yeasts).
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* '''SD''' is plain growth media.
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Characterizasing the response to the KOH we also found interesting to determinate the reproducibility of the process.
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* '''yeast -coe''' is our wild type luminiscent cell, without coelenterazine, the prosthetic group that is needed for the light emission.
 +
Three kinds of behaviour stem out of this experiment: no light at all, some light emission and full light emission.
 +
* ''no light at all'' is the case of '''SD +coe''','''SD''' and  '''yeast -coe''' where there is a lack of at least one of the compnents of the LEC.
 +
* ''some light emission'' is the case of '''mid1''', '''cch1''' and '''EDTA''' where all the LEC components are present, but free calcium in the cell is somehow constrained.
 +
* ''full light emission'' is the case of '''WT''' where all the components of the LEC are presents and calcium channels are fully open.
 +
<br>
 +
===Voltage===
 +
<br>
 +
Varying voltage and exciting time, we can have a variety of responses (arrows indicate electric current supply):
 +
<center>
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{|
 +
|[[Image:1,5V 10s.jpg|center|450px]]
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|-
 +
|[[Image:4,5V 5s.jpg|center|450px]]
 +
|}
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</center>
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[[Image:Repetibilitat KOH.jpg|600px]]
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===Supply time===
 +
<br>
 +
Varying the electric current time supply we can study the influence of this variable in our system. Here we see that if we apply too much time the system is overdosed. Figure on the right shows the behaviour of the three pixels, the third one would be a blow pixel as a consequence of an oversupply of voltage.
 +
<table>
 +
    <tr>
 +
        <td>[[Image:6V variats disc.jpg|410px|center]]</td>
 +
        <td>[[Image:Sixvolts.gif|360 px|center]]</td>
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    </tr>
 +
</table>
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By adding 30 microliters of KOH at the time where the rows indicates us, we discovered that before the first peak, cells couldn’t return to the basals levels, and every new shock make yeasts produce light in higher levels than the last one.
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===Repetition===
 +
<br>
 +
Our idea was to have a screen, that is '''to have several images sequentially''', in order to see animated pictures. So we studied the different voltage times in a sequence of power applications to one LEC.
 +
[[Image:Manteniment resposta disc.jpg|center|600px]]
 +
===Refreshing rate===
 +
<br>
 +
Finally, we were looking for the shortest refreshing rate as possible, so to have something close to a ''real'' screen.
 +
[[Image:24V 0,5s.jpg|center|500 px]]
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===Electrical input===
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We have accomplished a refreshing rate of 12 seconds with a supply of 24V in 0,5 seconds.
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+
-
 
+
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When the experiments with an alkali input showed us that yeasts were able to produce light because of their transformation, we tried with our ambitious goal: stimulate calcium channels with an electrical input.
+
-
 
+
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We reproduced the mentionated Arinyo’s protocol, incubating the transformed yeasts with coelenterazine, but changing the KOH by electricity. Surprisingly, we found that  light was also produce in a very similar way. We tryied with different times and voltages in order to find the optim conditions for a big peak of light. Some of our graphics are theese:
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+
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(Gràfiques)
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We realised that the time of exposure to the electrical stimulus was crucial, even more that the aplied voltage. That means, if we increased the voltage at very short times, cells could produce a more abrupt peak of light. But if we increased the time of exposure to the electricity, we observe a less defined response, with more flattened peaks. That’s probably because a big exposure time of electrical input damages and killes the yeasts, making them to release their components to the medium, including the aequorin-coelenterazine-Ca2+ complex, so the emission of light is more uniform in time, instead of the production of the flash produced by the Calcium enetering in the cell.
+
-
In the case of very little voltages (like 1,5V) this observation is not carried out by our yeasts. The reason must be that the electrical input is too low, so yeasts don’t die so easily as with more elevated voltage, and a better response is produce with a more prolongated electrical shock.
+
-
 
+
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[[Image:6V variats disc.jpg|500px]]
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This graphic clearly show us that using a same voltage, we obtain a better response with the shortest time of the electrical input.
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-
 
+
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Our controls discard the idea of an artifact. For example, light could be made by a spark produced during the discharge. It was not very probable, because the peak observed was produce near 400 seconds before of the stimulus. But, another time, when cells without coelenterazine or mutants are used, we see no light.
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-
 
+
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+
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(Gràfica del discontinu en els mutants, etc)
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+
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Studying the repetibility of the process, this is a little different from the chemical stimulus, but the system has a similar behaviour, and we can stimulate several times the same sample getting a response. However, every nex shock produces a fewer peak of light. We hace two hypothesis: one of them is that a part of our yeasts die meanwhile the electrical stimulus. The other one is that coelenterazine is not reusable, so a proportion of it runs down in every emission of light.
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-
 
+
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(Gràfica de la repetibilitat en electricitat)
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+
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===SCREEN===
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+
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Using that information and ability of our yeasts, we decided to design a bio-screen, where every single pixel was composed of a group of luminiscent cells and individualy stimulated with a cable. We could, then, control which pixel gets iluminated, forming the image/picture we want (whose resolution depends on the number of pixels we have).
+
-
 
+
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This simple mechanism is the first example of electronic communication between computers and single celled organisms. Thus, our engineered yeast are a state-of-art bioelectronic device.
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+
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'''It is just like a bacterial photographic system, but it's digital.''' Within seconds, instead of hours, you can get an image formed of living cells.
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+
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And, the chose the calcium signaling because it is the fastest known modality of signaling in biology, and will allow for a fast refreshing rate of the screen.
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+
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[[Image:10V 2s.jpg|600px]]
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[[Image:24V 0,5s.jpg|600px]]
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[[Image:Wt cont.jpg|600px]]
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[[Image:Wt-coe cont.jpg|600px]]
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[[Image:SD+coe cont.jpg|600px]]
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[[Image:Cch1 cont.jpg|700px]]
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[[Image:Combinació.jpg|700px]]
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[[Image:1,5V 5st.jpg|600px]]
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[[Image:1,5V 10s.jpg|600px]]
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+
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[[Image:4,5V 5s.jpg|600px]]
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+
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[[Image:10V 1s disc.jpg|600px]]
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+
-
[[Image:Comparació disc.jpg|600px]]
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-
[[Image:Exp Arinyo.jpg|600px]]
 
-
[[Image:Comparació koh.jpg|600px]]
+
Again, if you find yourself hungry of knowledge, please find much more information on the aequorin and the study we have made upon it on the [https://2009.igem.org/Team:Valencia/Parts/Characterization '''Parts Characterization'''] page or on the [http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2009&group=Valencia&Done=1 '''Registry'''].

Latest revision as of 03:13, 22 October 2009




Experimental results


Our ultimate goal was to make a bio-screen made with cellular pixels (LECs). But, before to be able to build this iLCD, we had to study the behaviour of one single LEC. Therefore, we focused on the electrical excitation of our transformed yeasts.

This is the behaviour that stands as the cornerstone of our project. With a serie of voltage inputs, we accomplished a series of light emissions. No exciting light was needed, only aequorin, coelenterazine (the prosthetic group) and Ca2+.

Valencia Grafica continu 2 pics.jpg
Bursts.gif


Arrows indicate when voltage was applied: 16V during 5 seconds applied at 300 seconds (5 minutes) and 600 seconds (10 minutes). Movie on the right shows how the response would be seen in a pixel.


  • WT indicates our LEC: our aequorin-engineered yeast, with coelenterazine and wild-type calcium channels
  • WT -coe indicates the same yeast, without the addition of coelenterazine.

As we can observe, only our LEC is excited when electric current is applied. Withour coelenterazine there is no response to that stimulus.

Conclusions of the study


We have investigated a set of different voltages and times in order to precisely know how our system respons to electric current. Knowing this, we could control it properly.

We will show and explain here the major results, for a thorough description of the behaviour of the aequorin light emission system please refer to the Characterization page or to the [http://partsregistry.org/wiki/index.php?title=Part:BBa_K222000 Registry].

Controls


In every single experiment in a molecular biology laboratory, one has to bear in mind the use of negative controls for every logical step of the hypothesis. Therefore, we have taken advantage of the different experimental designs that were available: we had several calcium channel knock outs (mid1 and ch1), as well as functional inhibitors of the calcium channels (KCl) and divalent ion quelant (EDTA).

All these designs were used in order to reject the idea of looking to an artifact.

Comparació disc.jpg

This graph shows the behaviour of a set of designs after the supply of a 4V shock.

  • WT is our wild type luminiscent cell, with coelenterazine and fully working calcium channels
  • mid1 is a knock out for a calcium channel. Light is not observed because Ca2+ can’t enter into the cell and bind to the aequorin-coelenterazine complex.
  • cch1 is another knock out mutant for a calcium channel, so the absence of light can be explainned in the same way.
  • EDTA is a divalent ion quelant, so Ca2+ is quenched and not useful for the light emission, although every compound necessary for the reaction is present.
  • SD +coe is growth media with coelenterazine, just to be sure that without cells we had no light.
  • SD is plain growth media.
  • yeast -coe is our wild type luminiscent cell, without coelenterazine, the prosthetic group that is needed for the light emission.

Three kinds of behaviour stem out of this experiment: no light at all, some light emission and full light emission.

  • no light at all is the case of SD +coe,SD and yeast -coe where there is a lack of at least one of the compnents of the LEC.
  • some light emission is the case of mid1, cch1 and EDTA where all the LEC components are present, but free calcium in the cell is somehow constrained.
  • full light emission is the case of WT where all the components of the LEC are presents and calcium channels are fully open.


Voltage


Varying voltage and exciting time, we can have a variety of responses (arrows indicate electric current supply):

1,5V 10s.jpg
4,5V 5s.jpg

Supply time


Varying the electric current time supply we can study the influence of this variable in our system. Here we see that if we apply too much time the system is overdosed. Figure on the right shows the behaviour of the three pixels, the third one would be a blow pixel as a consequence of an oversupply of voltage.

6V variats disc.jpg
Sixvolts.gif

Repetition


Our idea was to have a screen, that is to have several images sequentially, in order to see animated pictures. So we studied the different voltage times in a sequence of power applications to one LEC.

Manteniment resposta disc.jpg

Refreshing rate


Finally, we were looking for the shortest refreshing rate as possible, so to have something close to a real screen.

24V 0,5s.jpg

We have accomplished a refreshing rate of 12 seconds with a supply of 24V in 0,5 seconds.


Again, if you find yourself hungry of knowledge, please find much more information on the aequorin and the study we have made upon it on the Parts Characterization page or on the [http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2009&group=Valencia&Done=1 Registry].

Retrieved from "http://2009.igem.org/Team:Valencia/WetLab/YeastTeam/Results"