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-	<div style="position:relative; top:-250px; left:10px; width:700px; font-family: Verdana;" align="justify">	+	<div style="position:relative; margin-top:-295px; margin-left:190px; width:700px; font-family: Verdana;" align="justify">
	<br>		<br>
-	==Experimental results==	+	='''Experimental results'''=
-		+
	<br>		<br>
-
	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.		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 Ca<sup>2+</sup>.
-	Taking advantage of the different experimental designs we had at a glance: 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).	+	<table>
		+	<tr>
		+	<td>[[Image:Valencia_Grafica_continu_2_pics.jpg|500 px|left]]</td>
		+	<td>[[Image:Bursts.gif|100 px|center]]</td>
		+	</tr>
		+	</table>
		+	<br>
		+	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.
-	characterize the cell light emission if we wanted to control it better.	+	As we can observe, '''only our LEC is excited when electric current is applied'''. Withour coelenterazine there is no response to that stimulus.
-	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. we choose 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.	+
-	===Chemical input===	+	==Conclusions of the study==
	<br>		<br>
-	In order to make our yeasts produce light, we firstly reproduce experiments made by Viladevall et al, after a lot of different trials, we finally could characterize the luminiscence curve in a discontinuos luminometer.	+	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.
-	[[Image:Comparació koh.jpg|center|520px]]	+	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).
-	As we can see in the graph, a peak of light is emited about 450 seconds after the addition 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 preferred to make the same experiment with different kind of controls and make sure we were not observing any artiffact:	+	All these designs were used in order to reject the idea of looking to an artifact.
-	* '''Mid1''': one knock out mutant for a Calcium channel. Light is not observed because Ca2+ can’t enter into the cell and bind to the aequorin-coelenterazine complex.	+	[[Image:Comparació_disc.jpg|700 px||left]]
-	* '''Cch1''': another knock out mutant for Calcium channel, so the absence of light can be explainned in the same way.	+	This graph shows the behaviour of a set of designs after the supply of a 4V shock.
-	* '''EDTA''': Although 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.	+	* '''WT''' is our wild type luminiscent cell, with coelenterazine and fully working calcium channels
-	* '''KCl''': another negative control. The absence of the -OH group prevents the opening of calcium channels and makes yeast produce no light.	+	* '''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.
-	We wanted to characterize in detail this kind of response.	+	* '''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.
-	To complete the work with the chemical input, we though KOH amounts could influence in the quantity of emitted light, so we repited the experiment with different concentrations of KOH.	+
-	[[Image:Caracterització KOH.jpg|center|thumb|700px| Light emission under diferent concentrations of the chemical input]]	+	* '''SD +coe''' is growth media with coelenterazine, just to be sure that without cells we had no light.
-	As we can see, the volume of KOH added (from 15 microliters to 120) is related to the luminiscent peak. Although there is not linearly proportional, luminiscence intensity is increased when we increase the quantity of KOH we put in the sample (always 170 microliters of medium with yeasts).	+	* '''SD''' is plain growth media.
-	Characterising the response to the KOH we also found interesting to determinate the reproducibility of the process.	+	* '''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>
		+	{|
		+	|[[Image:1,5V 10s.jpg|center|450px]]
		+	|-
		+	|[[Image:4,5V 5s.jpg|center|450px]]
		+	|}
		+	</center>
-	[[Image:Repetibilitat KOH.jpg|700px]]	+	===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>
		+	</tr>
		+	</table>
-	By adding 30 microliters of KOH at certain times (arrows), we discovered that after 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.	+	===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]]
-	===Electrical input===	+	===Refreshing rate===
	<br>		<br>
-	When the experiments with an alkali input showed us that yeasts were able to produce light because of their transformation, we tried our ambitious goal: stimulate calcium channels with an electrical input.	+	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]]
-	'''We use two luminometers''', one luminometer discontinuous and the other is continuous. Each luminometer uses different units, depends on the manufacturer. For this reasons we can't compare directly the results obtained with one luminometer with the results of the other. According this, when we only compare results in the same graph if they were obtained with the same luminometer. However, an increase (or not) in the luminosity, means the same at two luminometers and the experiments are complementary and reaffirms our conclusions.	+
-		+
-	We reproduced the mentionated Viladevall et al's protocol, incubating the transformed yeasts with coelenterazine, but changing the KOH by electricity. Surprisingly, we found that light was also produced in a very similar way. We tried with different times and voltages in order to find the optim conditions for a big peak of light. Some of our graphics are theese:	+
-		+
-	[[Image:1,5V 5st.jpg|center|thumb|700px| Light emitted when 1,5V are applicated during 5 seconds]]	+
-		+
-	[[Image:1,5V 10s.jpg|center|thumb|700px| Light emitted when 1,5V are applicated during 10 seconds]]	+
-		+
-	[[Image:4,5V 5s.jpg|center|thumb|700px| Light emitted when 4,5V are applicated during 5 seconds]]	+
-		+
-	[[Image:10V 1s disc.jpg|center|thumb|700px| Light emitted when 10V are applicated during 1 second]]	+
-		+
-	[[Image:10V 2s.jpg|center|thumb|700px| Light emitted when 10V are applicated during 2 seconds]]	+
-		+
-	[[Image:24V 0,5s.jpg|center|thumb|700px| Light emitted when 24V are applicated during 0,5 seconds]]	+
-		+
-	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.	+
-		+
-	[[Image:6V variats disc.jpg|500px]]	+
-		+
-	This graph clearly show us that using a same voltage, we obtain a better response with the shortest time of the electrical input.	+
-		+
-	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 after the stimulus. But, another time, when cells without coelenterazine or mutants are used, we see no light.	+
-		+
-	[[Image:Wt cont.jpg|center|thumb|700px| Our yeasts with coelentrazine ]]	+
-		+
-	[[Image:Wt-coe cont.jpg|center|thumb|700px| Our yeastse without coelenterazine]]	+
-		+
-	[[Image:SD+coe cont.jpg|center|thumb|700px| Medium with coelenterazine]]	+
-		+
-	[[Image:Cch1 cont.jpg|center|thumb|700px| Mutant yeasts, deficients in calcium channels]]	+
-		+
-	[[Image:graf_compar.jpg|center|thumb|800px| Comparison of the results]]	+
-		+
-	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 next 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.	+	We have accomplished a refreshing rate of 12 seconds with a supply of 24V in 0,5 seconds.
-	[[Image:Manteniment resposta disc.jpg|center|thumb|700px| Here, we can see that the process can be repeat consecutively]]
-	<!--[[Image:Combinació.jpg|center|thumb|700px| Comparation of the repetibility between our yeasts, our yeasts without coelenterazine, medium whit coelenterazine and mutants]]	+	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⁺.

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.

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):

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.

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.

Refreshing rate

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

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].