Team:Valencia/Parts/Characterization

From 2009.igem.org

(Difference between revisions)

Revision as of 20:28, 20 October 2009

Aequorin characterization

AEQ is a gene which encodes for aequorin, our luminiscent protein.

It's a photoprotein isolated from luminescent jellyfish (like various Aequorea species like Aequorea victoria) and a variety of other marine organisms. It was originally isolated from the coelenterate by Osamu Shimomura, and it has been used as a reporter gene in different eukariotes. Nowadays, there are different aequorin types, depending on the target organism.
The aequorin which we are working with has been introduced in our yeasts by a plasmid called pEVP11/AEQ, which encondes the aequorin sequence showed below. Cells containing this plasmid are able to sintetize apoaequorin, the apoprotein of 22 kDa. That apoprotein cannot produce luminiscence by itself, but when it binds to its cofactor coelenterazine, in presence of Ca2+, full aequorin emits light.
The two components of aequorin reconstitute spontaneously, forming the functional protein. The protein bears three EF-hand motifs that function as binding sites for Ca2+ ions. When Ca2+ occupies such sites, the protein undergoes a conformational change and converts through oxidation its prosthetic group, coelenterazine, into excited coelenteramide and CO2 (as we explain in Wetlab overview). As the excited coelenteramide relaxes to the ground state, blue light (wavelength = 469 nm) is emitted.

Sequence

Aequorin sequence is (primer binding sites are underlined in green):

Characterization

The aequorin-coelenterazine complex needs calcium to produce light. In the process this calcium enters through a special type of calcium channels present in the cell’s plasma membrane which open or close in response to a change in the transmembrane potential, that is why they are called voltage-dependent calcium channels (VDCC). The equation which describes calcium current through these channels is the following:

Where g is the conductance associated with the channel, V is the transmembrane potential and ECa is the Nernst potential, related to the different ionic concentration inside and outside the cell. Considering that these channels are only permeable to calcium and have two states -open or closed-, the total conductance associated with the population of VDCCs can be expressed as the maximal conductance ( ) times the fraction of all channels that are open. This fraction is determined by hypothetical activation and inactivation variables m and h, which depend on voltage and time:

VDCCs are well characterized in excitable cells such as neurons, where the value of is known:

Budding yeast, Saccharomyces cerevisiae has homologous voltage-dependent channels in its plasma membrane. However, there is a lack of study of the properties of these channels. That is why we have determined their conductance according to our experimental results:

Having characterized calcium entry to the cell, we studied the aequorin production of light through a calcium-mediated response after two types of stimulation:

• Chemical stimulation Alkali shock (performed by the addition of KOH) induced a calcium entry to cytoplasm (where aequorin is located) through voltage-dependent calcium channels. These calcium ions bind the aequorin-coelenterazine complex and photons –light- are emitted during a short period of time. This graph shows yeasts’ response to the addition of 30 µL of KOH:

Furthermore, we made experiments with increasing KOH volumes (in microliters) and realized that light emission was higher: the more KOH arrives to cells, the more calcium ions get into the cytoplasm and more photons are emitted by aequorin.

It has to be noted that, in presence of suitable amounts of coelenterazine, the aequorin production of light can be repeated with several stimulations of 15 microliters each (where arrows show):

Finally, we have proved that calcium enters through VDCCs, as yeast strains lacking this calcium channels (cch1 and mid1) emit less amount of light. Two negative controls (addition of EDTA, a quelant of calcium ions, and KCl, which do not perform alkali shock) are also included in this graph:

• Electrical stimulation Calcium entrance to cytosol –and the consequent aequorin light emission- can also be perfomed by applying electricity to the yeast culture. This response was characterized at different voltage values and times of application. Again, the stimulation was repeatable (stimulus applied where arrows):

The following graph show data obtained with an ELISA plates reader which made discontinuous measures, one each 30 s. Therefore, we had not information about light production between dots.

Although the previous graphs show that the second stimulation induces a lower amount of light, varying the time of stimulus application, higher peaks could be obtained:

Similarly to chemical stimulation, yeast mutant strains lacking calcium channels had less light emission. Several negative controls were made to prove that light came from aequorin-transformed yeasts:

The following graphs show data obtained with a continuous luminometer. It has to be noted the sharp peak of light produced when stimulation is applied (we were not able to detect it with the discontinuous measurements)

Light emission under diferent concentrations of the chemical input

Electrical input

Light emitted when 1,5V are applicated during 5 seconds

Light emitted when 1,5V are applicated during 10 seconds

Light emitted when 4,5V are applicated during 5 seconds

Light emitted when 10V are applicated during 1 second

Light emitted when 10V are applicated during 2 seconds

Light emitted when 24V are applicated during 0,5 seconds

Our yeasts with coelentrazine

Our yeastse without coelenterazine

Medium with coelenterazine

Mutant yeasts, deficients in calcium channels

Comparation of the results

Here, we can see that the process can be repeat consecutively

Comparation of the repetibility between our yeasts, our yeasts without coelenterazine, medium whit coelenterazine and mutants

Retrieved from "http://2009.igem.org/Team:Valencia/Parts/Characterization"

@@ Line 1: / Line 1: @@
 {{Template:Valencia09iGEM23}}
-<div style="position:relative; top:-5px; left:70px; width:700px" align="justify">
-<br>
-==General information==
-<br>
 <div align="justify" style="position:relative; top:-5px; left:30px; width:700px; color:black; font-size:10pt; font-family: Verdana">
 <br>
+==Aequorin characterization==
 AEQ is a gene which encodes for aequorin, our luminiscent protein.
 [[Image:aequorin.GIF|300px|center]]
-It's a photoprotein isolated from luminescent jellyfish (like various Aequorea species like Aequorea victoria) and a variety of other marine organisms. It was originally isolated from the coelenterate by Osamu Shimomura, and it has been used as a reporter gene in different superior eukariotes. Nowadays, there are different aequorin types, depending on the target organism. <br>
+It's a photoprotein isolated from luminescent jellyfish (like various ''Aequorea species'' like ''Aequorea victoria'') and a variety of other marine organisms. It was originally isolated from the coelenterate by Osamu Shimomura, and it has been used as a reporter gene in different eukariotes. Nowadays, there are different aequorin types, depending on the target organism. <br>
-The aequorin which we are working with has been introduced in our yeasts by a plasmid called pEVP11/AEQ, wich encondes aequorin sequence showed below. Cells containing this plasmid are able to sintetize apoaequorin, the apoprotein of 22 kDa, and keep it in their citoplasm. So, that apoprotein can't produce luminiscence by itself, but when it binds to its cofactor coelenterazine, in presence of Ca2+, full aequorin emits light.<br>
+The aequorin which we are working with has been introduced in our yeasts by a plasmid called pEVP11/AEQ, which encondes the aequorin sequence showed below. Cells containing this plasmid are able to sintetize apoaequorin, the apoprotein of 22 kDa. That apoprotein cannot produce luminiscence by itself, but when it binds to its cofactor coelenterazine, in presence of Ca2+, full aequorin emits light.<br>
 The two components of aequorin reconstitute spontaneously, forming the functional protein. The protein bears three EF-hand motifs that function as binding sites for Ca2+ ions. When Ca2+ occupies such sites, the protein undergoes a conformational change and converts through oxidation its prosthetic group, coelenterazine, into excited coelenteramide and CO2 (as we explain in Wetlab overview). As the excited coelenteramide relaxes to the ground state, blue light (wavelength = 469 nm) is emitted.<br>
 ==Sequence==
 Aequorin sequence is (primer binding sites are underlined in green):<br>
@@ Line 24: / Line 18: @@
 ==Characterization==
-===Chemical input.===
+The aequorin-coelenterazine complex needs calcium to produce light. In the process this calcium enters through a special type of calcium channels present in the cell’s plasma membrane which open or close in response to a change in the transmembrane potential, that is why they are called voltage-dependent calcium channels (VDCC). The equation which describes calcium current through these channels is the following:
-<br>
-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.
+Where g is the conductance associated with the channel, V is the transmembrane potential and ECa is the Nernst potential, related to the different ionic concentration inside and outside the cell.
+Considering that these channels are only permeable to calcium and have two states -open or closed-, the total conductance associated with the population of VDCCs can be expressed as the maximal conductance (   ) times the fraction of all channels that are open. This fraction is determined by hypothetical activation and inactivation variables m and h, which depend on voltage and time:
-[[Image:Comparació koh.jpg|center|520px]]
+VDCCs are well characterized in excitable cells such as neurons, where the value of   is known:
-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:
+Budding yeast, Saccharomyces cerevisiae has homologous voltage-dependent channels in its  plasma membrane. However, there is a lack of study of the properties of these channels. That is why we have determined their conductance according to our experimental results:
-* '''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.
+Having characterized calcium entry to the cell, we studied the aequorin production of light through a calcium-mediated response after two types of stimulation:
-* '''Cch1''': another mutant for Calcium channel, so the absence of light can be explainned in the same way.
+•	Chemical stimulation
+Alkali shock (performed by the addition of KOH) induced a calcium entry to cytoplasm (where aequorin is located) through voltage-dependent calcium channels. These calcium ions bind the aequorin-coelenterazine complex and photons –light- are emitted during a short period of time. This graph shows yeasts’ response to the addition of 30 µL of KOH:
-* '''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.
+Furthermore, we made experiments with increasing KOH volumes (in microliters) and realized that light emission was higher: the more KOH arrives to cells, the more calcium ions get into the cytoplasm and more photons are emitted by aequorin.
-* '''KCl''': another negative control. The Absence of the -OH group prevents the oppening of calcium channels and makes yeasts produce no light.
+It has to be noted that, in presence of suitable amounts of coelenterazine, the aequorin production of light can be repeated with several stimulations of 15 microliters each (where arrows show):
+Finally, we have proved that calcium enters through VDCCs, as yeast strains lacking this calcium channels (cch1 and mid1) emit less amount of light. Two negative controls (addition of EDTA, a quelant of calcium ions, and KCl, which do not perform alkali shock) are also included in this graph:
+•	Electrical stimulation
+Calcium entrance to cytosol –and the consequent aequorin light emission- can also be perfomed by applying electricity to the yeast culture. This response was characterized at different voltage values and times of application. Again, the stimulation was repeatable (stimulus applied where arrows):
+*The following graph show data obtained with an ELISA plates reader which made discontinuous measures, one each 30 s. Therefore, we had not information about light production between dots.
-But we wanted to characterize in detail this kind of response.
+Although the previous graphs show that the second stimulation induces a lower amount of light, varying the time of stimulus application, higher peaks could be obtained:
-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.
-[[Image:Caracterització KOH.jpg|center|thumb|700px| Light emitted under diferent concentrations of the chemical input]]
+Similarly to chemical stimulation, yeast mutant strains lacking calcium channels had less light emission. Several negative controls were made to prove that light came from aequorin-transformed yeasts:
-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).
-Characterizasing the response to the KOH we also found interesting to determinate the reproducibility of the process.
+*The following graphs show data obtained with a continuous luminometer. It has to be noted the sharp peak of light produced when stimulation is applied (we were not able to detect it with the discontinuous measurements)
+[[Image:eq1.jpg|center]]
+[[Image:Comparació koh.jpg|center|520px]]
+[[Image:Caracterització KOH.jpg|center|thumb|700px| Light emission under diferent concentrations of the chemical input]]
 [[Image:Repetibilitat KOH.jpg|700px]]
-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.
 ===Electrical input===
 <br>
-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.
-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:
 [[Image:1,5V 5st.jpg|center|thumb|700px| Light emitted when 1,5V are applicated during 5 seconds]]
@@ Line 73: / Line 66: @@
 [[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 graphic 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 before of 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 ]]
@@ Line 95: / Line 78: @@
 [[Image:Comparació disc.jpg|center|thumb|700px| Comparation 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.
 [[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]]
-===SCREEN===
-<br>
-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).
-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.
-'''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.
-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.