Difference between revisions of "Team:Slovenia/Notebook/Protocols"

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<p>For successful execution of all experiments, described in the previous pages, we used the following protocols:</p>
 
<p>For successful execution of all experiments, described in the previous pages, we used the following protocols:</p>
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</div>
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<div class="main ui citing justified container"><h1 class = "ui left dividing header"><span class="section">&nbsp;</span>Inovation in measurement</h1>
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<div class = "ui segment" style = "background-color: #ebc7c7; ">
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<p><b><ul><li>A new, simple and reliable approach for real time measurement of the intracellular calcium concentration was developed.
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</ul></b></p>
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</div>
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<div class = "ui segment">
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Our project required a sensitive and fast method for detecting cytosolic calcium concentrations.
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This measurement is a fast-relay system, has low response at the physiological cytosolic level of calcium and works <i>in vivo</i> in mammalian cells.
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</div>
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<h3 style="clear:both">Sensor development and characterization</h3>
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<p>While calcium influx could be detected by exogenous fluorescent dyes such as Fura Red and Fluo-4, our project required a sensitive, genetically
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encoded calcium sensor that would couple a change in the calcium concentration to a biologically relevant output, such as the luciferase reporter or
 +
reconstitution of a split protease for the initiation of the signaling pathway. An ideal calcium sensor should be inactive at intracellular concentration
 +
of calcium and have a high response to calcium concentrations above physiological levels and should be detected by a quick and easy readout. For our
 +
intended application, the calcium sensor should also have the potential to act as the reconstitution mechanism for split proteins so that a conformational
 +
rearrangement in the presence of calcium would bring the two split protein fragments together and reconstitute the protein’s activity.</p>
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<p>A wide palette of genetically encoded calcium sensors have been used for mapping intracellular calcium concentration <x-ref>Whitaker2010</x-ref>,
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including calmodulin, troponin C and aequorin <x-ref>Wilms2014</x-ref>. These reporters are based on different mechanisms of detection. From this abundant
 +
collection we selected calmodulin (CaM)-based calcium sensors (in particular CaMeleons), since their mechanism is based on a large conformational change,
 +
allowing reconstitution of split proteins <x-ref>Whitaker2010</x-ref>.</p>
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<p>We replaced the FRET pair of CaMeleon2.12 by a split firefly luciferase (fLuc) as it provides a distinct signal even at small amounts and has a high
 +
signal-to-noise ratio. The new luciferase based calcium sensor was named fLuc2.12. The fLuc2.12 was tested on HEK293 cells, but we found that the sensor
 +
was active already in resting cells (<ref>1</ref>). We hypothesized the activation was a consequence of a close proximity of calmodulin and M13 in the
 +
fusion even in the absence of calcium binding. In order to resolve this problem we set out to test a similar sensor based on two separate molecules.
 +
Two-molecule-based CaM sensors have not been widely used, but lower leakage in comparison to a single molecule sensor has been reported by Miyawaki
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<i>et al.</i> <x-ref>Miyawaki1997</x-ref>.</p>
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<div style="float:left; width:50%">
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<figure data-ref="1">
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<img src="https://static.igem.org/mediawiki/2016/c/cf/T--Slovenia--3.6.1.png" >
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<figcaption><b>A luminescence calcium sensor based on the calmodulin-M13 fusion fLuc2.12 has high activity already at the resting levels of calcium.</b><br/>
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HEK293T cells were transfected with 50 ng fLuc2.12. 24 h after transfection luciferase activities were measured immediately after addition of calcium ionophore A23187 (10 µM). Scheme: The chimeric protein M13-calmodulin fused to N- and C- fragments of split luciferase changes conformation upon calcium binding.</figcaption>
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</figure>
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</div>
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</div>
 
</div>

Revision as of 18:49, 18 October 2016

Protocols

Project Achievements Medal requirements Idea

 Protocols

For successful execution of all experiments, described in the previous pages, we used the following protocols:

 Inovation in measurement

  • A new, simple and reliable approach for real time measurement of the intracellular calcium concentration was developed.

Our project required a sensitive and fast method for detecting cytosolic calcium concentrations. This measurement is a fast-relay system, has low response at the physiological cytosolic level of calcium and works in vivo in mammalian cells.

Sensor development and characterization

While calcium influx could be detected by exogenous fluorescent dyes such as Fura Red and Fluo-4, our project required a sensitive, genetically encoded calcium sensor that would couple a change in the calcium concentration to a biologically relevant output, such as the luciferase reporter or reconstitution of a split protease for the initiation of the signaling pathway. An ideal calcium sensor should be inactive at intracellular concentration of calcium and have a high response to calcium concentrations above physiological levels and should be detected by a quick and easy readout. For our intended application, the calcium sensor should also have the potential to act as the reconstitution mechanism for split proteins so that a conformational rearrangement in the presence of calcium would bring the two split protein fragments together and reconstitute the protein’s activity.

A wide palette of genetically encoded calcium sensors have been used for mapping intracellular calcium concentration Whitaker2010, including calmodulin, troponin C and aequorin Wilms2014. These reporters are based on different mechanisms of detection. From this abundant collection we selected calmodulin (CaM)-based calcium sensors (in particular CaMeleons), since their mechanism is based on a large conformational change, allowing reconstitution of split proteins Whitaker2010.

We replaced the FRET pair of CaMeleon2.12 by a split firefly luciferase (fLuc) as it provides a distinct signal even at small amounts and has a high signal-to-noise ratio. The new luciferase based calcium sensor was named fLuc2.12. The fLuc2.12 was tested on HEK293 cells, but we found that the sensor was active already in resting cells (1). We hypothesized the activation was a consequence of a close proximity of calmodulin and M13 in the fusion even in the absence of calcium binding. In order to resolve this problem we set out to test a similar sensor based on two separate molecules. Two-molecule-based CaM sensors have not been widely used, but lower leakage in comparison to a single molecule sensor has been reported by Miyawaki et al. Miyawaki1997.

A luminescence calcium sensor based on the calmodulin-M13 fusion fLuc2.12 has high activity already at the resting levels of calcium.
HEK293T cells were transfected with 50 ng fLuc2.12. 24 h after transfection luciferase activities were measured immediately after addition of calcium ionophore A23187 (10 µM). Scheme: The chimeric protein M13-calmodulin fused to N- and C- fragments of split luciferase changes conformation upon calcium binding.