Difference between revisions of "Team:Slovenia/Proof"

 
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                     <a class="item" href="//2016.igem.org/Team:Slovenia/Demonstrate">
 
                     <a class="item" href="//2016.igem.org/Team:Slovenia/Demonstrate">
 
                         <i class="chevron circle left icon"></i>
 
                         <i class="chevron circle left icon"></i>
                         <b>Protease inducible secretion</b>
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                         <b>Protease-based inducible secretion</b>
                    </a>
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                    <a class="item" href="" style="margin-left: 10%">
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                        <i class="selected radio icon"></i>
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                        <b>Project</b>
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                     </a>
 
                     </a>
 
                     <a class="item" href="//2016.igem.org/Team:Slovenia/Proof" style="color:#DB2828;">
 
                     <a class="item" href="//2016.igem.org/Team:Slovenia/Proof" style="color:#DB2828;">
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                 <!-- content goes here -->
 
                 <!-- content goes here -->
 
                 <div>
 
                 <div>
                     <div class="main ui citing justified container"><h1 class="ui left dividing header"><span
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                     <div class="main ui citing justified container">
                            class="section">&nbsp;</span>Touch painting</h1>
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<div>
                        <div class="ui segment" style="background-color: #ebc7c7; ">
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<h1 class="ui left dividing header"><span id = "ach" class="section colorize">&nbsp;</span>Touch painting</h1>
<h4><span id = "ach" class = "section colorize">&nbsp;</span></h4>
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<div class="ui segment" style="background-color: #ebc7c7; ">
                            <p><b>
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<b>
                                <ul>
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<ul>
                                    <li>Human cells with increased ultrasound sensitivity were shown to respond to other
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<li>Human cells with increased ultrasound sensitivity were shown to respond to other
                                        types of mechanical stimulation, such as shear stress and touch.
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types of mechanical stimulation, such as shear stress and touch.
                                    <li>Touch responsive cells were used as a canvas in our sci-art touch painting
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<li>Touch responsive cells were used as a canvas in our sci-art touch painting
                                        implementation.
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implementation.
                                </ul>
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</ul>
                            </b></p>
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</b>
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</div>
 
                         </div>
 
                         </div>
  
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                                 cells
 
                                 cells
 
                                 <x-ref>Seriani2016</x-ref>
 
                                 <x-ref>Seriani2016</x-ref>
                                 . Mechanosensing can cell proliferation and differentiation
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                                 . Mechanosensing can mediate cell proliferation and differentiation
 
                                 <x-ref>Shah2014</x-ref>
 
                                 <x-ref>Shah2014</x-ref>
 
                                 . In recent years, many methods of activation of
 
                                 . In recent years, many methods of activation of
 
                                 mechanosensitive channels have been implemented. Among the activators of mechanosensors,
 
                                 mechanosensitive channels have been implemented. Among the activators of mechanosensors,
 
                                 hyperosmolarity has been shown to trigger the activation of certain genes
 
                                 hyperosmolarity has been shown to trigger the activation of certain genes
                                 <x-ref>Veltmann</x-ref>
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                                 <x-ref>Veltmann</x-ref>. Additional mechanical stimuli such as fluid shear stress causes activation of
                                . Additional mechanical stimuli such as fluid shear stress causes activation of
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                                 embryonic endothelial cells
 
                                 embryonic endothelial cells
 
                                 <x-ref>Ranade2014</x-ref>
 
                                 <x-ref>Ranade2014</x-ref>
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                             <p>Therefore our system might be also activated by other mechanical stimuli. One of them was
 
                             <p>Therefore our system might be also activated by other mechanical stimuli. One of them was
 
                                 stimulation by direct contact that underlies the sense of touch. Our designed
 
                                 stimulation by direct contact that underlies the sense of touch. Our designed
                                 mechano-responsive system is composed of two modules. First we included modules to
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                                 mechano-responsive system is composed of two modules. Firstly we included modules to
 
                                 provide and increase the sensitivity to mechanical stress by
 
                                 provide and increase the sensitivity to mechanical stress by
 
                                 <a href="https://2016.igem.org/Team:Slovenia/Mechanosensing/Mechanosensitive_channels">mechanoresponsive
 
                                 <a href="https://2016.igem.org/Team:Slovenia/Mechanosensing/Mechanosensitive_channels">mechanoresponsive
 
                                     ion channels</a> and
 
                                     ion channels</a> and
 
                                 <a href="https://2016.igem.org/Team:Slovenia/Mechanosensing/Gas_vesicles#vesicles">gas
 
                                 <a href="https://2016.igem.org/Team:Slovenia/Mechanosensing/Gas_vesicles#vesicles">gas
                                     vesicles</a> and secondly, the influx of calcium is visualized by the
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                                     vesicles</a>. Secondly, the influx of calcium is visualized by the
 
                                 <a href="https://2016.igem.org/Team:Slovenia/Mechanosensing/CaDependent_mediator#split">dimerization
 
                                 <a href="https://2016.igem.org/Team:Slovenia/Mechanosensing/CaDependent_mediator#split">dimerization
 
                                     of calmodulin</a> (attached to the C terminus of split luciferase) and M13
 
                                     of calmodulin</a> (attached to the C terminus of split luciferase) and M13
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                                 medium around cells might be sensed by cells. The experiment to test this ability was
 
                                 medium around cells might be sensed by cells. The experiment to test this ability was
 
                                 performed by agitating the petri dish with attached cells. In order to identify
 
                                 performed by agitating the petri dish with attached cells. In order to identify
                                 if the engineered cells are able to respond to the sheaf flow we included control cells
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                                 if the engineered cells are able to respond to the shear flow we included control cells
 
                                 that either constitutively express the luciferase and cells that harbour only
 
                                 that either constitutively express the luciferase and cells that harbour only
 
                                 the calcium-dependent reporter without constructs to increase the mechanosensing along
 
                                 the calcium-dependent reporter without constructs to increase the mechanosensing along
 
                                 with cells expressing the gas vesicles and reporters.
 
                                 with cells expressing the gas vesicles and reporters.
 
                             </p>
 
                             </p>
                             <div style="float:left; width:100%">
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                             <div style = "float:right;width:50%">
 
                                 <figure data-ref="1">
 
                                 <figure data-ref="1">
 
                                     <img class="ui large"
 
                                     <img class="ui large"
 
                                         src="https://static.igem.org/mediawiki/2016/a/a4/T--Slovenia--6.1.a.png">
 
                                         src="https://static.igem.org/mediawiki/2016/a/a4/T--Slovenia--6.1.a.png">
 
                                     <figcaption><b>Mechanical stimulation of cells by manual agitation</b><br/>
 
                                     <figcaption><b>Mechanical stimulation of cells by manual agitation</b><br/>
                                         <p style="text-align:justify">Firefly luciferase activity before (A) and
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                                         <p style="text-align:justify">Firefly luciferase activity before and
                                             immediately after (B) manual agitation is shown. 24 hours after
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                                             immediately after manual agitation is shown. 24 hours after
 
                                             the transfection, luciferin and calcium were added to the cell medium. The
 
                                             the transfection, luciferin and calcium were added to the cell medium. The
 
                                             cells were shaken a few times and the response was measured via
 
                                             cells were shaken a few times and the response was measured via
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                             </div>
 
                             </div>
 
                             <p>Results show that the light was emitted only in cells with the constitutive luciferase if
 
                             <p>Results show that the light was emitted only in cells with the constitutive luciferase if
                                 the plate has not been moved (
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                                 the plate has not been moved (<ref>1</ref>). On the other hand shaking
                                <ref>1</ref>
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                                ). On the other hand shaking
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                                 triggered activation of the luciferase, with significantly higher level in cells that
 
                                 triggered activation of the luciferase, with significantly higher level in cells that
 
                                 harboured gas vesicles in addition to the reporter, demonstrating that activation
 
                                 harboured gas vesicles in addition to the reporter, demonstrating that activation
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                             </div>
 
                             </div>
  
                             <p>Light emitted from cells was recorded by an imager by capturing light for with 30 sec. We
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                             <p>Emitted light was recorded with an imager by capturing light for 30 sec. We
                                 observed that the lines that were drawn first were more fain that the those
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                                 observed that the drawn lines were fading with time, which suggested that the response is quite fast. Therefore, we sequentially recorded separately drawn letters to obtain presented images (<ref>2</ref>).
                                drawn later, which suggested that the response is quite fast. This was tested by
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                                sequentially drawing separate letters and recording the image after draing each
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                                letter.
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                             </p>
 
                             </p>
                             <div align="center">
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                             <div align="center" style = "width:100%;">
                                <video width="60%" controls loop align="center">
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<div style = "clear:both;float:left; width:60%;" >
                                    <source src="//2016.igem.org/wiki/images/9/90/T--Slovenia--Laura-drawing-on-cells.mp4"
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<video width="100%" controls loop align="center">
                                            type="video/mp4">
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<source src="//2016.igem.org/wiki/images/9/90/T--Slovenia--Laura-drawing-on-cells.mp4"
                                    Your browser does not support the video.
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type="video/mp4">
                                </video>
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Your browser does not support the video.
                                <p style="font-size:10px;">
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</video>
                                    <b>Video of the procedure of Laura drawing on cells and imaging of each letter on
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<p style="font-size:10px;">
                                        the imager.</b>
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<b>Video of the procedure of Laura drawing on cells and imaging of each letter on
                                </p>
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the imager.</b>
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</p>
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</div>
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<div style = "float:left; width:40%;">
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<img class="ui large circular centered image" src="//2016.igem.org/wiki/images/6/67/T--Slovenia--iGEM-gif.gif" style = "width:80%;">
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</div>
 
                             </div>
 
                             </div>
 
                             <!--MIHA insert video in gif (Folder Pictures, Laura paints on cells.mp4)
 
                             <!--MIHA insert video in gif (Folder Pictures, Laura paints on cells.mp4)
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                             <p style = "clear:both;">
 
                                 <br/>
 
                                 <br/>
 
                                 Laura prepared several stamps and we experimented with different methods of cell
 
                                 Laura prepared several stamps and we experimented with different methods of cell
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                         </div>
 
                         </div>
 
                         </div>
 
                         </div>
                         <h1 class="ui left dividing header"><span id="ref-title" class="section">&nbsp;</span>References
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                         <h3 class="ui left dividing header"><span id="ref-title" class="section">&nbsp;</span>References
                         </h1>
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                         </h3>
 
                         <div class="ui segment citing" id="references"></div>
 
                         <div class="ui segment citing" id="references"></div>
 
                     </div>
 
                     </div>

Latest revision as of 17:20, 19 October 2016

Touch painting

 Touch painting

  • Human cells with increased ultrasound sensitivity were shown to respond to other types of mechanical stimulation, such as shear stress and touch.
  • Touch responsive cells were used as a canvas in our sci-art touch painting implementation.

 

While ultrasound was used to stimulate mechanosensors in our project, since it is most appropriate for the stimulation of deep tissue, other mechanical stimuli could also trigger activation of mechanosensors. Mechanical stimulation appears to be a critical modulator for many aspects of biology, both of living tissue and cells Seriani2016 . Mechanosensing can mediate cell proliferation and differentiation Shah2014 . In recent years, many methods of activation of mechanosensitive channels have been implemented. Among the activators of mechanosensors, hyperosmolarity has been shown to trigger the activation of certain genes Veltmann. Additional mechanical stimuli such as fluid shear stress causes activation of embryonic endothelial cells Ranade2014 .

 Results

Therefore our system might be also activated by other mechanical stimuli. One of them was stimulation by direct contact that underlies the sense of touch. Our designed mechano-responsive system is composed of two modules. Firstly we included modules to provide and increase the sensitivity to mechanical stress by mechanoresponsive ion channels and gas vesicles. Secondly, the influx of calcium is visualized by the dimerization of calmodulin (attached to the C terminus of split luciferase) and M13 (attached to the N terminus of split luciferase) which results in bioluminescence after the reconstitution of split luciferase. This means that this system is able to convert the mechanical stimulus into light signal, or to put it differently, with our system we are able to see when cells are touched.

Shear flow also exerts mechanical force on cells, which also has relevance for the endothelial cells within blood vessels, therefore also the flow of the liquid medium around cells might be sensed by cells. The experiment to test this ability was performed by agitating the petri dish with attached cells. In order to identify if the engineered cells are able to respond to the shear flow we included control cells that either constitutively express the luciferase and cells that harbour only the calcium-dependent reporter without constructs to increase the mechanosensing along with cells expressing the gas vesicles and reporters.

Mechanical stimulation of cells by manual agitation

Firefly luciferase activity before and immediately after manual agitation is shown. 24 hours after the transfection, luciferin and calcium were added to the cell medium. The cells were shaken a few times and the response was measured via bioluminescence imaging.

Results show that the light was emitted only in cells with the constitutive luciferase if the plate has not been moved (1). On the other hand shaking triggered activation of the luciferase, with significantly higher level in cells that harboured gas vesicles in addition to the reporter, demonstrating that activation does not require high pressure on cells.

Next we wondered if cells could respond to a gentle touch. We were excited to see that already in the first experiment we could clearly see where cells have been touched by a glass rod. The best paintbush to stimulate cells without removing them from the plate has yet to be identified but we found that glass rod functions quite well. We realized that we could use touch to illuminate cells, effectively painting or drawing on cells by recording the trace with emitted light, which we called Touchpaint. At that time the Argentinian visual artist Laura Olalde, PhD, interested in arts and science symbiosis, and interested in connections between art and science visited our lab. She was immediately eager to test Touchpaint as the new artistic medium, which enables us to transfer the sense of touch into light.

The immediate results were visible from drawing of symbols of our project on cells that were immediately (within 5 min) visualized by imager.

Synthetic mechano-responsive calcium sensing system enables visualization of calcium influx after mechanical stimulation.

(A) Schematic of a cell with increased sensitivity to mechanical stimulation due to expression of mechanosensitive ion channels MscS and gas vesicle-forming proteins. Split calcium sensing system based on split firefly luciferase linked to M13 and calmodulin is shown. (B) Images of petri dishes seeded with HEK293 cells transfected with mechanosensing enhancers and luciferase reporters after stimulation of cells with glass rod. HEK293 cells were transfected with split calcium sensors. 24 h after transfection cells were stimulated by touching with a glass rod. Afterwards, camera images were taken in darkness with exposure time 30 s.

Emitted light was recorded with an imager by capturing light for 30 sec. We observed that the drawn lines were fading with time, which suggested that the response is quite fast. Therefore, we sequentially recorded separately drawn letters to obtain presented images (2).

Video of the procedure of Laura drawing on cells and imaging of each letter on the imager.


Laura prepared several stamps and we experimented with different methods of cell immobilization on the plate, however due to the time constraints of the iGEM project we have yet to explore the intricacies of Touchpaint techniques. Response of the system is quite fast as the sequential drawing of letters of the iGEM acronym already started to fade by drawing the text letter few minutes after the first one, therefore real time monitoring of cell drawing is a very realistic proposition. While Touchpaint is just one artistic implementation of the technology, there are many other scientific and applied uses of this technology, which are discussed in the Impact section.

 References