Difference between revisions of "Team:Slovenia/Idea/Solution"

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<a class="item" href="#intro" style="margin-left: 10%">
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<b>Home</b>
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<b>Challenge 1</b>
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<b>Project</b>
 
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<b>Challenge 2</b>
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<b>Achievements</b>
 
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<b>Challenge 3</b>
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<b>Medal requirements</b>
 
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<b>Solutions</b>
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<h1 class = "ui centered dividing header"><span class="section">nbsp;</span></h1>
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<h1 class = "ui centered dividing header"><span class="section">&nbsp;</span></h1>
 
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<h3><span id="c1" class = "section"> &nbsp; </span>Challenge 1: Noninvasive activation of cells in the tissue</h3>
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<h3>Enhanced sensitivity of mechano-sensors and their coupling to the signaling</h3>
<p>Among the signals that can be used to activate engineered cells, light seems to have the best properties – it can be instantly turned on and off, it can be  
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<div style="float:right">
restricted to the selected location and its intensity can be adjusted, which makes it in many ways superior to stimulation by chemicals, temperature or pH and is
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<figure data-ref="1">
responsible for the popularity of optogenetics. Its only apparent disadvantage is that most tissues are poorly transparent to light, which can therefore penetrate only
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<img class="ui medium image" src="https://static.igem.org/mediawiki/2016/0/03/T--Slovenia--S.2.1.png">
short distance. Therefore, to stimulate cells in deep tissue, such as in the brain, invasive procedures have to be used. We envisioned, as an attractive
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<figcaption><b> Enhancement of cell sensitivity to mechnostimuli by the ectopic expression of mechanosensing ion channels and gas vesicles.</b><br/>
alternative signal, induction of cells with ultrasound, which can readily penetrate deep into tissues, has been proven harmless unless used at very high power, can be
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Gas vesicles sensitize cells to respond to ultrasound which triggers opening of mechanosensitive calcium channels. Influx of free calcium ions
regulated in duration and intensity similar to light, and can also be focused. Ultrasound has been reported to activate mechanoreceptors; therefore the challenge was
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activates the calcium-responsive reporters and reconstitutes a mediator to activate the downstream signaling pathway.</figcaption>
to <b>increase the sensitivity of cellular mechanosensing and then detect the activation of mechanosensors and couple it to selected signaling processes.</b> Achieving these milestones would <b>usher in the era of mechanogenetics/sonogenetics.</b>
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</figure>
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</div>
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<p>The challenge of increasing the sensitivity of cells to ultrasound or other mechanic stimuli could be solved by overexpression of ultrasound responsive ion channels.
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Mechanosensitive promoters have been investigated by the <a href="https://2010.igem.org/Team:MIT_mammalian_Mechanosensation">MIT 2010 team</a>, however we were interested
 +
in mechanosensitive receptors. There are reports in the literature that some TRP family mammalian channels and bacterial channels (MscS and MscL) are responsive to  
 +
osmotic shock, which lead us to hypothesize that they should also be able to respond to ultrasound. An additional idea for the enhancement of the mechanosensors occurred
 +
to us through browsing through some previous iGEM projects. Protein gas vesicles have been used in bacteria by several iGEM teams
 +
(<a href="https://2007.igem.org/wiki/index.php/Melbourne/Gas_vesicles">Melbourne 2007</a>, <a href="https://2009.igem.org/Team:Groningen/Project/Vesicle">Groningen 2009
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</a>, <a href="https://2012.igem.org/Team:University_College_London/Module_4">UCL2012</a>, <a href="https://2014.igem.org/Team:Glasgow/Project/Measurements/Gas_Vesicles">
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Glasgow2014</a>) and <a href="https://2012.igem.org/Team:OUC-China/Project/GVP/GasandBackground">OUC-China team</a> reported in 2012 that they could reconstitute gas
 +
vesicles in E. coli from only two components. Gas vesicles, in contrast to cytosol, are compressible, so application of pressure waves to the gas vesicles should
 +
strongly affect the cellular cytoskeleton and the membrane of cells containing such gas vesicles. If we could successfully reconstitute gas vesicles in mammalian cells
 +
this could strongly increase the response of cells to mechanical stimuli and ultrasound.
 
</p>
 
</p>
</div>
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<p>Activation of mechanosensing receptors usually leads to an influx of calcium ions into cells, which might thereby be coupled to reporters or selected
<div class = "ui segment">
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signaling pathways via formation of a calcium dependent complex. Several calcium responsive protein probes have been reported in the past, such as those based on  
<h3><span id="c2" class = "section"> &nbsp; </span>Challenge 2: Engineering fast cellular response</h3>
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calmodulin, and we set out to identify the design that would efficiently respond within the range of calcium ion concentrations triggered by mechanosensor stimulation
<p>One of the important challenges of synthetic biology is to achieve faster response of cells to different stimuli. While most engineered genetic circuits are based
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(<ref>1</ref>).
on transcriptional regulation, which has a delay due to the transcription/activation, cells are also able to respond faster using signaling pathways based on protein
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interactions or their modifications. Fast response is particularly important, for example, for the release of hormones such as insulin or neurotransmitters, where the
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response is required within minutes. Engineering new orthogonal signaling pathways without interfering with the normal cellular processes may be quite demanding,
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particularly for a complex response combining several inputs. Simple transfer of a signaling pathway from another organism may be feasible; however, engineering the
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desired response by adapting an existing signaling pathway may be difficult. The challenge was therefore to <b>build a modular protein interaction/modification-based
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signaling and logic processing pathway that would provide a faster response than transcriptional regulation.</b>
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</p>
 
</p>
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</div>
 
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<h3><span id="c3" class = "section"> &nbsp; </span>Challenge 3: Fast secretion of target protein from cells</h3>
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<h3>Proteolysis based signaling pathway and logic function processing</h3>
<p>Fast signal processing may not be sufficient to result in a fast benefit to the biological system. In addition to detection by reporters cells need to rapidly
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<div style = "float:right">
demonstrate a new phenotype or send output signals to the environment. Therefore a fast response should not depend on the new synthesis of molecules but rather on  
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<figure data-ref="2">
release or modification of already synthesized molecules. The challenge was therefore to design a system where the <b>already synthesized proteins would be released
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<img class="ui large image" src="https://static.igem.org/mediawiki/2016/6/67/T--Slovenia--S.2.2.png">
from the cells upon introduction of a signal. </b> This has been reported before to function through the addition of chemicals to disassemble protein aggregates,
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<figcaption style="justified"><b>Site specific orthogonal split proteases can be used to design a protease-based signaling pathway or complex logic function processing
however we wanted the <b>complete system to be genetically encoded.</b>
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cellular circuit. Proteases can also trigger the controlled rapid release of protein from endoplasmic reticulum</b><br/></figcaption>
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</figure>
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</div>
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<p>Our idea was to use highly specific proteolysis to mediate protein signaling. In comparison to phosphorylation, proteolysis is irreversible, but could nevertheless
 +
lead to a response as fast as the one based on phosphorylation, regardless of the longer time to replenish the degraded proteins once the signal is no longer present.
 +
This is in fact sufficient for most envisioned therapeutic applications. Cleavage of a protein can either lead to activation or inactivation of the selected protein and
 +
could be engineered by introduction of a target cleavage site into the protein of interest. Some proteases have highly specific recognition sites, such as for example
 +
thrombin or the Tobacco etch virus protease (TEVp). This high specificity prevents toxicity of activated proteases to cells due to degradation of essential proteins and
 +
activation of cell death.  To use proteases as signal mediators, we need the activation of the proteases to be triggered by a signal. We reasoned this could be achieved
 +
by reconstitution of split proteases, either by an external signal or by cleavage of peptides preventing reconstitution. The latter would also allow the engineering of
 +
a cascade of the proteolytic activity with each enzyme activating a downstream protein with an introduced specific target site, functioning as a linear signaling pathway.
 
</p>
 
</p>
 +
<p>Split proteases could also be used to combine several input signals. To test this hypothesis we would need to expand the toolbox of highly specific proteases
 +
(similar to TEVp) that should be highly orthogonal and harmless to cells, design appropriate split variants of these proteases and design a system where proteolytic
 +
cleavage results in activation. Such a system was previously designed based on competition between linked coiled-coil domains, however it has only been reported in
 +
vitro <x-ref>Shekhawat2009</x-ref>. Furthermore, the reported system was not based on split protease reconstitution and was therefore not responsive to external inputs.
 +
</p>
 +
<p>Finally, we realized that site specific proteolysis could also be used to solve the challenge of the release of molecules from cells. Secretion of proteins from cells
 +
occurs via the ER and the secretory pathway, while the retention of proteins in the ER occurs via the ER retrieval signals. Proteolytic cleavage of the retrieval peptide
 +
could release the selected cargo proteins or peptides into the secretory pathway, and result in protein secretion from the pool of already synthesized
 +
proteins <ref>2</ref>.
 +
</p>
 +
  
 
 
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Revision as of 20:42, 17 October 2016

Solution

 

Enhanced sensitivity of mechano-sensors and their coupling to the signaling

Enhancement of cell sensitivity to mechnostimuli by the ectopic expression of mechanosensing ion channels and gas vesicles.
Gas vesicles sensitize cells to respond to ultrasound which triggers opening of mechanosensitive calcium channels. Influx of free calcium ions activates the calcium-responsive reporters and reconstitutes a mediator to activate the downstream signaling pathway.

The challenge of increasing the sensitivity of cells to ultrasound or other mechanic stimuli could be solved by overexpression of ultrasound responsive ion channels. Mechanosensitive promoters have been investigated by the MIT 2010 team, however we were interested in mechanosensitive receptors. There are reports in the literature that some TRP family mammalian channels and bacterial channels (MscS and MscL) are responsive to osmotic shock, which lead us to hypothesize that they should also be able to respond to ultrasound. An additional idea for the enhancement of the mechanosensors occurred to us through browsing through some previous iGEM projects. Protein gas vesicles have been used in bacteria by several iGEM teams (Melbourne 2007, Groningen 2009 , UCL2012, Glasgow2014) and OUC-China team reported in 2012 that they could reconstitute gas vesicles in E. coli from only two components. Gas vesicles, in contrast to cytosol, are compressible, so application of pressure waves to the gas vesicles should strongly affect the cellular cytoskeleton and the membrane of cells containing such gas vesicles. If we could successfully reconstitute gas vesicles in mammalian cells this could strongly increase the response of cells to mechanical stimuli and ultrasound.

Activation of mechanosensing receptors usually leads to an influx of calcium ions into cells, which might thereby be coupled to reporters or selected signaling pathways via formation of a calcium dependent complex. Several calcium responsive protein probes have been reported in the past, such as those based on calmodulin, and we set out to identify the design that would efficiently respond within the range of calcium ion concentrations triggered by mechanosensor stimulation (1).

Proteolysis based signaling pathway and logic function processing

Site specific orthogonal split proteases can be used to design a protease-based signaling pathway or complex logic function processing cellular circuit. Proteases can also trigger the controlled rapid release of protein from endoplasmic reticulum

Our idea was to use highly specific proteolysis to mediate protein signaling. In comparison to phosphorylation, proteolysis is irreversible, but could nevertheless lead to a response as fast as the one based on phosphorylation, regardless of the longer time to replenish the degraded proteins once the signal is no longer present. This is in fact sufficient for most envisioned therapeutic applications. Cleavage of a protein can either lead to activation or inactivation of the selected protein and could be engineered by introduction of a target cleavage site into the protein of interest. Some proteases have highly specific recognition sites, such as for example thrombin or the Tobacco etch virus protease (TEVp). This high specificity prevents toxicity of activated proteases to cells due to degradation of essential proteins and activation of cell death. To use proteases as signal mediators, we need the activation of the proteases to be triggered by a signal. We reasoned this could be achieved by reconstitution of split proteases, either by an external signal or by cleavage of peptides preventing reconstitution. The latter would also allow the engineering of a cascade of the proteolytic activity with each enzyme activating a downstream protein with an introduced specific target site, functioning as a linear signaling pathway.

Split proteases could also be used to combine several input signals. To test this hypothesis we would need to expand the toolbox of highly specific proteases (similar to TEVp) that should be highly orthogonal and harmless to cells, design appropriate split variants of these proteases and design a system where proteolytic cleavage results in activation. Such a system was previously designed based on competition between linked coiled-coil domains, however it has only been reported in vitro Shekhawat2009. Furthermore, the reported system was not based on split protease reconstitution and was therefore not responsive to external inputs.

Finally, we realized that site specific proteolysis could also be used to solve the challenge of the release of molecules from cells. Secretion of proteins from cells occurs via the ER and the secretory pathway, while the retention of proteins in the ER occurs via the ER retrieval signals. Proteolytic cleavage of the retrieval peptide could release the selected cargo proteins or peptides into the secretory pathway, and result in protein secretion from the pool of already synthesized proteins 2.