(5 intermediate revisions by the same user not shown) | |||
Line 73: | Line 73: | ||
<!-- menu goes here --> | <!-- menu goes here --> | ||
<!-- content goes here --> | <!-- content goes here --> | ||
− | <div class="main ui citing justified container"><h1 class = "ui left dividing header"><span class="section colorize"> </span> | + | <div class="main ui citing justified container"><h1 class = "ui left dividing header"><span class="section colorize"> </span>Candidate for best new basic part</h1> |
<div class = "ui segment"> | <div class = "ui segment"> | ||
Line 84: | Line 84: | ||
<p>MscS can be described as an important receptor, involved in the response to ultrasound stimulation. We used the MscS channel as a source of Ca<sup>2+</sup> influx | <p>MscS can be described as an important receptor, involved in the response to ultrasound stimulation. We used the MscS channel as a source of Ca<sup>2+</sup> influx | ||
when stimulated with ultrasound <ref>1</ref>. </p> | when stimulated with ultrasound <ref>1</ref>. </p> | ||
− | <div style=" | + | <div style="margin-left:auto; margin-right:auto; width:50%"> |
<figure data-ref="1"> | <figure data-ref="1"> | ||
<img onclick="resize(this)" src="https://static.igem.org/mediawiki/2016/f/f2/T--Slovenia--S.3.1.1.png"> | <img onclick="resize(this)" src="https://static.igem.org/mediawiki/2016/f/f2/T--Slovenia--S.3.1.1.png"> | ||
Line 141: | Line 141: | ||
− | <p>In an attempt to improve calcium influx, we co-transfected HEK293 cells with the MscS channel and gas vesicle-forming proteins GvpC (BBa_K1965003) | + | <p style="clear:both">In an attempt to improve calcium influx, we co-transfected HEK293 cells with the MscS channel and gas vesicle-forming proteins GvpC (BBa_K1965003) |
and GvpA (BBa_K1965004). The voltage of ultrasound stimulation was decreased to 450 Vpp as higher voltage also causes calcium influx in cells expressing | and GvpA (BBa_K1965004). The voltage of ultrasound stimulation was decreased to 450 Vpp as higher voltage also causes calcium influx in cells expressing | ||
only gas vesicle-forming proteins <a href="https://2016.igem.org/Team:Slovenia/Mechanosensing/Gas_vesicles(">Read more</a>) | only gas vesicle-forming proteins <a href="https://2016.igem.org/Team:Slovenia/Mechanosensing/Gas_vesicles(">Read more</a>) | ||
Line 169: | Line 169: | ||
ultrasound stimulation, confirming that cell response was indeed dependent on the activity of mechanosensitive channels.</p> | ultrasound stimulation, confirming that cell response was indeed dependent on the activity of mechanosensitive channels.</p> | ||
− | <div style="float:left;width: | + | <div style="float:left;width:100%"> |
<figure data-ref="5"> | <figure data-ref="5"> | ||
<img src="https://static.igem.org/mediawiki/2016/4/4c/T--Slovenia--3.3.8.png"> | <img src="https://static.igem.org/mediawiki/2016/4/4c/T--Slovenia--3.3.8.png"> | ||
Line 184: | Line 184: | ||
</div> | </div> | ||
− | <p>The construct was further caracterized under <a href="https://2016.igem.org/Team:Slovenia/Mechanosensing/Mechanosensitive_channels">Mechanosensitive | + | <p style="clear:both">The construct was further caracterized under <a href="https://2016.igem.org/Team:Slovenia/Mechanosensing/Mechanosensitive_channels">Mechanosensitive |
channels</a>, <a href="https://2016.igem.org/Team:Slovenia/Mechanosensing/Gas_vesicles#vesicles">Gas vesicles</a> and <a href="https://2016.igem.org/Team:Slovenia/Proof">Touch | channels</a>, <a href="https://2016.igem.org/Team:Slovenia/Mechanosensing/Gas_vesicles#vesicles">Gas vesicles</a> and <a href="https://2016.igem.org/Team:Slovenia/Proof">Touch | ||
painitng</a>.</p> | painitng</a>.</p> |
Latest revision as of 13:25, 19 October 2016
Candidate for best new basic part
The BioBrick nominated for best part is MscS (BBa_K1965000) from
our Mechanosensing Collection. It contains the coding sequence for the E.coli small-conductance
mechanosensitive channel, MscS. Its role is to mediate turgor regulation in bacteria and it is activated by changes in the osmotic pressure
MscS can be described as an important receptor, involved in the response to ultrasound stimulation. We used the MscS channel as a source of Ca2+ influx when stimulated with ultrasound 1.
Characterization
Expression and subcellular localization of MscS channels in HEK293T cells was inspected. HEK293T cells were transfected with plasmids encoding HA-tagged MscS channel and protein expression was confirmed by western blot analysis, while protein localization was investigated by confocal microscopy.
After confirming MscS expression in HEK293 cells, we stimulated the transfected cells with ultrasound to verify and characterize channel activity. Our experimental setup included an in-house built hardware MODUSON connected to unfocused transducer Olympus V318-SU. To monitor cell response in situ and in real time we used standard ratiometric fluorescent calcium indicators Fura Red, AM and Fluo-4, AM, which can be easily detected with confocal microscopy. When activated, mechanosensitive channels open, leading to calcium influx, which in turn binds the fluorescent calcium indicators. The indicator conformation changes upon calcium binding, resulting in an increase or a decrease of fluorescence.
When cells transfected with a mock plasmid were stimulated with ultrasound, we did not observe calcium influx. On the contrary, when cells transfected with the plasmid, encoding the MscS channel were stimulated with ultrasound, we detected a significant increase in calcium influx 3.
In an attempt to improve calcium influx, we co-transfected HEK293 cells with the MscS channel and gas vesicle-forming proteins GvpC (BBa_K1965003) and GvpA (BBa_K1965004). The voltage of ultrasound stimulation was decreased to 450 Vpp as higher voltage also causes calcium influx in cells expressing only gas vesicle-forming proteins Read more) . By decreasing the voltage of ultrasound stimulation we successfully showed that only cells expressing both the MscS channel and the gas vesicle-forming proteins were activated as a result of ultrasound stimulation 4.
To verify that calcium influx was indeed a result of mechanosensitive channel activity , we used gadolinium (Gd3+), an inhibitor of ion channels, which
is a trivalent ion of the lanthanide series. Due to its high charge density and similar ionic radius to Ca2+
The construct was further caracterized under Mechanosensitive channels, Gas vesicles and Touch painitng.