Difference between revisions of "Part:BBa K2541301"
Line 2: Line 2:
 
__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_K2541301 short</partinfo>
 
<partinfo>BBa_K2541301 short</partinfo>
 +
<partinfo>BBa_K2541101 short</partinfo>
 +
<h5>
 +
<P style="text-indent:2em;">
 +
A RNA-based thermosensor that can be used for temperature sensitive post-transcriptional regulation which is based on the change of RNA advanced structure. The cold-inducible RNA-based thermosensors can initiate translation of downstream genes at low temperatures.
 +
</p>
 +
</h5>
  
−
A RNA thermosensor that can be used for temperature sensitive post-transcriptional regulation which is based on the change of RNA advanced structure. The cold-inducible RNA thermosensors can initiate translation of downstream genes at low temperatures.
+
<h1>'''1. Usage and Biology'''</h1>
−
<!-- -->
+
<h5>
 +
<P style="text-indent:2em;">
 +
There are multiple families of cold-inducible proteins in prokaryotes, the most widely studied of which are the Csp family of cold shock proteins in E. coli. CspA family is represented by cspA, which has been quite extensively investigated. There is a temperature-sensing region in the 5’untranslated region (5'UTR) of CspA mRNA, which can regulate the accessibility of the translation initiation region by altering the advanced structure of RNA, thereby regulating the initiation of translation.
 +
</p>
 +
<P style="text-indent:2em;">
 +
At low temperatures (<20℃), 5’UTR of cspA mRNA can form an advanced structure called pseudoknot, which is more efficiently translated because the conformation exposes the Shine–Dalgarno (SD) sequence, it is beneficial to recruit ribosomes and somewhat less susceptible to degradation. At normal temperatures, due to thermodynamic instability, pseudoknot unfolds. 5’UTR forms a secondary structure masking Shine–Dalgarno (SD) sequence to block translation initiation region, which impedes translation. We designed a series of cold-inducible RNA-based thermosensors with different melting temperature, intensity and sensitivity based on the pseudoknot structure.
 +
</p>
 +
<P style="text-indent:2em;">
 +
Our team designed synthetic cold-inducible RNA-based thermosensors that are considerably simpler than naturally occurring cspA thermosensors and can be exploited as convenient on/off switches of gene expression.
 +
</p>
 +
</h5>
 +
<html><center>
 +
<div class="stem-loop cold-induced">
  
−
<h1>'''Usage and Biology'''</h1>
+
    <input id="checked_3" type="checkbox" class="switch" />
−
There are multiple families of cold-inducible proteins in prokaryotes, the most widely studied of which are the Csp family of cold shock proteins in E. coli. CspA family is represented by cspA, which has been quite extensively investigated. There is a temperature-sensing region in the 5'UTR of CspA mRNA, which can regulate the accessibility of the translation initiation region by altering the advanced structure of RNA, thereby regulating the initiation of translation. At low temperatures (<20℃), 5’UTR of cspA mRNA can form an advanced structure called pseudoknot, which is more efficiently translated because the conformation exposes the Shine–Dalgarno (SD) sequence, it is beneficial to recruit ribosomes and somewhat less susceptible to degradation. At normal temperatures, due to thermodynamic instability, pseudoknot unfolds. 5’UTR forms a secondary structure masking Shine–Dalgarno (SD) sequence to block translation initiation region, which impedes translation. In our design, we deleted the conserved region called the cold box upstream of the 5'UTR of cspA mRNA, so that the expression of CspA is not regulated by its own negative feedback. The pseudoknot in the cspA mRNA contains four sets of base pairings, and its stability is temperature-regulated. We increase base pairing or increase GC content, which may increase the temperature threshold for pseudoknot unfolding; we reduce base pairing or reduce GC content, which may cause the temperature threshold for pseudoknot unfolding to drop. Our team designed synthetic cold-inducible RNA thermosensors that are considerably simpler than naturally occurring cspA thermosensors and can be exploited as convenient on/off switches of gene expression.
+
  
−
<!-- -->
+
  <svg class="svg_animate" xmlns="http://www.w3.org/2000/svg" width="50%" viewBox="0 0 374.12 206.26">
−
<h1>'''Characterization'''</h1>
+
  <defs>
−
The thermosensor is constructed on the pSB1C3 vector by GoldenGate assembly. As shown below, the measurement device is composed of Anderson promotor (BBa_J23100), thermosensor (BBa_K2541301) and sfGFP(BBa_K2541400). We measured the sfGFP expression to get the actual melting temperature of the cold-inducible RNA thermosensor.
+
    <style>
  
 +
.switch::before{
 +
content:"";
 +
display:block;
 +
height:2rem;
 +
width:4rem;
 +
position:absolute;
 +
}
 +
.switch{
 +
display:block;
 +
height:2rem;
 +
width:4rem;
 +
opacity:1;
 +
border-radius:2rem;
 +
background: #00B8FF;
 +
-webkit-appearance: none;
 +
cursor:pointer;
 +
border:0;
 +
position:relative;
 +
transition:all 0.3s ease;
 +
margin:2rem;
 +
}
 +
.switch::after{
 +
content:"";
 +
position:absolute;
 +
display:block;
 +
height:2rem;
 +
width:2rem;
 +
border-radius:2rem;
 +
background: white;
 +
box-shadow: 0 0 1rem rgba(0,0,0,0.2);
 +
box-sizing:border-box;
 +
transition:all 0.3s ease;
 +
transform:translateX(0rem);
 +
}
 +
.switch:checked::after{
 +
transform:translateX(2rem);
 +
}
 +
.switch:checked{
 +
background: #FFAB63;
 +
}
  
−
As shown in the figure, the thermosensor melting temperature range is [   ]. Our data show that efficient RNA thermosensors can be built from RNA advanced structure masking the ribosome binding site, thus providing useful RNA-based toolkit for the regulation of gene expression.
+
      .cold-induced .a,.cold-induced .b,.cold-induced .c {
−
<!-- -->
+
        fill: none;
 +
        stroke-linecap: round;
 +
        stroke-linejoin: round;
 +
        stroke-width: 11px;
 +
      }
 +
.cold-induced .a {
 +
        stroke: #036;
 +
      }
 +
.cold-induced .b {
 +
        stroke: #060;
 +
      }
 +
.cold-induced .c {
 +
        stroke: #600;
 +
      }
 +
      .cold-induced .l{
 +
        fill: none;
 +
        stroke-linecap: round;
 +
        stroke-linejoin: round;
 +
stroke: #000;
 +
        stroke-width: 11px;
 +
      }
 +
      .hydrogen-bond {
 +
        opacity: 1;
 +
transition: all 0.3s ease 1s;
 +
      }
 +
.rotate{
 +
transition: all 1s ease;
 +
transform: rotate(90deg);
 +
}
 +
.r1{
 +
transform-origin: 41% 85.7%;
 +
transition-delay:0.5s;
 +
}
 +
.r2{
 +
transform-origin: 54% 14.5%;
 +
}
 +
.cold-induced.svg_checked .rotate{
 +
transform: rotate(30deg);
 +
}
 +
.cold-induced.svg_checked .hydrogen-bond{
 +
opacity:0;
 +
transition: all 0.3s ease 0s;
 +
}
 +
 +
    </style>
 +
   </defs>
 +
  <g>
 +
    <path class="l rotate r1" d="M177.06,176.54a24.23,24.23,0,0,1-24.23,24.22H5.5"/>
 +
    <path class="l" d="M201.28,5.5a24.22,24.22,0,0,0-24.22,24.22V176.54a24.23,24.23,0,0,1-24.23,24.22"/>
 +
    <path class="l rotate r2" d="M348.62,5.5H201.28a24.22,24.22,0,0,0-24.22,24.22"/>
 +
    <g class="hydrogen-bond">
 +
      <line class="a" x1="219.21" y1="86.31" x2="183.35" y2="86.31"/>
 +
      <line class="b" x1="219.21" y1="67.42" x2="183.35" y2="67.42"/>
 +
      <line class="c" x1="219.21" y1="48.54" x2="183.35" y2="48.54"/>
 +
    </g>
 +
    <g class="hydrogen-bond">
 +
      <line class="a" x1="170.76" y1="150.31" x2="134.9" y2="150.31"/>
 +
      <line class="b" x1="170.76" y1="169.2" x2="134.9" y2="169.2"/>
 +
      <line class="c" x1="170.76" y1="131.42" x2="134.9" y2="131.42"/>
 +
      <line class="a" x1="170.76" y1="112.54" x2="134.9" y2="112.54"/>
 +
    </g>
 +
  </g>
 +
  <p>Figure 1. Mechanism of cold-inducible RNA-based thermosensors.</p>
 +
</svg>
  
  
−
<span class='h3bb'>Sequence and Features</span>
+
  <script type="text/javascript">
−
<partinfo>BBa_K2541301 SequenceAndFeatures</partinfo>
+
    $(document).ready(function(){
 +
      $("#checked_3").click(function(){
 +
        if($(this).is(':checked')){
 +
          $(".cold-induced").addClass("svg_checked");
 +
        }else{
 +
          $(".cold-induced").removeClass("svg_checked");
 +
      }
 +
      });
 +
    });
 +
  </script>
 +
  </div></center>
 +
</html>
  
  
 +
<span class='h3bb'>Sequence and Features</span>
 +
<partinfo>BBa_K2541301 SequenceAndFeatures</partinfo>
 
<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  
 
===Functional Parameters===
 
===Functional Parameters===
 
<partinfo>BBa_K2541301 parameters</partinfo>
 
<partinfo>BBa_K2541301 parameters</partinfo>
 
<!-- -->
 
<!-- -->

Revision as of 06:41, 13 October 2018


Cold-inducible RNA-based thermosensor-1 Heat-repressible RNA-based thermosensor-1

A RNA-based thermosensor that can be used for temperature sensitive post-transcriptional regulation which is based on the change of RNA advanced structure. The cold-inducible RNA-based thermosensors can initiate translation of downstream genes at low temperatures.

1. Usage and Biology

There are multiple families of cold-inducible proteins in prokaryotes, the most widely studied of which are the Csp family of cold shock proteins in E. coli. CspA family is represented by cspA, which has been quite extensively investigated. There is a temperature-sensing region in the 5’untranslated region (5'UTR) of CspA mRNA, which can regulate the accessibility of the translation initiation region by altering the advanced structure of RNA, thereby regulating the initiation of translation.

At low temperatures (<20℃), 5’UTR of cspA mRNA can form an advanced structure called pseudoknot, which is more efficiently translated because the conformation exposes the Shine–Dalgarno (SD) sequence, it is beneficial to recruit ribosomes and somewhat less susceptible to degradation. At normal temperatures, due to thermodynamic instability, pseudoknot unfolds. 5’UTR forms a secondary structure masking Shine–Dalgarno (SD) sequence to block translation initiation region, which impedes translation. We designed a series of cold-inducible RNA-based thermosensors with different melting temperature, intensity and sensitivity based on the pseudoknot structure.

Our team designed synthetic cold-inducible RNA-based thermosensors that are considerably simpler than naturally occurring cspA thermosensors and can be exploited as convenient on/off switches of gene expression.

Figure 1. Mechanism of cold-inducible RNA-based thermosensors.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]