Difference between revisions of "Part:BBa K2541101"
Line 2: Line 2:
 
__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_K2541101 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 sencondary structure. The heat-repressible RNA-based thermosensors can repress translation of downstream genes at high temperatures.
 +
</p>
 +
</h5>
  
−
A RNA thermosensor that can be used for temperature sensitive post-transcriptional regulation which is based on the change of RNA sencondary structure. The heat-repressible RNA thermosensors can repress translation of downstream genes at high temperatures.
+
<h1>'''1. Usage and Biology'''</h1>
 +
<h5>
 +
<P style="text-indent:2em;">
 +
RNA-based temperature sensing is common in bacteria that live in fluctuating environments. Most naturally occurring RNA-based thermosensors are heat inducible, have long sequences, and function by sequestering the Shine–Dalgarno (SD) sequence in a stem-loop structure at lower temperatures.
 +
</p>
 +
<P style="text-indent:2em;">
 +
Here, we designed short, heat-repressible RNA-based thermosensors. These thermosensors contain a single-strand RNA cleavage (RC) site for RNase E, an enzyme native to Escherichia coli and many other organisms. Each heat-repressible RNA-based thermosensor sequence was inserted downstream of the transcription start site and upstream of the SD sequence. At high temperatures, the RNase E cleavage site (RC) is exposed, mRNA was cleaved by RNase E, and expression is ‘OFF’. At low temperatures, the RC binds to the anti-RNase E cleavage site (ARC) and forms a stem-loop. This structure sequesters the RC, and expression is ‘ON’.
 +
</p>
 +
<P style="text-indent:2em;">
 +
These short, modular heat-repressible RNA-based thermosensors can be applied to the construction of complex genetic circuits, facilitating rational reprogramming of cellular processes for synthetic biology applications.
 +
</p>
 +
</h5>
 +
<html><center>
 +
  <div class="stem-loop hot-repressive">
  
−
<h1>'''Usage and Biology'''</h1>
+
    <input id="checked_2" type="checkbox" class="switch" />
−
RNA-based temperature sensing is common in bacteria that live in fluctuating environments. Most naturally-occurring RNA thermosensors are heat-inducible, have long sequences, and function by sequestering the ribosome binding site in a stem-loop structure at lower temperatures. Here, we designed short, heat-repressible RNA thermosensors. These thermosensors contain a single-strand RNA cleavage site for RNase E, an enzyme native to Escherichia coli and many other organisms, in the 5' untranslated region of the target gene. At low temperatures, the cleavage site is sequestered in a stem-loop, and gene expression is unobstructed. At elevated temperatures, the stem-loop unfolds, allowing for mRNA degradation and turning off expression. These short, modular heat-repressible RNA thermosensors can be applied to the construction of complex genetic circuits, facilitating rational reprogramming of cellular processes for synthetic biology applications.
+
  
−
<!-- -->
 
−
<h1>'''Characterization'''</h1>
 
−
The thermosensor is constructed on the pSB1C3 vector by GoldenGate assembly. As shown below, the measurement device is composed of Anderson promotor (BBa_J23106), thermosensor (BBa_K2541101) and sfGFP(BBa_K2541400). We measured the sfGFP expression to get the state of the heat-repressible RNA thermosensor at different temperatures.
 
  
 +
  <svg class="svg_animate" xmlns="http://www.w3.org/2000/svg" viewBox="0 0 599.75 278.7">
 +
    <defs>
 +
    <style>
  
−
As shown in the figure, the thermosensor is "off" at [  ]. Our data show that efficient RNA thermosensors can be built from a single small RNA stem-loop structure masking the ribosome binding site, thus providing useful RNA-based toolkit for the regulation of gene expression.
+
.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;
 +
}
  
 +
      .hot-repressive .a, .b, .c, .f {
 +
    fill: none;
 +
    stroke-width: 11px;
 +
      }
  
−
<span class='h3bb'>Sequence and Features</span>
+
      .hot-repressive .a {
−
<partinfo>BBa_K2541101 SequenceAndFeatures</partinfo>
+
    stroke: #036;
 +
      }
  
 +
      .hot-repressive .a,.hot-repressive  .b,.hot-repressive  .c,.hot-repressive  .f,.hot-repressive  .g {
 +
    stroke-linecap: round;
 +
    stroke-linejoin: round;
 +
      }
  
 +
      .hot-repressive .b {
 +
    stroke: #060;
 +
      }
 +
 +
      .hot-repressive .c {
 +
    stroke: #600;
 +
      }
 +
 +
      .hot-repressive .d,.hot-repressive  .e,.hot-repressive  .h{
 +
    font-size: 17px;
 +
    font-family: Quicksand-Medium, Quicksand;
 +
      }
 +
 +
      .hot-repressive .d {
 +
    fill: #006837;
 +
      }
 +
 +
      .hot-repressive .e {
 +
    fill: #900;
 +
      }
 +
 +
      .hot-repressive .f {
 +
    stroke: #000;
 +
      }
 +
 +
    .hot-repressive .h {
 +
    fill: #930;
 +
      }
 +
 +
      .hot-repressive .g {
 +
    fill: #fff794;
 +
    stroke: #fbb03b;
 +
    stroke-width: 7px;
 +
      }
 +
    .hot-repressive .hydrogen-bond{
 +
      opacity:1;
 +
      transition: all 0.3s linear 1s;
 +
    }
 +
    .hot-repressive #RNAse_E{
 +
      transform: translateY(-60px);
 +
      opacity:0;
 +
      transition: all 1s ease-out 0s;
 +
    }
 +
    .hot-repressive #ribosome_text{
 +
      opacity:0;
 +
      transition: all 0.3s linear 0s;
 +
    }
 +
    .hot-repressive #text{
 +
      transform: translateY(0px);
 +
      transition: all 1s ease-out;
 +
    }
 +
    .hot-repressive #loop{
 +
      transform: translateY(0px);
 +
      transition: all 2s ease-out;
 +
    }
 +
    .hot-repressive #left,#right{
 +
      stroke-dasharray:130,500;
 +
      transition: all 2s ease-out;
 +
    }
 +
    .hot-repressive #left{
 +
      stroke-dashoffset:0;
 +
      stroke:red;
 +
    }
 +
    .hot-repressive #right{
 +
      stroke-dashoffset:-277;
 +
      stroke:green;
 +
    }
 +
    .hot-repressive #left_s,#right_s{
 +
      stroke-dasharray:60,500;
 +
      color:black;
 +
      transition: all 2s ease-out;
 +
    }
 +
    .hot-repressive #left_s{
 +
      stroke-dashoffset:-130;
 +
    }
 +
    .hot-repressive #right_s{
 +
      stroke-dashoffset:-217;
 +
    }
 +
 +
    .hot-repressive.svg_checked .hydrogen-bond{
 +
      opacity:0;
 +
      transition: all 0.3s linear 0s;
 +
    }
 +
    .hot-repressive.svg_checked #RNAse_E{
 +
      transform: translateY(0px);
 +
      opacity:1;
 +
      transition: all 0.5s ease-out 2s;
 +
    }
 +
    .hot-repressive.svg_checked #ribosome_text{
 +
      opacity:1;
 +
      transition: all 0.5s ease-out 2s;
 +
    }
 +
    .hot-repressive.svg_checked #text{
 +
      transform: translateY(-60px);
 +
    }
 +
    .hot-repressive.svg_checked #loop{
 +
      transform: translateY(130px);
 +
    }
 +
    .hot-repressive.svg_checked #left{
 +
      stroke-dashoffset:-135;
 +
    }
 +
    .hot-repressive.svg_checked #right{
 +
      stroke-dashoffset:-142;
 +
    }
 +
    .hot-repressive.svg_checked #left_s,#right_s{
 +
      stroke-dasharray:60,500;
 +
    }
 +
    .hot-repressive.svg_checked #left_s{
 +
      stroke-dashoffset:-265;
 +
    }
 +
    .hot-repressive.svg_checked #right_s{
 +
      stroke-dashoffset:-82;
 +
    }
 +
 +
    </style>
 +
    </defs>
 +
    <g>
 +
    <g class="hydrogen-bond">
 +
      <line class="a" x1="284.86" y1="144.21" x2="320.72" y2="144.21"/>
 +
      <line class="b" x1="284.86" y1="125.32" x2="320.72" y2="125.32"/>
 +
      <line class="c" x1="284.86" y1="163.09" x2="320.72" y2="163.09"/>
 +
      <line class="a" x1="284.86" y1="181.98" x2="320.72" y2="181.98"/>
 +
      <line class="b" x1="284.86" y1="200.87" x2="320.72" y2="200.87"/>
 +
      <line class="c" x1="284.86" y1="219.75" x2="320.72" y2="219.75"/>
 +
    </g>
 +
    <g id="text">
 +
      <text class="d" transform="translate(429.01 185.45)">RC</text>
 +
      <text class="e" transform="translate(123.8 185.45)">anti-RC</text>
 +
 +
    </g>
 +
 +
    <text id="ribosome_text" class="h" transform="translate(385.48 175.45)">RNAse E</text>
 +
 +
    <path id="loop" class="f" d="M327,101.42c0-21.25,19.2-20.39,19.2-52.51,0-24-16.62-43.41-43.42-43.41s-43.41,19.44-43.41,43.41c0,28.79,19.19,29.92,19.19,52.51"/>
 +
 +
    <g>
 +
      <path id="right_s" class="f" d="M594.25,247.5l-252.2.21a15,15,0,0,1-15-15V101.42"/>
 +
      <path id="left_s" class="f" d="M278.57,101.42V232.67a15,15,0,0,1-15.05,15H5.5"/>
 +
    </g>
 +
    <g>
 +
      <path id="right" class="f" d="M594.25,247.5l-252.2.21a15,15,0,0,1-15-15V101.42"/>
 +
      <path id="left" class="f" d="M278.57,101.42V232.67a15,15,0,0,1-15.05,15H5.5"/>
 +
    </g> 
 +
<g id="RNAse_E">
 +
 +
      <path class="g" d="M417,187.26a40.56,40.56,0,0,0-39.11,51.23L417,229.77l-24.18,30.57A40.54,40.54,0,1,0,417,187.26Z"/>
 +
  <circle class="g" cx="402.84" cy="214.07" r="1.00"/>
 +
    </g>
 +
    </g>
 +
    <p>Figure 1. Mechanism of heat repressible RNA-based thermosensors.</p>
 +
  </svg>
 +
 +
 +
  <script type="text/javascript">
 +
    $(document).ready(function(){
 +
      $("#checked_2").click(function(){
 +
        if($(this).is(':checked')){
 +
          $(".hot-repressive").addClass("svg_checked");
 +
        }else{
 +
          $(".hot-repressive").removeClass("svg_checked");
 +
      }
 +
      });
 +
    });
 +
  </script>
 +
  </div></center>
 +
</html>
 +
 +
<h1>'''2. Design'''</h1>
 +
<h5>
 +
<P style="text-indent:2em;">
 +
The RC and ARC were considered to be the two separate strands. We keep the RC sequence conserved whie changing the ARC sequence. In order to obtain heat repressible RNA-based thermosensores with different melting temperatures, intensity and sensitivity, we change the stem length, loop size and the mismatches or bulges in the stem.
 +
</p>
 +
<P style="text-indent:2em;">
 +
Stem length is determined by ARC sequence because the RC sequence is conserved. Adding stem length can optimize heat repressible RNA-based thermosensors to more high temperature, while decreasing stem length has the opposite effect. The stem length is X base paring in K2541101.
 +
Loop size can moderate thermosensors melting temperature to a suitable temperature. In K2541101, the loop sequence is (选一个) (AAUAA,AAAUAA, AAAAUAUAAA,AAAAAAUAUAAA, AAAAAUAUAUAUAAAA).
 +
Furthermore, we change base composition in ARC sequence to decrease response temperature. We make a mismatch in the sixth base by changing it from A to T. (or We make a bulge in the sixth base by deleting or inserting A.)
 +
Finally, we get the sequence as the figure 2.
 +
</p>
 +
</h5>
 +
[[File:K2541101 f2.png|center|K2541101 f2]]
 +
Figure 2. Design of synthetic heat repressible RNA-based thermosensor. (A) The RNA secondary structure is predictred by mFOLD. (B) Thermosensor sequence, ARC sequence, loop sequence, the site of mismatch or bulge in the stem, △G and Tm are in the table.
 +
 +
<h1>'''3.Characterization'''</h1>
 +
<h5>
 +
<P style="text-indent:2em;">
 +
The thermosensor sequence is constructed on the pSB1C3 vector by GoldenGate assembly. The measurement device is composed of Anderson promotor (BBa_J23106), thermosensor (BBa_K2541101), sfGFP_optimism (BBa_K2541400) and terminator (BBa_B0015). We select a medium constitutive Anderson promoter J23106 as a suitable promoter by doing pre-experiment. The sfGFP_optimism has faster folding speed and higher fluorescence intensity. The B0015 is a double terminator that can reduce leakge (Figure 3). We characterized RNA-based thermosensors in E.coli DH5a.
 +
</p>
 +
</h5>
 +
[[File:measurement device figure3.png|center|caption]]
 +
<html><center>
 +
Figure 3. The measurement device.
 +
  </script></center>
 +
</html>
 +
----
 +
 +
 +
<h5>
 +
<P style="text-indent:2em;">
 +
On the left side of the figure 4 is K2541101. On the right of figure 4 is positive control. K2415029, K2514013, K2514037 are three heat inducible RNA-based thermosensors with different intensity. The final normalized fluorescence was calculated as follows: normalized fluorescence = [(GFP/Abs)TS - (GFP/Abs)neg] / [(GFP/Abs)pos - (GFP/Abs)neg] ( TS = thermosensor, pos = positive control, and neg = E.coli DH5a ). As shown in the figure 4, the thermosensor K2541001 melting temperature range is ‘off’ from 29°C to 37°C. Its intensity is about equal to K2541037.
 +
</p>
 +
</h5>
 +
[[File:K2541101 f4.png|center|K2541101 f4]]
 +
Figure 4. Characteristics of synthetic heat repressible RNA-based thermosensors. Each set of three bars represents the activity level of a different thermosensor. The bar colors purple, yellow and red represent the temperatures 29, 37 and 42°C, respectively. The height of the bars corresponds to the normalized fluorescence.
 +
----
 +
 +
 +
<h5>
 +
<P style="text-indent:2em;">
 +
This year, our objective is to design a collection of RNA-based thermosensors with different melting temperatures, intensity and sensitivity. We used a combination of experimental measurements and computations of RNA secondary structures to achieve this objective. We studied a set of measured synthetic RNA-based thermosensors, finding consistency with measured results and among our experimental and computational analyses.
 +
</p>
 +
</h5>
 +
[[File:K2514101 f5.png|center|K2514101 f5]]
 +
Figure 5. Temperature response of the heat repressible RNA-based thermosensor. The green solid line represents the activity of the RNA-based thermosensor as a function of temperature. Key quantitative features of the response such as threshold and sensitivity are emphasized.
 +
----
 +
 +
 +
[[File:RE figure6.png|center|RE figure6]]
 +
Figure 6. Experimental measurements of the collection of heat repressible RNA-based thermosensors show a variety of responses. (A) Rows represent activity levels of different thermosensors. (B) Each set of three bars represents the activity level of a different thermosensor. The bar colors purple, yellow and red represent the temperatures 29, 37 and 42°C, respectively. The height of the bars corresponds to the normalized fluorescence.
 +
 +
 +
<h1>'''4. Conclusion'''</h1>
 +
<h5>
 +
<P style="text-indent:2em;">
 +
Our data show that efficient RNA-based thermosensors with different melting temperatures, intensity and sensitivity can be built from a single small RNA stem-loop structure, thus providing useful RNA-based toolkit for the regulation of gene expression.
 +
</p>
 +
</h5>
 +
 +
 +
<span class='h3bb'>Sequence and Features</span>
 +
<partinfo>BBa_K2541101 SequenceAndFeatures</partinfo>
 
<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  
 
===Functional Parameters===
 
===Functional Parameters===
 
<partinfo>BBa_K2541101 parameters</partinfo>
 
<partinfo>BBa_K2541101 parameters</partinfo>
 
<!-- -->
 
<!-- -->

Revision as of 04:02, 13 October 2018


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 sencondary structure. The heat-repressible RNA-based thermosensors can repress translation of downstream genes at high temperatures.

1. Usage and Biology

RNA-based temperature sensing is common in bacteria that live in fluctuating environments. Most naturally occurring RNA-based thermosensors are heat inducible, have long sequences, and function by sequestering the Shine–Dalgarno (SD) sequence in a stem-loop structure at lower temperatures.

Here, we designed short, heat-repressible RNA-based thermosensors. These thermosensors contain a single-strand RNA cleavage (RC) site for RNase E, an enzyme native to Escherichia coli and many other organisms. Each heat-repressible RNA-based thermosensor sequence was inserted downstream of the transcription start site and upstream of the SD sequence. At high temperatures, the RNase E cleavage site (RC) is exposed, mRNA was cleaved by RNase E, and expression is ‘OFF’. At low temperatures, the RC binds to the anti-RNase E cleavage site (ARC) and forms a stem-loop. This structure sequesters the RC, and expression is ‘ON’.

These short, modular heat-repressible RNA-based thermosensors can be applied to the construction of complex genetic circuits, facilitating rational reprogramming of cellular processes for synthetic biology applications.

RC anti-RC RNAse E

Figure 1. Mechanism of heat repressible RNA-based thermosensors.

2. Design

The RC and ARC were considered to be the two separate strands. We keep the RC sequence conserved whie changing the ARC sequence. In order to obtain heat repressible RNA-based thermosensores with different melting temperatures, intensity and sensitivity, we change the stem length, loop size and the mismatches or bulges in the stem.

Stem length is determined by ARC sequence because the RC sequence is conserved. Adding stem length can optimize heat repressible RNA-based thermosensors to more high temperature, while decreasing stem length has the opposite effect. The stem length is X base paring in K2541101. Loop size can moderate thermosensors melting temperature to a suitable temperature. In K2541101, the loop sequence is (选一个) (AAUAA,AAAUAA, AAAAUAUAAA,AAAAAAUAUAAA, AAAAAUAUAUAUAAAA). Furthermore, we change base composition in ARC sequence to decrease response temperature. We make a mismatch in the sixth base by changing it from A to T. (or We make a bulge in the sixth base by deleting or inserting A.) Finally, we get the sequence as the figure 2.

K2541101 f2

Figure 2. Design of synthetic heat repressible RNA-based thermosensor. (A) The RNA secondary structure is predictred by mFOLD. (B) Thermosensor sequence, ARC sequence, loop sequence, the site of mismatch or bulge in the stem, △G and Tm are in the table.

3.Characterization

The thermosensor sequence is constructed on the pSB1C3 vector by GoldenGate assembly. The measurement device is composed of Anderson promotor (BBa_J23106), thermosensor (BBa_K2541101), sfGFP_optimism (BBa_K2541400) and terminator (BBa_B0015). We select a medium constitutive Anderson promoter J23106 as a suitable promoter by doing pre-experiment. The sfGFP_optimism has faster folding speed and higher fluorescence intensity. The B0015 is a double terminator that can reduce leakge (Figure 3). We characterized RNA-based thermosensors in E.coli DH5a.

caption

Figure 3. The measurement device.


On the left side of the figure 4 is K2541101. On the right of figure 4 is positive control. K2415029, K2514013, K2514037 are three heat inducible RNA-based thermosensors with different intensity. The final normalized fluorescence was calculated as follows: normalized fluorescence = [(GFP/Abs)TS - (GFP/Abs)neg] / [(GFP/Abs)pos - (GFP/Abs)neg] ( TS = thermosensor, pos = positive control, and neg = E.coli DH5a ). As shown in the figure 4, the thermosensor K2541001 melting temperature range is ‘off’ from 29°C to 37°C. Its intensity is about equal to K2541037.

K2541101 f4

Figure 4. Characteristics of synthetic heat repressible RNA-based thermosensors. Each set of three bars represents the activity level of a different thermosensor. The bar colors purple, yellow and red represent the temperatures 29, 37 and 42°C, respectively. The height of the bars corresponds to the normalized fluorescence.


This year, our objective is to design a collection of RNA-based thermosensors with different melting temperatures, intensity and sensitivity. We used a combination of experimental measurements and computations of RNA secondary structures to achieve this objective. We studied a set of measured synthetic RNA-based thermosensors, finding consistency with measured results and among our experimental and computational analyses.

K2514101 f5

Figure 5. Temperature response of the heat repressible RNA-based thermosensor. The green solid line represents the activity of the RNA-based thermosensor as a function of temperature. Key quantitative features of the response such as threshold and sensitivity are emphasized.


RE figure6

Figure 6. Experimental measurements of the collection of heat repressible RNA-based thermosensors show a variety of responses. (A) Rows represent activity levels of different thermosensors. (B) Each set of three bars represents the activity level of a different thermosensor. The bar colors purple, yellow and red represent the temperatures 29, 37 and 42°C, respectively. The height of the bars corresponds to the normalized fluorescence.


4. Conclusion

Our data show that efficient RNA-based thermosensors with different melting temperatures, intensity and sensitivity can be built from a single small RNA stem-loop structure, thus providing useful RNA-based toolkit for the regulation of gene expression.


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
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 31