Difference between revisions of "Part:BBa K2541408"
 
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__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_K2541408 short</partinfo>
 
<partinfo>BBa_K2541408 short</partinfo>
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<h5>
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<P style="text-indent:2em;">
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A RNA-based thermosensor that can be used for temperature sensitive translational 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. The composite part is a measurement device, consisting of Anderson promoter (BBa_J23106), cold-inducible RNA-based thermosensor-1 (BBa_K2541301), sfGFP_optimism (BBa_K2541400) and double terminator (BBa_B0010 and BBa_B0012).
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</p>
 +
</h5>
  
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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. The composite part is composed of promoter BBa_J23100, cold-inducible RNA thermosensor-1 BBa_K2541301, reporetr protein sfGFP BBa_K2541400 and terminator BBa_B0015.
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<h1>'''1. Usage and Biology'''</h1>
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<!-- -->
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<h5>
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<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 represents CspA family, which has been quite extensively studied for the mechanism of its cold response. 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 SD sequence to block translation initiation region, which impedes translation. We designed a series of cold-inducible RNA-based thermosensors with different melting temperatures, 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.
 +
</p>
 +
<P style="text-indent:2em;">
 +
Green fluorescent protein (GFP) is commonly used as a reporter gene in intact cells and organisms. This year we select sfGFP (BBa_K2541400), a robustly folded version of GFP, called superfolder GFP as a reporter protein. Compared to superfolder GFP (BBa_I746916), sfGFP_optimism (BBa_K2541400) is BbsI restriction site free, so it can be used in GoldenGate assembly to achieve efficient and rapid assembly of gene fragments. And sfGFP_optimism (BBa_K2541400) has stronger fluorescence intensity than superfolder GFP (BBa_I746916).
 +
</p>
 +
</h5>
 +
<html><center>
 +
<div class="stem-loop cold-induced">
  
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<h1>'''Usage and Biology'''</h1>
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    <input id="checked_3" type="checkbox" class="switch" />
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== Cold-inducible RNA thermosensor-1 ==
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−
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.
+
  
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== sfGFP ==
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  <svg class="svg_animate" xmlns="http://www.w3.org/2000/svg" width="50%" viewBox="0 0 374.12 206.26">
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Green fluorescent protein (GFP) exhibits intrinsic fluorescence and is commonly used as a reporter gene in intact cells and organisms. Many mutants of the protein with either modified spectral properties, increased fluorescence intensity, or improved folding properties have been reported.  
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  <defs>
 +
    <style>
  
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GFP often misfold when expressed as fusions with other proteins, while a robustly folded version of GFP, called superfolder GFP, was developed and described by Pédelacq et al at 2006 that folds well even when fused to poorly folded polypeptides. There is another superfolder GFP designed by Overkamp W et al at 2013, which is codon optimized for S. pneumoniae. It was be used in Escherichia coli by Segall-Shapiro T H et al at 2018.
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transition:all 0.3s ease;
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display:block;
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border-radius:2rem;
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background: white;
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box-shadow: 0 0 1rem rgba(0,0,0,0.2);
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box-sizing:border-box;
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transition:all 0.3s ease;
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transform:translateX(0rem);
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}
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.switch:checked::after{
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transform:translateX(2rem);
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}
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.switch:checked{
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background: #FFAB63;
 +
}
  
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This year our team registered the superfolder GFP designed by Overkamp W et al with a BBa_K2541400 (called sfGFP). Compared to superfolder GFP(BBa_I746916), sfGFP (BBa_K2541400) is BbsI restriction site free, so it can be used in GoldenGate assembly to achieve efficient and
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      .cold-induced .a,.cold-induced .b,.cold-induced .c {
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rapid assembly of gene fragments. And sfGFP (BBa_K2541400) has stronger fluorescence intensity than superfolder GFP(BBa_I746916).
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transition: all 0.3s ease 1s;
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      }
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.rotate{
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transition: all 1s ease;
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transform: rotate(90deg);
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}
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.r1{
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transform-origin: 41% 85.7%;
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transition-delay:0.5s;
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}
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.r2{
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}
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.cold-induced.svg_checked .rotate{
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transform: rotate(30deg);
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}
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.cold-induced.svg_checked .hydrogen-bond{
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opacity:0;
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transition: all 0.3s ease 0s;
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}
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 +
    </style>
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  </defs>
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  <g>
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    <path class="l rotate r1" d="M177.06,176.54a24.23,24.23,0,0,1-24.23,24.22H5.5"/>
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    <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"/>
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    <path class="l rotate r2" d="M348.62,5.5H201.28a24.22,24.22,0,0,0-24.22,24.22"/>
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      <line class="a" x1="219.21" y1="86.31" x2="183.35" y2="86.31"/>
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      <line class="c" x1="219.21" y1="48.54" x2="183.35" y2="48.54"/>
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      <line class="a" x1="170.76" y1="150.31" x2="134.9" y2="150.31"/>
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    </g>
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  </g>
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  <p>Figure 1. Mechanism of cold-inducible RNA-based thermosensors.</p>
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</svg>
  
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== Conclusion ==
 
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The composite part can be used as a measurement device for different cold-inducible RNA thermosensors. Wu use GoldenGate assembly to change differrnt thermosensors to measure their melting temperature.
 
  
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<!-- -->
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  <script type="text/javascript">
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<h1>'''Characterization'''</h1>
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    $(document).ready(function(){
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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) and terminator (BBa_B0015). We measured the sfGFP expression to get the actual melting temperature of the cold-inducible RNA thermosensor.
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      $("#checked_3").click(function(){
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        if($(this).is(':checked')){
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          $(".cold-induced").addClass("svg_checked");
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        }else{
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          $(".cold-induced").removeClass("svg_checked");
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      }
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      });
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    });
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  </script>
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  </div></center>
 +
</html>
  
 +
<h1>'''2. Design'''</h1>
 +
<h5>
 +
<P style="text-indent:2em;">
 +
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. Several structural parameters come into consideration to optimize cold-inducible RNA-based thermosensors: base pairing, base pair position and GC content in pseudoknot region. Increasing base pairing can raise the melting temperature of pseudoknot, thus enlarging the temperature range of open conformation. Increasing GC content can also raise the melting temperature of pseudoknot. We also change base pair at different position. What’s more, we delete the cold box which is at the upstream of ''CspA'' 5’UTR. In K2541301, we added two base pairs (A:U) in the pseudoknot region (figure 2).
 +
</p>
 +
</h5>
 +
[[File:K2541301 f2.png|center|K2541301 f2]]
 +
<center>Figure 2. Design of K2541301.</center>
  
−
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.
+
<h1>'''3.Characterization'''</h1>
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<!-- -->
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<h3>3.1 Measurement device</h3>
 +
<h5>
 +
<P style="text-indent:2em;">
 +
The thermosensor sequence is constructed on the pSB1C3 vector by Golden Gate assembly. The measurement device is composed of Anderson promoter (BBa_J23100), thermosensor (BBa_K2541301), sfGFP_optimism (BBa_K2541400) and double terminator (BBa_B0010 and BBa_B0012). We select a constitutive Anderson promoter J23100 as an appropriate promoter by pre-experiment. The sfGFP_optimism has faster folding speed and higher fluorescence intensity. The double terminator can reduce leakage (Figure 3). We characterized RNA-based thermosensors in ''E.coli'' DH5a.
 +
</p>
 +
</h5>
 +
[[File:measurement device 123.png|center|caption]]
 +
<center>Figure 3. The measurement device.</center>
 +
----
  
 +
<h3>3.2 Measurement results</h3>
 +
<h5>
 +
<P style="text-indent:2em;">
 +
In figure 4, there are eight different cold-inducible RNA-based thermosensors. pos.control is positive control. The final normalized fluorescence was calculated as follows: normalized fluorescence = [(Fluorescence/Abs<sub>600</sub>)<sub>TS</sub> - (Fluorescence/Abs<sub>600</sub>)<sub>neg</sub>] / [(Fluorescence/Abs<sub>600</sub>)<sub>pos</sub> - (Fluorescence/Abs<sub>600</sub>)<sub>neg</sub>] ( TS = thermosensor, pos = positive control, and neg = BBa_J364007 ). As shown in figure 4, the fluorescence intensity of K2541301 increases with decreased temperature.
 +
</p>
 +
</h5>
 +
[[File:K2541301 f4.png|center|K2541301 f4]]
 +
Figure 4. Characteristics of K2541301. Each set of three bars represents the activity level of a different thermosensor. The bar colors purple, yellow and red represent the temperatures 15, 20 and 25°C, respectively. The height of the bars corresponds to the normalized fluorescence.
  
 +
<h1>'''4. Collection of cold-inducible RNA-based thermosensors '''</h1>
 +
[[File:cspA figure6 new.png|center|cspA figure6 new]]
 +
Figure 5. Experimental measurements of the collection of cold-inducible 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 15, 20 and 25°C, respectively. The height of the bars corresponds to the normalized fluorescence.
  
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<span class='h3bb'>Sequence and Features</span>
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<h1>'''5. Conclusion'''</h1>
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<partinfo>BBa_K2541408 SequenceAndFeatures</partinfo>
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<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 RNA advanced structure, thus providing useful SynRT toolkit for the regulation of gene expression.
 +
</p>
 +
</h5>
  
  
 +
 +
<span class='h3bb'>Sequence and Features</span>
 +
<partinfo>BBa_K2541408 SequenceAndFeatures</partinfo>
 
<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  
 
===Functional Parameters===
 
===Functional Parameters===
 
<partinfo>BBa_K2541408 parameters</partinfo>
 
<partinfo>BBa_K2541408 parameters</partinfo>
 
<!-- -->
 
<!-- -->

Latest revision as of 09:10, 16 October 2018


Cold-inducible RNA thermosensor measurement device

A RNA-based thermosensor that can be used for temperature sensitive translational 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. The composite part is a measurement device, consisting of Anderson promoter (BBa_J23106), cold-inducible RNA-based thermosensor-1 (BBa_K2541301), sfGFP_optimism (BBa_K2541400) and double terminator (BBa_B0010 and BBa_B0012).

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 represents CspA family, which has been quite extensively studied for the mechanism of its cold response. 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 SD sequence to block translation initiation region, which impedes translation. We designed a series of cold-inducible RNA-based thermosensors with different melting temperatures, 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.

Green fluorescent protein (GFP) is commonly used as a reporter gene in intact cells and organisms. This year we select sfGFP (BBa_K2541400), a robustly folded version of GFP, called superfolder GFP as a reporter protein. Compared to superfolder GFP (BBa_I746916), sfGFP_optimism (BBa_K2541400) is BbsI restriction site free, so it can be used in GoldenGate assembly to achieve efficient and rapid assembly of gene fragments. And sfGFP_optimism (BBa_K2541400) has stronger fluorescence intensity than superfolder GFP (BBa_I746916).

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

2. Design

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. Several structural parameters come into consideration to optimize cold-inducible RNA-based thermosensors: base pairing, base pair position and GC content in pseudoknot region. Increasing base pairing can raise the melting temperature of pseudoknot, thus enlarging the temperature range of open conformation. Increasing GC content can also raise the melting temperature of pseudoknot. We also change base pair at different position. What’s more, we delete the cold box which is at the upstream of CspA 5’UTR. In K2541301, we added two base pairs (A:U) in the pseudoknot region (figure 2).

K2541301 f2
Figure 2. Design of K2541301.

3.Characterization

3.1 Measurement device

The thermosensor sequence is constructed on the pSB1C3 vector by Golden Gate assembly. The measurement device is composed of Anderson promoter (BBa_J23100), thermosensor (BBa_K2541301), sfGFP_optimism (BBa_K2541400) and double terminator (BBa_B0010 and BBa_B0012). We select a constitutive Anderson promoter J23100 as an appropriate promoter by pre-experiment. The sfGFP_optimism has faster folding speed and higher fluorescence intensity. The double terminator can reduce leakage (Figure 3). We characterized RNA-based thermosensors in E.coli DH5a.

caption
Figure 3. The measurement device.

3.2 Measurement results

In figure 4, there are eight different cold-inducible RNA-based thermosensors. pos.control is positive control. The final normalized fluorescence was calculated as follows: normalized fluorescence = [(Fluorescence/Abs600)TS - (Fluorescence/Abs600)neg] / [(Fluorescence/Abs600)pos - (Fluorescence/Abs600)neg] ( TS = thermosensor, pos = positive control, and neg = BBa_J364007 ). As shown in figure 4, the fluorescence intensity of K2541301 increases with decreased temperature.

K2541301 f4

Figure 4. Characteristics of K2541301. Each set of three bars represents the activity level of a different thermosensor. The bar colors purple, yellow and red represent the temperatures 15, 20 and 25°C, respectively. The height of the bars corresponds to the normalized fluorescence.

4. Collection of cold-inducible RNA-based thermosensors

cspA figure6 new

Figure 5. Experimental measurements of the collection of cold-inducible 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 15, 20 and 25°C, respectively. The height of the bars corresponds to the normalized fluorescence.

5. Conclusion

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


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 639
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]