Difference between revisions of "Team:Tokyo Tech/Experiment/RNA thermometer assay"

 
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     </div>
 
     </div>
 
     <div id="titlebottom">
 
     <div id="titlebottom">
    <h4 class="subtitle"><strong>We have characterized previous parts.</strong></h4>
 
 
     </div>
 
     </div>
 
   <div class="texttop">
 
   <div class="texttop">
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       <h3 class="link"><a href="#Introduction">1. Introduction</a></h3>
 
       <h3 class="link"><a href="#Introduction">1. Introduction</a></h3>
 
       <h3 class="link"><a href="#Summary">2. Summary of the Experiment</a></h3>
 
       <h3 class="link"><a href="#Summary">2. Summary of the Experiment</a></h3>
       <h3 class="link"><a href="#Results">3. Results</a></h3>  
+
       <h3 class="link"><a href="#Results">3. Results</a></h3>
       <h3 class="link"><a href="#Discussion">4. Discussion</a></h3>                
+
      <h3 class="link2"><a href="#fluorescence">3.1. The fluorescence intensities of RFP</a></h3>
 +
      <h3 class="link2"><a href="#standardized">3.2. The standardized fluorescence intensities of RFP</a></h3>
 +
        <h3 class="link3"><a href="#standardized2">3.2.1. The standardized fluorescence intensities of RFP</a></h3>
 +
        <h3 class="link3"><a href="#standardized3">3.2.2. The standardized fluorescence intensities of RFP<br>
 +
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;after subtracting the background derived from the Negative control cell</a></h3>  
 +
       <h3 class="link"><a href="#Discussion">4. Discussion</a></h3>            
 
       <h3 class="link"><a href="#Materials">5. Materials and Methods</a></h3>
 
       <h3 class="link"><a href="#Materials">5. Materials and Methods</a></h3>
 
       <h3 class="link2"><a href="#Const">5.1.  Construction</a></h3>
 
       <h3 class="link2"><a href="#Const">5.1.  Construction</a></h3>
 
       <h3 class="link2"><a href="#Protocol">5.2. Assay Protocol</a></h3>
 
       <h3 class="link2"><a href="#Protocol">5.2. Assay Protocol</a></h3>
        <h3 class="link3"><a href="#Protol1">5.2.1. Protocol1</a></h3>
 
        <h3 class="link3"><a href="#Protol2">5.2.2. Protocol2</a></h3>
 
 
       <h3 class="link"><a href="#Reference">6. Reference</a></h3>
 
       <h3 class="link"><a href="#Reference">6. Reference</a></h3>
 
       <br>
 
       <br>
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   <div class="textarea">
 
   <div class="textarea">
 
           <h2 id="Introduction" class="smalltitle">1. Introduction</h2>
 
           <h2 id="Introduction" class="smalltitle">1. Introduction</h2>
      <p class="text">ここにコピペ。</p>
+
      <p class="text">Temperature increase is required for an RNA thermometer in the enhanced expression of <a href="http://parts.igem.org/Part:BBa_K1333309:Experience">BBa_K1333309</a> (J23119_K115002_E1010) constructed by iGEM 2014 SYSU-China.  The RNA thermometers are located in the 5’-untranslated region (5’-UTR) and block the Shine-Dalgarno (SD) sequence by base pairing.  Translation initiating temperature allows the disconnection of the coupling of the hydrogen bonds, which block the SD sequence at low temperature.  Therefore, RNA thermometers change their conformations to the open state so that the ribosome could access the SD sequence and to initiate translation.</p><br>&nbsp;&nbsp;&nbsp;
 
+
                    <p class="text">We improved characterization of <a href="http://parts.igem.org/Part:BBa_K1333309:Experience">BBa_K1333309</a> by <b>(1)</b> measuring the function of the part at 42ºC, <b>(2)</b> explicating the way to deal with the background derived from Negative control, <b>(3)</b> measuring with the flow cytometer. </p><br>
 +
      <p class="text">We think that this experiment is meets Gold medal criteria. <a href="https://2015.igem.org/Team:Tokyo_Tech/Description">Description</a></p>
 
           <h2 id="Summary" class="smalltitle">2. Summary of the Experiment</h2>
 
           <h2 id="Summary" class="smalltitle">2. Summary of the Experiment</h2>
      <p class="text">ここにコピペ。</p>
+
      <p class="text">Our purpose is to confirm the behavior of the RNA thermometer by setting Positive control and Negative control and to characterize the temperature dependency of the RNA thermometer at 30ºC, 37ºC and 42ºC by using the flow cytometer.  We prepared the samples as shown below.</p>
 +
<li><p class="list"><a href="http://parts.igem.org/Part:BBa_K1333309:Experience">BBa_K1333309</a>: Pcon_RNA thermometer_<i>rfp</i> (pSB1C3)</p></li>
 +
          <li><p class="list">Positive control: Plac_<i>rfp</i>_TT (pSB1C3)</p></li>
 +
          <li><p class="list">Negative control: RNA thermometer_<i>rfp</i> (pSB1C3) (Promoter-less control)</p></li>
 +
<br>
 +
    <table width="980 px" border="0px">
 +
      <tr>
 +
      <td width="980px"><div align="center"><img src="https://static.igem.org/mediawiki/2015/f/f9/Tokyo_Tech_RNA-thermometer_summary.png" />
 +
      </td>
 +
      </tr>
 +
      <tr>
 +
      <td width="980px">
 +
      <h4 align="center" class="fig"><strong>Fig. 3-7-2-1.</strong>&nbsp;Parts that we used</h4>
 +
      <td>
 +
      </tr>
 +
      </table>
  
 +
   
 
           <h2 id="Results" class="smalltitle">3. Results</h2>
 
           <h2 id="Results" class="smalltitle">3. Results</h2>
      <p class="text">ここにコピペ。</p>
+
      <p class="text">We measured each sample at 30ºC, 37ºC and 42ºC.  The translation initiating temperature is 37ºC.  Little background from medium affect results for flow cytometer.  Although <a href="https://2014.igem.org/Team:SYSU-China">iGEM 2014 SYSU-China</a> confirmed the function of Pcon_RNA thermometer_<i>rfp</i> at these temperatures, we additionally measured each sample at 42ºC, which is higher than the translation initiating temperature. </p>
 +
              <h3 id="fluorescence" class="sub5">3.1. The fluorescence intensities of RFP</h3>
 +
        <p class="text2">We found that the fluorescence intensities of both Pcon_RNA thermometer_<i>rfp</i> and Plac_<i>rfp</i> increased along with the rise of the temperature (Fig. 3-7-3-1).<p><br>
 +
                <table width="940 px" border="0px">
 +
                <tr>
 +
                <td width="940px"><div align="center"><img src="https://static.igem.org/mediawiki/2015/8/85/Tokyo_Tech_RNA_thermometer_Result1.png" width="600px" />
 +
                </td>
 +
                </tr>
 +
                <tr>
 +
                <td width="940px">
 +
                <h4 align="center" class="fig"><strong>Fig. 3-7-3-1.</strong>&nbsp;RAW data<br></h4>
 +
                <td>
 +
                </tr>
 +
      </table><br>
 +
        <p class="text2">The error bar represents the standard deviation of two samples which derived from two different colonies, respectively.</p>
 +
              <h3 id="standardized" class="sub5">3.2.  The standardized fluorescence intensities of RFP</h3>
 +
              <h3 id="standardized2" class="sub6">3.2.1. The standardized fluorescence intensities of RFP</h3>
 +
                <p class="text3">We obtained increasing ratios of fluorescence intensities at 37ºC and at 42ºC by dividing the each of the raw fluorescence intensities (Fig. 3-7-3-1) by those at 30ºC.  The increasing ratios of the Plac_<i>rfp</i> at 37ºC and 42ºC show that the fluorescence intensities, even without the RNA thermometer, increased dependent on temperature.  We further evaluated the increasing ratios of Pcon_RNA thermometer_<i>rfp</i> at 37ºC and 42ºC.  Compared to the increasing ratios of Plac_<i>rfp</i>, those of the Pcon_RNA thermometer_<i>rfp</i> were higher at respective temperatures.  This comparison shows not only the increase in the fluorescence intensities dependent on temperature, (Fig. 3-7-3-1) but also the increase in the fluorescence intensities due to the function of the RNA thermometer (Table. 3-7-3-1).  The increasing ratio of Pcon_RNA thermometer_<i>rfp</i> was 1.3 times higher than that of Plac_<i>rfp</i> at 37ºC.  Furthermore, the increasing ratio of Pcon_RNA thermometer_<i>rfp</i> was 3.2 times higher than that of Plac_<i>rfp</i> at 42ºC.  These differences of the increasing ratios were dependent on the function of the RNA thermometer.  We concluded that the RNA thermometer shows higher function at 42ºC compared to 37ºC.</p>
 +
                <table width="940 px" border="0px">
 +
                <tr>
 +
                <td width="940px">
 +
                <h4 align="center" class="fig"><strong>Table. 3-7-3-1.</strong>&nbsp;The increasing ratios<br></h4>
 +
                <td>
 +
                </tr>
 +
                <tr>
 +
                <td width="940px"><div align="center"><img src="https://static.igem.org/mediawiki/2015/6/65/Tokyo_Tech_RNA_thermometer_Result2.png" width="400px" />
 +
                </td>
 +
                </tr>
 +
      </table><br>
 +
              <h3 id="standardized3" class="sub6">3.2.2. The standardized fluorescence intensities of RFP <br>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;after subtracting the background derived from the Negative control cell</h3>
 +
                <p class="text3">In this section, we obtained processed fluorescence intensities by subtracting the fluorescence intensity of a negative control, the RNA thermometer_<i>rfp</i> (Fig. 3-7-3-1) from both the fluorescence intensities of Plac_<i>rfp</i> and Pcon_RNA thermometer_<i>rfp</i> (Fig. 3-7-3-1), each at the same temperature.  We then obtained increasing ratios with background subtraction at 37ºC and at 42ºC by dividing the each of the processed fluorescence intensities by those at 30ºC (Table. 3-7-3-2).  The increasing ratios of the Plac_<i>rfp</i> with background subtraction at 37ºC and at 42ºC show that the fluorescence intensities increased dependently on temperature.  We evaluated the increasing ratios of Pcon_RNA thermometer_<i>rfp</i> with background subtraction at 37ºC and at 42ºC.  Compared to the increasing ratios of Plac_<i>rfp</i> with background subtraction, those of the Pcon_RNA thermometer_<i>rfp</i> were higher at respective temperatures.  We observed again the increase in the fluorescence intensities due to the function of the RNA thermometer (Table. 3-7-3-2).  The increasing ratio of Pcon_RNA thermometer_<i>rfp</i> at 37ºC with background subtraction was 2.6 times higher than that of Plac_<i>rfp</i> at 37ºC.  Furthermore, the increasing ratio of Pcon_RNA thermometer_<i>rfp</i> with background subtraction was 6.8 times higher than that of Plac_<i>rfp</i> at 42ºC.  These differences in the increasing ratios were dependent on the function of the RNA thermometer.  We concluded that the RNA thermometer shows higher function at 42ºC compared to 37ºC.</p><br>
 +
                <table width="940 px" border="0px">
 +
<tr>
 +
                <td width="940px">
 +
                <h4 align="center" class="fig"><strong>Table. 3-7-3-2.</strong> The increasing ratios with background subtraction</h4>
 +
                <td>
 +
                </tr>
 +
                <tr>
 +
                <td width="940px"><div align="center"><img src="https://static.igem.org/mediawiki/2015/e/ef/Tokyo_Tech_RNA_thermometer_Result3.png" width="400px" />
 +
                </td>
 +
                </tr>
 +
                      </table><br>
 +
 
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           <h2 id="Discussion" class="smalltitle">4. Discussion</h2>
 
           <h2 id="Discussion" class="smalltitle">4. Discussion</h2>
      <p class="text">ここにコピペ。</p>
+
      <p class="text">We examined that the reason the function of the RNA thermometer was worse at 37ºC than at 42ºC was that the amount of hydrogen bonds forming RNA thermometer was not enough to change in the structure of the RNA thermometer at 37ºC.<br>&nbsp;&nbsp;
 +
Furthermore, differences between the increasing ratios with and without background subtraction disclose the importance of background treatment.  At 30ºC, Pcon_RNA thermometer_<i>rfp</i> wasn’t translated enough and showed little expression of RFP.  Since the fluorescence intensity of Pcon_RNA thermometer_<i>rfp</i> at 30ºC was small, the increasing ratios of the Pcon_RNA thermometer with and without background subtraction greatly differ in both 37ºC and 42ºC (Table. 3-7-4-1).  Therefore, it is important to clarify whether the background derived from Negative control was processed or not.
 +
</p><br>
 +
                <table width="940 px" border="0px">
 +
<tr>
 +
                <td width="940px">
 +
                <h4 align="center" class="fig"><strong>Table. 3-7-4-1. </strong>The difference the increasing ratios with and without background subtraction</h4>
 +
                <td>
 +
                </tr>
 +
 
 +
                <tr>
 +
                <td width="940px"><div align="center"><img src="https://static.igem.org/mediawiki/2015/2/23/Tokyo_Tech_RNA_thermometer_Result4.png" width="600px" />
 +
                </td>
 +
                </tr>
 +
                      </table>
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           <h2 id="Materials" class="smalltitle">5. Materials and Methods</h2>
 
           <h2 id="Materials" class="smalltitle">5. Materials and Methods</h2>
 
               <h3 id="Const" class="sub5">5.1.  Construction</h3>
 
               <h3 id="Const" class="sub5">5.1.  Construction</h3>
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          <p class="text2">All the samples were DH5alpha strain.</p>
 
          <p class="text2">All the samples were DH5alpha strain.</p>
 
               <h3 class="sub5">-Plasmids</h3>
 
               <h3 class="sub5">-Plasmids</h3>
          <p class="text2">Device 1: J23101 + I13504(pSB1C3)
+
          <p class="text2">(1)<a href="http://parts.igem.org/Part:BBa_K1333309:Experience">BBa_K1333309</a>: Pcon_RNA thermometer_<i>rfp</i> (pSB1C3)</p>
 
                 <table width="980 px" border="0px">
 
                 <table width="980 px" border="0px">
 
                   <tr>
 
                   <tr>
                   <td width="980px"><div align="center"><img src="https://static.igem.org/mediawiki/2015/1/1c/Tokyo_Tech_Device1.png"/>
+
                   <td width="980px"><div align="center"><img src="https://static.igem.org/mediawiki/2015/a/a0/Tokyo_Tech_J23119_RNAthermometer_rfp.png"/>
 
       </td>
 
       </td>
 
       </tr>
 
       </tr>
 
       <tr>
 
       <tr>
 
       <td width="980px">
 
       <td width="980px">
       <h4 align="center" class="fig"><strong>Fig.3-7-4-1.</strong></h4>
+
       <h4 align="center" class="fig"><strong>Fig.&nbsp;3-7-5-1.</strong></h4>
 
       <td>
 
       <td>
 
       </tr>
 
       </tr>
 
       </table><br>
 
       </table><br>
 +
          <p class="text2"><p class="text2">(2) Positive control: Plac_<i>rfp</i>_TT (pSB1C3)</p>
 +
                <table width="980 px" border="0px">
 +
                  <tr>
 +
                  <td width="980px"><div align="center"><img src="https://static.igem.org/mediawiki/2015/a/a0/Tokyo_Tech_Plac_RFP_TT.png"/>
 +
      </td>
 +
      </tr>
 +
      <tr>
 +
      <td width="980px">
 +
      <h4 align="center" class="fig"><strong>Fig.&nbsp;3-7-5-2.</strong></h4>
 +
      <td>
 +
      </tr>
 +
      </table><br>
 +
          <p class="text2">(3) Negative Control:RNA thermometer_<i>rfp</i> (pSB1C3)
 +
                <table width="980 px" border="0px">
 +
                  <tr>
 +
                  <td width="980px"><div align="center"><img src="https://static.igem.org/mediawiki/2015/0/06/Tokyo_Tech_RNAthermomer_RFP.png"/>
 +
      </td>
 +
      </tr>
 +
      <tr>
 +
      <td width="980px">
 +
      <h4 align="center" class="fig"><strong>Fig.&nbsp;3-7-5-3.</strong></h4>
 +
      <td>
 +
      </tr>
 +
      </table><br>
 +
 +
 +
 
               <h3 id="Protocol" class="sub5">5.2. Assay Protocol</h3>
 
               <h3 id="Protocol" class="sub5">5.2. Assay Protocol</h3>
              <h3 id="Protol1" class="sub6">5.2.1. Protocol1</h3>
 
                <p class="text2"></p>
 
                  <p class="text3"></p>
 
              <h3 id="reader" class="sub6">4.3.1. Plate reader</h3>
 
 
                     <p class="text4">
 
                     <p class="text4">
1. Prepare 3 over night cultures for each sample Device1〜Device3, Positive control and Negative control in 3 mL LB medium containing chloramphenicol (35 microg / mL) at 37 °C for 17h and shake at 180 rpm.<br>
+
1. Prepare 2 over night cultures for each sample in 3 mL LB medium containing chloramphenicol (25 microg / mL) at 37ºC for 12 h.<br>
2 .Measured the OD590 of each sample and diluted each sample to adjust OD590 within 5% of 0.5.<br>
+
2. Dilute the overnight cultures to 1/100 in fresh LB medium (3 mL) containing chloramphenicol (25 microg / mL ) in triplicate (fresh culture).<br>
3. Set the plate reader to measure GFP.<br>
+
3. Incubate the triplicated fresh cultures each at 30ºC, 37ºC and 42ºC for each sample for 8 h.<br>
4. Take 1 mL of the samples, and centrifuge at 9000x g, 1 min, 4°C.<br>
+
4. Centrifuge the samples at 9000x g, 1 min, 4ºC.<br>
5. Remove the supernatants by using P1000 pipette. <br>
+
5. Remove the supernatants by using P100 pipette and suspend the samples with 1 mL of filtered PBS (phosphate-buffered saline).<br>
6. Add 1 mL of filtered PBS (phosphate-buffered saline) and suspend.<br>
+
7. Place 200 μL of each sample into the 96-well plate as described in Table. 3-7-4-1.<br>
+
8. Measure the fluorescence intensity with plate reader.<br>
+
9. Rotate the 96-well plate 180 degrees horizontally and measure the fluorescence intensity again.<br></p>
+
                <table width="940 px" border="0px">
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                <tr>
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                <td width="940px"><div align="center"><img src="https://static.igem.org/mediawiki/2015/f/f7/Tokyo_Tech_Interlab_Table.3-7-4-1.png" width="700px"/>
+
                </td>
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                </tr>
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                <tr>
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                <td width="940px">
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                <h4 align="center" class="fig"><strong>Table. 3-7-4-1.</strong>&nbsp;Position of samples in 96-well plate</h4>
+
                <td>
+
                </tr>
+
      </table><br>
+
              <h3 id="meter" class="sub6">4.3.2. Flow cytometer</h3>
+
                    <p class="text4">
+
1. Prepare 3 over night cultures for each sample Device1〜Device3, Positive control and Negative control in 3m LB medium containing chloramphenicol (35 microg / mL) at 37°C for 17h and shake at 180 rpm.<br>
+
2. Start preparing the flow cytometer 1 h before the end of incubation.<br>
+
3. Measure the OD590 and adjust the volume of each sample to centrifuge so that the amount of pellet will be about the same for every sample.<br>
+
4. Centrifuge the samples at 9000x g, 1min, 4°C.<br>
+
5. Remove the supernatants by using P1000 pipette and suspend the samples with 1mL of filtered PBS (phosphate-buffered saline).<br>
+
 
6. Dispense all of each suspension into a disposable tube through a cell strainer.<br>
 
6. Dispense all of each suspension into a disposable tube through a cell strainer.<br>
7. Measure fluorescence intensity with flow cytometer.<br><br>
+
7. Measure fluorescence intensity with flow cytometer.<br></p>
              <h3 id="How" class="sub5">4.4. How to process the data</h3>
+
          <h2 id="Reference" class="smalltitle">6. Reference</h2>
              <h3 id="Pr" class="sub6">4.4.1. Plate reader</h3>
+
      <p class="text">1. Stassen, Oscar MJA, <i>et al</i>., Toward tunable RNA thermo-switches for temperature dependent gene expression. arXiv preprint arXiv:1109.5402 (2011).</p>
                    <p class="text4">
+
            <p class="text">2. <a href="https://2014.igem.org/Team:SYSU-China/content.html#Project/Result/RNAT">SYSU-China 2014</a></p><br>
<strong>-How to draw the calibration curve</strong><br>
+
1. Place 200 μL of various concentrations of sodium fluorescein (500, 375, 250, 125, 50, 25, 10, 5 ng / mL and PBS only) into the 96-well plate in triplicate.<br>
+
2. Measure the fluorescence intensity with the plate reader.<br>
+
3. Rotate the 96-well plate 180 degrees horizontally.<br>
+
4. Measure the fluorescence intensity again.<br>
+
5. Determine the auto-fluorescence of PBS by calculating the arithmetic mean of fluorescence intensity of PBS added in triplicate and use this value as the background fluorescence. <br>
+
6. Subtract background fluorescence from each fluorescence intensity value of each well containing sodium fluorescein.<br>
+
7. Take the arithmetic mean of the three technical replicates of sodium fluorescein of each concentration.<br>
+
8. Draw the calibration curve.<br> </p><br>
+
                    <p class="text4">
+
<strong>-How to obtain the absolute unit of fluorescence intensity</strong><br>
+
1. Measure the fluorescence intensity with the plate reader.<br>
+
2. Rotate the 96-well plate 180 degrees horizontally and measure the fluorescence intensity again.<br>
+
3. Calculate the arithmetic mean of these two results.<br>
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4. Determine the auto-florescence of PBS by calculating the arithmetic mean of fluorescence intensity of PBS added in triplicate and use this value as the background fluorescence.<br>
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5. Subtract the background fluorescence from each well containing the samples.<br>
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6. Divide them by the value of OD590 of each sample.<br>
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7. Calculate the ng / mL fluorescence per OD590 unit by the formula we obtained from drawing the calibration curve.<br>
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              <h3 id="Flow cytometer" class="sub6">4.4.2. Flow cytometer</h3>
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                    <p class="text3">Cells were gated according to the side scatter (SSC) and the forward scatter (FCS) to exclude cell debris and impurities.</p><br><br>
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              <h3 id="Individuals" class="sub5">4.5. Individuals responsible for conducting Interlab study</h3>
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          <p class="text2">
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Misa Minegishi : Measured the devices and processed the data.<br>
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&nbsp;&nbsp;&nbsp;Yuta Yamazaki : Measured the devices and processed the data.<br>
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&nbsp;&nbsp;&nbsp;Hiraku Tokuma : Created the devices.<br>
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&nbsp;&nbsp;&nbsp;Riku Shinohara: Created the devices.<br>
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          <h2 id="Reference" class="smalltitle">6.. Reference</h2>
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      <p class="text">ここにコピペ。<p><br><br><br>
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Latest revision as of 00:46, 19 September 2015

RNA thermometer assay

  
  

1. Introduction

      

Temperature increase is required for an RNA thermometer in the enhanced expression of BBa_K1333309 (J23119_K115002_E1010) constructed by iGEM 2014 SYSU-China. The RNA thermometers are located in the 5’-untranslated region (5’-UTR) and block the Shine-Dalgarno (SD) sequence by base pairing. Translation initiating temperature allows the disconnection of the coupling of the hydrogen bonds, which block the SD sequence at low temperature. Therefore, RNA thermometers change their conformations to the open state so that the ribosome could access the SD sequence and to initiate translation.


   

We improved characterization of BBa_K1333309 by (1) measuring the function of the part at 42ºC, (2) explicating the way to deal with the background derived from Negative control, (3) measuring with the flow cytometer.


      

We think that this experiment is meets Gold medal criteria. Description

2. Summary of the Experiment

      

Our purpose is to confirm the behavior of the RNA thermometer by setting Positive control and Negative control and to characterize the temperature dependency of the RNA thermometer at 30ºC, 37ºC and 42ºC by using the flow cytometer. We prepared the samples as shown below.

  • BBa_K1333309: Pcon_RNA thermometer_rfp (pSB1C3)

  • Positive control: Plac_rfp_TT (pSB1C3)

  • Negative control: RNA thermometer_rfp (pSB1C3) (Promoter-less control)


  • Fig. 3-7-2-1. Parts that we used

    3. Results

          

    We measured each sample at 30ºC, 37ºC and 42ºC. The translation initiating temperature is 37ºC. Little background from medium affect results for flow cytometer. Although iGEM 2014 SYSU-China confirmed the function of Pcon_RNA thermometer_rfp at these temperatures, we additionally measured each sample at 42ºC, which is higher than the translation initiating temperature.

    3.1. The fluorescence intensities of RFP

          

    We found that the fluorescence intensities of both Pcon_RNA thermometer_rfp and Plac_rfp increased along with the rise of the temperature (Fig. 3-7-3-1).


    Fig. 3-7-3-1. RAW data


          

    The error bar represents the standard deviation of two samples which derived from two different colonies, respectively.

    3.2. The standardized fluorescence intensities of RFP

    3.2.1. The standardized fluorescence intensities of RFP

    We obtained increasing ratios of fluorescence intensities at 37ºC and at 42ºC by dividing the each of the raw fluorescence intensities (Fig. 3-7-3-1) by those at 30ºC. The increasing ratios of the Plac_rfp at 37ºC and 42ºC show that the fluorescence intensities, even without the RNA thermometer, increased dependent on temperature. We further evaluated the increasing ratios of Pcon_RNA thermometer_rfp at 37ºC and 42ºC. Compared to the increasing ratios of Plac_rfp, those of the Pcon_RNA thermometer_rfp were higher at respective temperatures. This comparison shows not only the increase in the fluorescence intensities dependent on temperature, (Fig. 3-7-3-1) but also the increase in the fluorescence intensities due to the function of the RNA thermometer (Table. 3-7-3-1). The increasing ratio of Pcon_RNA thermometer_rfp was 1.3 times higher than that of Plac_rfp at 37ºC. Furthermore, the increasing ratio of Pcon_RNA thermometer_rfp was 3.2 times higher than that of Plac_rfp at 42ºC. These differences of the increasing ratios were dependent on the function of the RNA thermometer. We concluded that the RNA thermometer shows higher function at 42ºC compared to 37ºC.

    Table. 3-7-3-1. The increasing ratios


    3.2.2. The standardized fluorescence intensities of RFP
              after subtracting the background derived from the Negative control cell

    In this section, we obtained processed fluorescence intensities by subtracting the fluorescence intensity of a negative control, the RNA thermometer_rfp (Fig. 3-7-3-1) from both the fluorescence intensities of Plac_rfp and Pcon_RNA thermometer_rfp (Fig. 3-7-3-1), each at the same temperature. We then obtained increasing ratios with background subtraction at 37ºC and at 42ºC by dividing the each of the processed fluorescence intensities by those at 30ºC (Table. 3-7-3-2). The increasing ratios of the Plac_rfp with background subtraction at 37ºC and at 42ºC show that the fluorescence intensities increased dependently on temperature. We evaluated the increasing ratios of Pcon_RNA thermometer_rfp with background subtraction at 37ºC and at 42ºC. Compared to the increasing ratios of Plac_rfp with background subtraction, those of the Pcon_RNA thermometer_rfp were higher at respective temperatures. We observed again the increase in the fluorescence intensities due to the function of the RNA thermometer (Table. 3-7-3-2). The increasing ratio of Pcon_RNA thermometer_rfp at 37ºC with background subtraction was 2.6 times higher than that of Plac_rfp at 37ºC. Furthermore, the increasing ratio of Pcon_RNA thermometer_rfp with background subtraction was 6.8 times higher than that of Plac_rfp at 42ºC. These differences in the increasing ratios were dependent on the function of the RNA thermometer. We concluded that the RNA thermometer shows higher function at 42ºC compared to 37ºC.


    Table. 3-7-3-2. The increasing ratios with background subtraction


    4. Discussion

          

    We examined that the reason the function of the RNA thermometer was worse at 37ºC than at 42ºC was that the amount of hydrogen bonds forming RNA thermometer was not enough to change in the structure of the RNA thermometer at 37ºC.
       Furthermore, differences between the increasing ratios with and without background subtraction disclose the importance of background treatment. At 30ºC, Pcon_RNA thermometer_rfp wasn’t translated enough and showed little expression of RFP. Since the fluorescence intensity of Pcon_RNA thermometer_rfp at 30ºC was small, the increasing ratios of the Pcon_RNA thermometer with and without background subtraction greatly differ in both 37ºC and 42ºC (Table. 3-7-4-1). Therefore, it is important to clarify whether the background derived from Negative control was processed or not.


    Table. 3-7-4-1. The difference the increasing ratios with and without background subtraction

    5. Materials and Methods

    5.1. Construction

    -Strain

          

    All the samples were DH5alpha strain.

    -Plasmids

          

    (1)BBa_K1333309: Pcon_RNA thermometer_rfp (pSB1C3)

    Fig. 3-7-5-1.


          

    (2) Positive control: Plac_rfp_TT (pSB1C3)

    Fig. 3-7-5-2.


          

    (3) Negative Control:RNA thermometer_rfp (pSB1C3)

    Fig. 3-7-5-3.


    5.2. Assay Protocol

    1. Prepare 2 over night cultures for each sample in 3 mL LB medium containing chloramphenicol (25 microg / mL) at 37ºC for 12 h.
    2. Dilute the overnight cultures to 1/100 in fresh LB medium (3 mL) containing chloramphenicol (25 microg / mL ) in triplicate (fresh culture).
    3. Incubate the triplicated fresh cultures each at 30ºC, 37ºC and 42ºC for each sample for 8 h.
    4. Centrifuge the samples at 9000x g, 1 min, 4ºC.
    5. Remove the supernatants by using P100 pipette and suspend the samples with 1 mL of filtered PBS (phosphate-buffered saline).
    6. Dispense all of each suspension into a disposable tube through a cell strainer.
    7. Measure fluorescence intensity with flow cytometer.

    6. Reference

          

    1. Stassen, Oscar MJA, et al., Toward tunable RNA thermo-switches for temperature dependent gene expression. arXiv preprint arXiv:1109.5402 (2011).

    2. SYSU-China 2014