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

 
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    <h4 class="subtitle"><strong>We have characterized previous parts.</strong></h4>
 
 
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         <h3 class="link3"><a href="#standardized2">3.2.1. 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>
 
         <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 processing the background derived from the Negative control</a></h3>  
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&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="#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>
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           <h2 id="Introduction" class="smalltitle">1. Introduction</h2>
 
           <h2 id="Introduction" class="smalltitle">1. Introduction</h2>
      <p class="text">Transcription of <a href="http://parts.igem.org/Part:BBa_K1333309">BBa_K1333309</a> (J23119_K115002_E1010) constructed by iGEM 2014 SYU-China is initiated at 37 ºC.  RNA thermometers are located in the 5’-untranslated region (5’-UTR) and block the Shine-Dalgarno (SD) sequence by base pairing.  At the transcription initiating temperature, the disconnection of the coupling of the hydrogen bonds, which block the SD sequence, initiates.  Therefore, RNA thermometers change their conformations to the open state so that the ribosome could access the SD sequence and the transcription of SD sequence is initiated.
+
      <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;
We improved characterization of <a href="http://parts.igem.org/Part:BBa_K1333309">BBa_K1333309</a> by <b>(1)</b> measuring the function of the part at 42 ºC, <b>(2)</b> explicating the way to deal with background derived from Negative control, <b>(3)</b> measuring with the flow cytometer. To express the feeling of the <i>E. coli</i> which fell into dilemma, we measured the temperature dependence of the RNA thermometer.
+
                    <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>
+
      <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>
      <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>
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           <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">Our purpose is to confirm the behavior of the RNA thermometer by setting Positive control and Negative control and to characterize the temperature dependence of the RNA thermometer at 30 ºC, 37ºC and 42 ºC by using the flow cytometer.  We prepared the samples as shown below.</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">Sample: <a href="http://parts.igem.org/Part:BBa_K1333309">BBa_K1333309</a>:Pcon_RNA thermometer_<i>rfp</i>(pSB1C3)</p></li>
+
<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">Positive control: Plac_<i>rfp</i>_TT (pSB1C3)</p></li>
           <li><p class="list">Negative control: RNA thermometer_<i>rfp</i>(pSB1C3)</p></li>
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           <li><p class="list">Negative control: RNA thermometer_<i>rfp</i> (pSB1C3) (Promoter-less control)</p></li>
 
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       <h4 align="center" class="fig"><strong>Fig. 3-8-2-1.</strong>&nbsp;Parts that we used</h4>
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       <h4 align="center" class="fig"><strong>Fig. 3-7-2-1.</strong>&nbsp;Parts that we used</h4>
 
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           <h2 id="Results" class="smalltitle">3. Results</h2>
 
           <h2 id="Results" class="smalltitle">3. Results</h2>
      <p class="text">We measured each sample at 30 ºC, 37 ℃ and 42 ℃37 ℃ is the transcription initiating temperature.  Although <a href="https://2014.igem.org/Team:SYSU-China">iGEM 2014 SYU-China</a> confirmed the function of Pcon_RNA thermometer_<i>rfp</i> at these temperatures, we measured each sample at 42 ºC, which is higher than the transcription initiating temperature. </p>
+
      <p class="text">We measured each sample at 30ºC, 37ºC and 42ºCThe 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>
 
               <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. 3-8-3-1).<p><br>
+
        <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>
 
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                 <h4 align="center" class="fig"><strong>Fig. 3-8-3-1.</strong>&nbsp;RAW data<br></h4>
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                 <h4 align="center" class="fig"><strong>Fig. 3-7-3-1.</strong>&nbsp;RAW data<br></h4>
 
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       </table><br>
        <p class="text2">The error bar represents the standard deviation for each sample calculated from the two values of all two colonies.</p>
+
        <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="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>
 
               <h3 id="standardized2" class="sub6">3.2.1. The standardized fluorescence intensities of RFP</h3>
                 <p class="text3">We got the increasing ratios of fluorescence intensities at 37 ℃ and at 42 ℃ by dividing the each of the raw fluorescence intensities (Fig. 3-8-3-1) by those at 30℃.  The increasing ratios of the Plac_<i>rfp</i> at 37 ℃ and 42 ℃ show that the fluorescence intensities increased dependent on temperature.  We evaluated the increasing ratios of Pcon_RNA thermometer_<i>rfp</i> at 37 ℃ and 42 ℃.  Compared to the increasing ratios of Plac_<i>rfp</i>, those of the Pcon_RNA thermometer_<i>rfp</i> were higher at respective temperatureWe observed not only the increase in the fluorescence intensities dependent on temperature but also the increase in the fluorescence intensities due to the function of the RNA thermometer (Table. 3-8-3-1).  The increasing ratio of Pcon_RNA thermometer_<i>rfp</i> was 1.3 times higher than that of Plac_<i>rfp</i> at 37 ℃.  Furthermore, the increasing ratio of Pcon_RNA thermometer_<i>rfp</i> was 3.2 times higher that of Plac_<i>rfp</i> at 42 ℃.  These differences of the increase ratios were dependent on the function of the RNA thermometer.  We concluded that the RNA thermometer shows higher function at 42 ℃ compared to 37 ℃.</p>
+
                 <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 temperaturesThis 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>
 
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                 <h4 align="center" class="fig"><strong>Table. 3-8-3-1</strong>&nbsp;The increasing ratios<br></h4>
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                 <h4 align="center" class="fig"><strong>Table. 3-7-3-1.</strong>&nbsp;The increasing ratios<br></h4>
 
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               <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 processing the background derived from the Negative control</h3>
+
               <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">By subtracting the fluorescence intensity of the RNA thermometer_<i>rfp</i> (Fig. 3-8-3-1) from both the fluorescence intensities of Plac_<i>rfp</i> and Pcon_RNA thermometer_<i>rfp</i> (Fig. 3-8-3-1), each at the same temperature, we processed the background derived from the Negative control.  We followed the increasing ratios (which backgrounds have been processed) as the section of the standardized fluorescence intensities of RFP, except this time we processed the backgrounds of all the fluorescence intensities.  The increasing ratios (which backgrounds have been processed) of the Plac_<i>rfp</i> at 37 ℃ and 42 ℃ show that the fluorescence intensities increased dependent on temperature.  We evaluated the increasing ratios (which backgrounds have been processed) of Pcon_RNA thermometer_<i>rfp</i> at 37 ℃ and 42 ℃.  Compared to the increasing ratios (which backgrounds have been processed) of Plac_<i>rfp</i>, those of the Pcon_RNA thermometer_<i>rfp</i> were higher at respective temperature.  We observed not only the increase in the fluorescence intensities dependent on temperature but also the increase in the fluorescence intensities due to the function of the RNA thermometer (Table. 3-8-3-2).  The increasing ratio (which background has been processed) of Pcon_RNA thermometer_<i>rfp</i> was 2.7 times higher than that of Plac_<i>rfp</i> at 37 ℃.  Furthermore, the increasing ratio (which background has been processed) of Pcon_RNA thermometer_<i>rfp</i> was 5.3 times higher that of Plac_<i>rfp</i> at 42 ℃.  These differences of the increase ratios were dependent on the function of the RNA thermometer.  We concluded that the RNA thermometer shows higher function at 42 ℃ compared to 37 ℃.</p><br>
+
                 <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>
 
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                 <h4 align="center" class="fig"><strong>Table. 3-8-3-2.</strong> The increasing ratios (which backgrounds have been processed)</h4>
+
                 <h4 align="center" class="fig"><strong>Table. 3-7-3-2.</strong> The increasing ratios with background subtraction</h4>
 
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           <h2 id="Discussion" class="smalltitle">4. Discussion</h2>
 
           <h2 id="Discussion" class="smalltitle">4. Discussion</h2>
      <p class="text">We examined that the reason the function of the RNA thermometer was worse at 37 ºC than at 42 ºC was that hydrogen bonds forming RNA thermometer was not enough to change in the structure of the RNA thermometer at 37 ºC.<br>&nbsp;&nbsp;
+
      <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, there were differences between the increasing ratios and the increasing ratios (which backgrounds have been processed). At 30 ºC, Pcon_RNA thermometer_<i>rfp</i> wasn’t transcribed and did not show expression of RFP. Since the fluorescence intensity of Pcon_RNA thermometer_<i>rfp</i> at 30 ℃ was small, the increasing ratios of between the Pcon_RNA thermometer which background has been processed and the Pcon_RNA thermometer which background has not been processed, greatly differ in both 37 ℃ and 42 ℃(Table. 3-8-4-1). Therefore, it is important to clarify whether the background derived from Negative control was processed or not.  
+
  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.  
 
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                 <h4 align="center" class="fig"><strong>Table. 3-8-4-1. </strong>The difference the increasing ratios and the increasing ratios (which backgrounds have been processed)</h4>
+
                 <h4 align="center" class="fig"><strong>Table. 3-7-4-1. </strong>The difference the increasing ratios with and without background subtraction</h4>
 
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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">(1) Sample: <a href="http://parts.igem.org/Part:BBa_K1333309">BBa_K1333309</a>:Pcon_RNA thermometer_<i>rfp</i>(pSB1C3)</p>
+
          <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>
 
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       <h4 align="center" class="fig"><strong>Fig.&nbsp;3-8-5-1.</strong></h4>
+
       <h4 align="center" class="fig"><strong>Fig.&nbsp;3-7-5-1.</strong></h4>
 
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          <p class="text2"><p class="text2">(2) Positive control: Plac_<i>rfp</i>_TT(pSB1C3)</p>
+
          <p class="text2"><p class="text2">(2) Positive control: Plac_<i>rfp</i>_TT (pSB1C3)</p>
 
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       <h4 align="center" class="fig"><strong>Fig.&nbsp;3-8-5-2.</strong></h4>
+
       <h4 align="center" class="fig"><strong>Fig.&nbsp;3-7-5-2.</strong></h4>
 
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          <p class="text2">(3) Negative Control:RNA thermometer(FourU)_<i>rfp</i>(pSB1C3)
+
          <p class="text2">(3) Negative Control:RNA thermometer_<i>rfp</i> (pSB1C3)
 
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       <h4 align="center" class="fig"><strong>Fig.&nbsp;3-8-5-3.</strong></h4>
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       <h4 align="center" class="fig"><strong>Fig.&nbsp;3-7-5-3.</strong></h4>
 
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               <h3 id="Protocol" class="sub5">5.2. Assay Protocol</h3>
 
               <h3 id="Protocol" class="sub5">5.2. Assay Protocol</h3>
 
                     <p class="text4">
 
                     <p class="text4">
1. Prepare 2 over night cultures for each sample in 3 mL LB medium containing chloramphenicol (25 microg / mL) at 37 ºC for 12 h.<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. Dilute the overnight cultures to 1/100 in fresh LB medium (3 mL) containing chloramphenicol (25 microg / mL) (fresh culture).<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. Incubate the fresh cultures at 37℃ for 8 h.<br>
+
3. Incubate the triplicated fresh cultures each at 30ºC, 37ºC and 42ºC for each sample for 8 h.<br>
4. Centrifuge the samples at 9000x g, 1 min , 4℃.<br>
+
4. Centrifuge the samples at 9000x g, 1 min, 4ºC.<br>
 
5. Remove the supernatants by using P100 pipette and suspend the samples with 1 mL of filtered PBS (phosphate-buffered saline).<br>
 
5. Remove the supernatants by using P100 pipette and suspend the samples with 1 mL 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>

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