Difference between revisions of "Team:Tokyo Tech/Promoter Assay/Pcold"

 
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<div id="contents_menu">
 
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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>
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<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="link"><a href="#discussion">4. Discussion</a></h3>
<h3 class="link"><a href="#methods">5. Materials and Methods</a></h3>
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<h3 class="link"><a href="#methods">5. Materials and methods</a></h3>
 
<h3 class="link"><a href="#construction"><font size="2.7">&nbsp;&nbsp;&nbsp;5-1. Construction</font></a></h3>
 
<h3 class="link"><a href="#construction"><font size="2.7">&nbsp;&nbsp;&nbsp;5-1. Construction</font></a></h3>
<h3 class="link"><a href="#protocol"><font size="2.7">&nbsp;&nbsp;&nbsp;5-2. Assay Protocol</font></a></h3>
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<h3 class="link"><a href="#protocol"><font size="2.7">&nbsp;&nbsp;&nbsp;5-2. Assay protocol</font></a></h3>
 
<h3 class="link"><a href="#reference">6. Reference</a></h3>
 
<h3 class="link"><a href="#reference">6. Reference</a></h3>
 
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<div id="introduction_contents" class="container_contents">
 
<div id="introduction_contents" class="container_contents">
<p class="normal_text">“Magic Mirror on the wall, who is the fairest one of all?”<br>
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<p class="normal_text">“Magic Mirror on the wall, who is the fairest one of all?”
Snow White begins with the scene where Magic Mirror replies that “Snow White is.” If the magic mirror didn’t reply as above, Snow White would not begin.</p>
+
Snow White begins with the scene where Magic Mirror replies that “Snow White is the fairest one.” If Magic Mirror did not reply as above, Snow White would not begin.
 +
</p>
 
                                 <p class="normal_text">We analyzed the cold inducible promoter (Pcold: <a href="http://parts.igem.org/Part:BBa_K1949000">BBa_K1949000</a>) as a starter of Snow White and found that expression from Pcold was induced at 18°C compared to 37°C.</p>
 
                                 <p class="normal_text">We analyzed the cold inducible promoter (Pcold: <a href="http://parts.igem.org/Part:BBa_K1949000">BBa_K1949000</a>) as a starter of Snow White and found that expression from Pcold was induced at 18°C compared to 37°C.</p>
  
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<div id="summary" class="container">
 
<div id="summary" class="container">
 
<div id="id_header" class="container_header">
 
<div id="id_header" class="container_header">
<h2><span>2. Summary of the Experiment</span></h2>
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<h2><span>2. Summary of the experiment</span></h2>
 
</div><!-- /_header -->
 
</div><!-- /_header -->
 
<div id="summary_contents" class="container_contents">
 
<div id="summary_contents" class="container_contents">
<p class="normal_text">The purpose of this experiment is to characterize temperature dependence of Pcold by comparing the expression level of a reporter gene, <i>gfp</i>, at 18°C to that at 37°C. We prepared three samples shown below.<br>
+
<p class="normal_text">The purpose of this experiment is to characterize temperature-dependence of Pcold by comparing the expression level of a reporter gene, gfp, at 18°C with that at 37°C. We prepared three transformants shown below.<br>
<p class="normal_text">A. Pcold - <span style="font-style : italic">gfp</span> (pSB1C3)<br>
+
<p class="normal_text">A. Pcold &#8208; <span style="font-style : italic">gfp</span> (pSB1C3)<br>
  
 
<div align="center"><img src="https://static.igem.org/mediawiki/2016/0/0b/Fig.3-4-1-2-1.png"height="150"><br></div>
 
<div align="center"><img src="https://static.igem.org/mediawiki/2016/0/0b/Fig.3-4-1-2-1.png"height="150"><br></div>
<div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig. 3-4-1-2-1 </span>Pcon - <i>rbs - gfp</i> (pSB1C3)</p></div>
+
<div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig. 3-4-1-2-1 </span>Pcon &#8208; <i>gfp</i> (pSB1C3)</p></div>
  
<p class="normal_text">B. Positive Cntrol : Pcon - <span style="font-style : italic">rbs - gfp</span> (pSB1C3)<br>
+
<p class="normal_text">B. Positive Cntrol : Pcon &#8208; <span style="font-style : italic">rbs &#8208; gfp</span> (pSB1C3)<br>
  
 
<div align="center"><img src="https://static.igem.org/mediawiki/2016/b/bf/Fig.3-4-1-2-2.png"height="150"><br></div>
 
<div align="center"><img src="https://static.igem.org/mediawiki/2016/b/bf/Fig.3-4-1-2-2.png"height="150"><br></div>
<div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig. 3-4-1-2-2 </span>Pcon - <i>rbs - gfp</i> (pSB1C3)</p></div>
+
<div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig. 3-4-1-2-2 </span>Pcon &#8208; <i>rbs &#8208; gfp</i> (pSB1C3)</p></div>
  
 
<p class="normal_text">C. Negative Control : empty vector (pSB1C3)
 
<p class="normal_text">C. Negative Control : empty vector (pSB1C3)
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<div align="center"><img src="https://static.igem.org/mediawiki/2016/9/9e/Fig.3-4-1-3-1.png"height="300"><br></div>
 
<div align="center"><img src="https://static.igem.org/mediawiki/2016/9/9e/Fig.3-4-1-3-1.png"height="300"><br></div>
 
<div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig. 3-4-1-3-1 </span>Pcold is a cold inducible promoter</p></div>
 
<div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig. 3-4-1-3-1 </span>Pcold is a cold inducible promoter</p></div>
<div align="center"><img src="https://static.igem.org/mediawiki/2016/4/4c/Fig.3-4-1-3-2.png"height="300"><br></div>
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<div align="center"><img src="https://static.igem.org/mediawiki/2016/archive/4/4c/20161019082614%21Fig.3-4-1-3-2.png"height="300"><br></div>
<div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig. 3-4-1-3-2 </span>title</p></div>
+
<div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig. 3-4-1-3-2 </span>Activity of Pcon</p></div>
  
<p class="normal_text">These graphs show RFU of GFP / Turbidity per cells every 1 h for 3 h after temperature induction. The error bar represents the standard deviation of three samples which are derived from three different colonies, respectively.<br>
+
<p class="normal_text">These graphs show RFU of GFP / Turbidity per cells at indicated time points after the temperature down-shift. The error bar represents the standard deviation from the triplicated data which are derived from three independent colonies, respectively.<br>
RFU of GFP / Turbidity of GFP induced by the promoter was calculated by subtracting RFU of GFP / Turbidity of negative control at each temperature from the fluorescence intensities of Pcold -<span style="font-style : italic"> gfp</span> and Pcon -<span style="font-style : italic">rbs - gfp</span>. The proportion of RFU of GFP / Turbidity at 18°C to RFU of GFP / Turbidity at 37°C about each sample was calculated.</p>
+
RFU of GFP / Turbidity of GFP induced by the promoter was calculated by subtracting RFU of GFP / Turbidity of negative control at each temperature from the fluorescence intensities of Pcold &#8208;<span style="font-style : italic"> gfp</span> and Pcon &#8208;<span style="font-style : italic">rbs &#8208; gfp</span>. The proportion of RFU of GFP / Turbidity at 18°C to RFU of GFP / Turbidity at 37°C about each sample was calculated.</p>
<p class="normal_text"> Concerning Pcold, it was found that RFU of GFP / Turbidity at 18°C was about eight times as much as that at 37°C. On the other hand, concerning Pcon, RFU of GFP / Turbidity at 18°C was about twice as much as RFU of GFP / Turbidity at 37°C. It is concluded that Pcold is a cold inducible promoter compared with Pcon from which expression is caused constantly.</p>
+
<p class="normal_text">Concerning Pcold, it was found that RFU of GFP / Turbidity at 18°C was about eight times as much as that at 37°C. On the other hand, concerning Pcon, RFU of GFP / Turbidity at 18°C was about twice as much as RFU of GFP / Turbidity at 37°C. It is concluded that Pcold is a cold inducible promoter compared with Pcon of which expression is caused constantly.</p>
  
 
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<div id="discussion_contents" class="container_contents">
 
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<p class="normal_text">
 
<p class="normal_text">
Snow White project attempts to introduce a Pcold-<span style="font-style : italic">rhlI</span> plasmid into the Magic Mirror <i>coli</i> and to use the Magic Mirror <i>coli</i> as a starter of the story. The Magic Mirror <i>coli</i> cultured at 37°C hardly produces RhlI protein and C4, which is produced by RhlI. C4 starts to accumulate when Snow White <i>coli</i> is cultured at low temperatures like 18°C. In this way, Pcold works as expected when it is introduced into the Magic Mirror <span style="font-style : italic">coli</span>.
+
Snow White project attempts to insert a Pcold <span style="font-style : italic">rhlI</span> plasmid into the Magic Mirror <span style="font-style : italic">coli</span> and to use the Magic Mirror <span style="font-style : italic">coli</span> as a starter of the story. The Magic Mirror <span style="font-style : italic">coli</span> cultured at 37°C hardly produces RhlI protein and C4, which is produced by RhlI. C4 starts to accumulate when Snow White <span style="font-style : italic">coli</span> is cultured at low temperatures like 18°C. In this way, Pcold works as expected when it is introduced into the Magic Mirror <span style="font-style : italic">coli</span>.  
  
 
</p>
 
</p>
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<div id="methods" class="container">
 
<div id="methods" class="container">
 
<div id="methods_header" class="container_header">
 
<div id="methods_header" class="container_header">
<h2><span>5. Materials and Methods</span></h2>
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<h2><span>5. Materials and methods</span></h2>
 
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All the sample were BL21(DE3) strain</p>
 
All the sample were BL21(DE3) strain</p>
 
<p class="normal_text">-Plasmids<br>
 
<p class="normal_text">-Plasmids<br>
-Pcold - <span style="font-style : italic">gfp</span> (pSB1C3)(<a href="http://parts.igem.org/Part:BBa_K1949001">BBa_K1949001</a>)<br>
+
-Pcold &#8208; <span style="font-style : italic">gfp</span> (pSB1C3)(<a href="http://parts.igem.org/Part:BBa_K1949001">BBa_K1949001</a>)<br>
-Positive Control: Pcon - <span style="font-style : italic">rbs - gfp</span> (pSB1C3)<br>
+
-Positive Control: Pcon &#8208; <span style="font-style : italic">rbs &#8208; gfp</span> (pSB1C3)<br>
 
-Negative Control: empty vector (pSB1C3)
 
-Negative Control: empty vector (pSB1C3)
  
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<div id="protocol">
 
<div id="protocol">
 
<div id="protocol_header">
 
<div id="protocol_header">
<h3><span>5-2. Assay Protocol</span></h3>
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<h3><span>5-2. Assay protocol</span></h3>
 
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2. Dilute the overnight cultures in 3 mL fresh LB medium containing chloramphenicol (34 microg/mL) so that the turbidity becomes around 0.05 in triplicate (fresh culture).<br> <br>
 
2. Dilute the overnight cultures in 3 mL fresh LB medium containing chloramphenicol (34 microg/mL) so that the turbidity becomes around 0.05 in triplicate (fresh culture).<br> <br>
 
3. Incubate the triplicated fresh cultures each at 28ºC so that the turbidity reaches 0.1 to 0.12<br><br>
 
3. Incubate the triplicated fresh cultures each at 28ºC so that the turbidity reaches 0.1 to 0.12<br><br>
4. Incubate the triplicated fresh cultures each in water and ice bath for 2min.<br><br>
+
4. Incubate the triplicated fresh cultures each in water and ice bath for 2 min.<br><br>
 
5. Incubate the triplicated fresh cultures each at 18ºC, and 37ºC for each sample for 3 h.<br><br>
 
5. Incubate the triplicated fresh cultures each at 18ºC, and 37ºC for each sample for 3 h.<br><br>
 
6. Measure the turbidity and RFU of GFP / Turbidity.
 
6. Measure the turbidity and RFU of GFP / Turbidity.
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<p class="normal_text">Jump to <a href="https://2016.igem.org/Team:Tokyo_Tech/Promoter_Assay/Pheat">Pheat assay</a> page.</p>
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Latest revision as of 12:20, 6 November 2016

1. Introduction

“Magic Mirror on the wall, who is the fairest one of all?” Snow White begins with the scene where Magic Mirror replies that “Snow White is the fairest one.” If Magic Mirror did not reply as above, Snow White would not begin.

We analyzed the cold inducible promoter (Pcold: BBa_K1949000) as a starter of Snow White and found that expression from Pcold was induced at 18°C compared to 37°C.

2. Summary of the experiment

The purpose of this experiment is to characterize temperature-dependence of Pcold by comparing the expression level of a reporter gene, gfp, at 18°C with that at 37°C. We prepared three transformants shown below.

A. Pcold ‐ gfp (pSB1C3)


Fig. 3-4-1-2-1 Pcon ‐ gfp (pSB1C3)

B. Positive Cntrol : Pcon ‐ rbs ‐ gfp (pSB1C3)


Fig. 3-4-1-2-2 Pcon ‐ rbs ‐ gfp (pSB1C3)

C. Negative Control : empty vector (pSB1C3)


Fig. 3-4-1-2-3 empty vector (pSB1C3)

3. Results

RFU of GFP / Turbidity of each sample is measured at 18°C and 37°C. The results are shown below.


Fig. 3-4-1-3-1 Pcold is a cold inducible promoter


Fig. 3-4-1-3-2 Activity of Pcon

These graphs show RFU of GFP / Turbidity per cells at indicated time points after the temperature down-shift. The error bar represents the standard deviation from the triplicated data which are derived from three independent colonies, respectively.
RFU of GFP / Turbidity of GFP induced by the promoter was calculated by subtracting RFU of GFP / Turbidity of negative control at each temperature from the fluorescence intensities of Pcold ‐ gfp and Pcon ‐rbs ‐ gfp. The proportion of RFU of GFP / Turbidity at 18°C to RFU of GFP / Turbidity at 37°C about each sample was calculated.

Concerning Pcold, it was found that RFU of GFP / Turbidity at 18°C was about eight times as much as that at 37°C. On the other hand, concerning Pcon, RFU of GFP / Turbidity at 18°C was about twice as much as RFU of GFP / Turbidity at 37°C. It is concluded that Pcold is a cold inducible promoter compared with Pcon of which expression is caused constantly.

4. Discussion

Snow White project attempts to insert a Pcold ‐ rhlI plasmid into the Magic Mirror coli and to use the Magic Mirror coli as a starter of the story. The Magic Mirror coli cultured at 37°C hardly produces RhlI protein and C4, which is produced by RhlI. C4 starts to accumulate when Snow White coli is cultured at low temperatures like 18°C. In this way, Pcold works as expected when it is introduced into the Magic Mirror coli.

5. Materials and methods

5-1. Construction

-Strain
All the sample were BL21(DE3) strain

-Plasmids
-Pcold ‐ gfp (pSB1C3)(BBa_K1949001)
-Positive Control: Pcon ‐ rbs ‐ gfp (pSB1C3)
-Negative Control: empty vector (pSB1C3)





5-2. Assay protocol


1. Prepare overnight cultures for each sample in 3 mL LB medium containing chloramphenicol (34 microg / mL) at 37ºC for 12 h.

2. Dilute the overnight cultures in 3 mL fresh LB medium containing chloramphenicol (34 microg/mL) so that the turbidity becomes around 0.05 in triplicate (fresh culture).

3. Incubate the triplicated fresh cultures each at 28ºC so that the turbidity reaches 0.1 to 0.12

4. Incubate the triplicated fresh cultures each in water and ice bath for 2 min.

5. Incubate the triplicated fresh cultures each at 18ºC, and 37ºC for each sample for 3 h.

6. Measure the turbidity and RFU of GFP / Turbidity.

6. Reference


[1] Nakashima N and Tamura T. Cell-free protein synthesis using cell extract of Pseudomonas fluorescence and CspA promoter. Biochemical and Biophysical Research Communications. Biochem Biophys Res Commun. 2004 Jun 25;319(2):671-6.

Jump to Pheat assay page.