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

 
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<h3 class="link"><a href="#introduction">1. Introduction</a></h3>
 
<h3 class="link"><a href="#introduction">1. Introduction</a></h3>
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<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>
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<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>
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<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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<p class="normal_text">We tested that the expression level of cold-inducible promoter (Pcold: <a href="http://parts.igem.org/Part:BBa_K1949000">BBa_K1949000</a>) that controls the start of the story. What happens if this story is set in a warm country? We tested that the expression level of temperature sensitive promoter (Pheat: <a href="http://parts.igem.org/Part:BBa_K608351">BBa_K608351</a>), which is heat-inducible promoter. </p>
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<p class="normal_text">In the previous section, the expression level of the cold-inducible promoter (Pcold: <a href="http://parts.igem.org/Part:BBa_K1949000">BBa_K1949000</a>) that triggers the start of the story was examined. What happens if this story is set in warm countries? Therefore, in this section, the expression level of the temperature sensitive promoter (Pheat: <a href="http://parts.igem.org/Part:BBa_K608351">BBa_K608351</a>), which is heat-inducible, was examined.</p>
  
 
 
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<h2><span>2. Summary of the Experiment</span></h2>
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<h2><span>2. Summary of the experiment</span></h2>
 
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<p class="normal_text">Our objective is to characterize temperature dependency of Pheat at 28°C and 37°C with positive control and negative control. We prepared three samples shown below. After temperature induction, we measured the RFU of GFP at regular time intervals.<br>
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<p class="normal_text">The objective of the experiment here is to characterize temperature dependency of Pheat;  28°C and 37°C are non-inducible and inducible conditions for this promoter. We prepared three samples shown below. After temperature up-shift, the RFU of GFP at regular time intervals was measured.<br>
 
<p class="normal_text">A. Pheat &#8208; <span style="font-style : italic">rbs &#8208; gfp</span> (pSB1C3)<br>
 
<p class="normal_text">A. Pheat &#8208; <span style="font-style : italic">rbs &#8208; gfp</span> (pSB1C3)<br>
  
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<p class="normal_text"> The RFU of GFP of each sample is measured at 28&deg;C and 37&deg;C. The results are shown below.</p>
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<p class="normal_text"> The RFU of GFP of each sample was measured at 28°C and 37°C. The results are shown below.</p>
 
<div align="center"><img src="https://static.igem.org/mediawiki/2016/b/b3/Fig.3-4-2-3-1.png"height="300"><br></div>
 
<div align="center"><img src="https://static.igem.org/mediawiki/2016/b/b3/Fig.3-4-2-3-1.png"height="300"><br></div>
 
<div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig. 3-4-2-3-1 </span>Pheat is a heat inducible promoter</p></div>
 
<div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig. 3-4-2-3-1 </span>Pheat is a heat inducible promoter</p></div>
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<p class="normal_text">This graph shows RFU of GFP / Turbidity every 1 h for 3 h that after temperature induction. The error bar represents the standard deviation of two samples which derived from two different colonies, respectively.<br>
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<p class="normal_text">This graph shows RFU of GFP / Turbidity at the indicated time points after temperature up-shift. The error bar represents the standard deviation of three samples which derived from three different colonies, respectively.<br>
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<p class="normal_text">The RFU of GFP induced by the promoter is calculated by subtracted the RFU of GFP of negative control at each temperature from the RFU of GFP of Pheat &#8208; <span style="font-style : italic">rbs &#8208; gfp</span>. It is confirmed that GFP expression can be expressed when cultivating at 37&deg;C but not at 28&deg;C. Therefore, it is concluded that Pheat is a heat-inducible promoter.</p>
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<p class="normal_text">The RFU of GFP induced by the promoter was calculated by subtracting the RFU of GFP of negative control at each temperature from the RFU of GFP of Pheat &#8208; <span style="font-style : italic">rbs &#8208; gfp</span>. It was confirmed that GFP expression was induced when cultivating at 37°C but not at 28°C. Therefore, it is concluded that Pheat is a heat-inducible promoter.</p>
 
 
 
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GFP is not induced of Pheat &#8208; <span style="font-style : italic">rbs &#8208; gfp</span> under low temperature and is induced under high temprature condition. This result is consistent with reference [1]. Therefore this experiment result is considered reasonable.
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GFP was not induced from the Pheat &#8208; <span style="font-style : italic">rbs &#8208; gfp</span> plasmid at low temperatures and was induced at high temperatures. This result is consistent with reference [1]. Therefore, this experimental result is considered to be reasonable.  
  
 
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Latest revision as of 12:23, 6 November 2016

2-4-2 Pheat assay

1. Introduction

In the previous section, the expression level of the cold-inducible promoter (Pcold: BBa_K1949000) that triggers the start of the story was examined. What happens if this story is set in warm countries? Therefore, in this section, the expression level of the temperature sensitive promoter (Pheat: BBa_K608351), which is heat-inducible, was examined.

2. Summary of the experiment

The objective of the experiment here is to characterize temperature dependency of Pheat; 28°C and 37°C are non-inducible and inducible conditions for this promoter. We prepared three samples shown below. After temperature up-shift, the RFU of GFP at regular time intervals was measured.

A. Pheat ‐ rbs ‐ gfp (pSB1C3)


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

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


Fig. 3-4-2-2-2 pSB1C3

C. Negative Control : empty vector (pSB1C3)

3. Results

The RFU of GFP of each sample was measured at 28°C and 37°C. The results are shown below.


Fig. 3-4-2-3-1 Pheat is a heat inducible promoter

This graph shows RFU of GFP / Turbidity at the indicated time points after temperature up-shift. The error bar represents the standard deviation of three samples which derived from three different colonies, respectively.

The RFU of GFP induced by the promoter was calculated by subtracting the RFU of GFP of negative control at each temperature from the RFU of GFP of Pheat ‐ rbs ‐ gfp. It was confirmed that GFP expression was induced when cultivating at 37°C but not at 28°C. Therefore, it is concluded that Pheat is a heat-inducible promoter.

4. Discussion

GFP was not induced from the Pheat ‐ rbs ‐ gfp plasmid at low temperatures and was induced at high temperatures. This result is consistent with reference [1]. Therefore, this experimental result is considered to be reasonable.

5. Materials and methods

5-1. Construction

-Strain
All the sample were BL21(DE3) strain

-Plasmids
-Pheat ‐ rbs ‐ gfp (pSB1C3)
-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 28°C 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 28℃ so that the turbidity reaches 0.1 to 0.12.

4. Incubate the triplicated fresh cultures for each sample at 28ºC and 37ºC for 3 h.

5. Measure the turbidity and the RFU of GFP.

6. Reference


[1] M Mieschendahl, B Müller-Hill. F'-coded, temperature-sensitive lambda cI857 repressor gene for easy construction and regulation of lambda promoter-dependent expression systems. J Bacteriol. 1985 Dec;164(3):1366-9.
Freiburg 2011

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