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

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<p class="normal_text">“Magic Mirror on the wall, who is the fairest one of all?”<br>
 
<p class="normal_text">“Magic Mirror on the wall, who is the fairest one of all?”<br>
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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 wouldn’t begin. We tested cold inducible promoter (Pcold) as a starter of Snow White.  As a result, we found that cold inducible promoter (Pcold) was induced more at 18°C than at 37°C.
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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. We analyzed the cold inducible promoter (Pcold) as a starter of Snow White and found that expression from Pcold was induced at 18°C compared to 37°C.
  
 
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<p class="normal_text">Our purpose of this experiment is to characterize temperature dependence of cold inducible promoter (Pcold) by comparing the fluorescence at 18°C with the fluorescence at 37°C using positive control and negative control.  We prepared three samples shown below.<br>
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<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>
 
・Pcold-<span style="font-style : italic">gfp</span> (pSB1C3)<br>
 
・Pcold-<span style="font-style : italic">gfp</span> (pSB1C3)<br>
 
・Positive Cntrol : Pcon-<span style="font-style : italic">rbs-gfp</span> (pSB1C3)<br>
 
・Positive Cntrol : Pcon-<span style="font-style : italic">rbs-gfp</span> (pSB1C3)<br>
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<div align="center"><p class="caption" style="font-size: 16px; text-align: center;"><span style="font-weight: bold;">Fig. 3-4-1- </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- </span>title</p></div>
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<p class="normal_text"> It is found that RFU of GFP / Turbidity of GFP induced by Pcold at 18°C was about eight times as much as RFU of GFP / Turbidity of GFP induced by Pcold at 37°C, and RFU of GFP / Turbidity of GFP induced by Pcon at 18°C was about twice as much as RFU of GFP / Turbidity of GFP induced by Pcon at 37°C. It is concluded that Pcold is a cold inducible promoter compared with Pcon which is induced constantly.</p>
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<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>
  
 
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<p class="normal_text">At high temperature condition, GFP was not induced by Pcold. At low temperature condition, GFP was induced by Pcold. This result is consistent with the reference. Therefore, the result is reasonable.<br>
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<p class="normal_text">
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Snow White project attempts to transform Pcold-<span style="font-style : italic">rhlI</span> into the Magic Mirror coli and to use the Magic Mirror coli as a starter of the story. The Magic Mirror coli cultivated at 37°C will produce RhlI protein and C4HSL and it will be constructed when Snow White begins at low temperature 18°C. In this way, this cold inducible promoter (Pcold) works as expected when it is transformed into the Magic Mirror <span style="font-style : italic">coli</span>.
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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>.
  
 
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Revision as of 10:59, 16 October 2016

2-4-1 Pcold assay

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.” If the magic mirror didn’t reply as above, Snow White would not begin. We analyzed the cold inducible promoter (Pcold) 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 to that at 37°C. We prepared three samples shown below.
・Pcold-gfp (pSB1C3)
・Positive Cntrol : Pcon-rbs-gfp (pSB1C3)
・Negative Control : 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- title

This graph shows 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.
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-rbs-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.


Fig. 3-4-1- title

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.

4. Discussion

Snow White project attempts to introduce 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)
-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 37 ºC for 12h.

2. Dilute the overnight cultures in 3 mL fresh LB medium containing chloramphenicol (34 microg/mL) so that the OD600 becomes around 0.05 in triplicate (fresh culture).

3. Incubate the triplicated fresh cultures each at 28ºC so that the OD600 reaches 0.3 to 0.4

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

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

6. Measure the OD 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.