Difference between revisions of "Team:Tartu TUIT/Results"

(Prototype team page)
 
Line 1: Line 1:
−
{{Tartu_TUIT}}
+
{{Tartu_TUIT/header}}
 +
{{Tartu_TUIT/reset}}
 +
{{Tartu_TUIT/style}}
 +
{{Tartu_TUIT/nav}}
 
<html>
 
<html>
 +
<section id="banner" class="banner">
 +
    <div class="title text-glow-blue">RESULTS</div>
 +
</section>
 +
<i id="header-trigger"></i>
 +
<section class="article" >
 +
    <div class="content">
 +
        <h2>Introduction</h2>
 +
        <p>In order to determine the strength of pRPL18B promoter, we have conducted the following experiment. First of all, we have constructed 2 plasmids where EGFP is expressed under different constitutive promoters. We have chosen pTDH3 as a positive control due to its wide use in yeast research. The empty plasmid was used as a negative control.</p>
 +
        <h2>MATERIAL AND METHOD</h2>
 +
       
 +
        <p>Plasmids used to conduct the experiment are listed in table 1.</p>
 +
        <p>Table 1. Plasmids created</p>
 +
<table>
 +
<tr><td colspan="2">&nbsp;</td><td colspan="3">Insert</td><td>&nbsp;</td></tr>
 +
<tr><td>number</td><td>Plasmid name</td><td>Promoter</td><td>Gene</td><td>Terminator</td><td>backbone</td></tr>
 +
<tr><td>1</td><td>pRS306 pTDH3-EGFP-tCYC1</td><td>TDH3</td><td>EGFP</td><td>tCYC1</td><td>pRS306</td></td>
 +
<tr><td>2</td><td>pRS306 pRPL18B-EGFP-tCYC1</td><td>pRPL18B</td><td>EGFP</td><td>tCYC1</td><td>pRS306</td></tr>
 +
<tr><td>3</td><td>pRS306</td><td>-</td><td>-</td><td>-</td><td>pRS306</td></tr>
 +
</table>
  
 +
        <p>After construction of plasmids was finished, we have transformed S. cerevisiae CEN.PK-2-1C strain with plasmids listed above to create the strains (table 2).</p>
 +
        <p>Table 2. S. cerevisiae strains created.</p>
 +
<table>
 +
<tr><td>&nbsp;</td><td>Strain name</td><td>Genotype</td><td>Plasmid integrated</td></tr>
 +
<tr><td>positive control</td><td>YST1</td><td>CEN.PK-2-1C: MATa ura3-52 trp1-289 leu2-3,112 his3 ∆ SUC2</td><td>pRS306 pTDH3-EGFP-tCYC1</td></tr>
 +
<tr><td>Negative control</td><td>YST2</td><td>CEN.PK-2-1C: MATa ura3-52 trp1-289 leu2-3,112 his3 ∆ SUC2</td><td>pRS306</td></tr>
 +
<td>Test</td><td>YST3</td><td>CEN.PK-2-1C: MATa ura3-52 trp1-289 leu2-3,112 his3 ∆ SUC2</td><td>pRS306 pRPL18B-EGFP-tCYC1</td></tr>
 +
</table>
  
−
<div class="column full_size">
+
       
−
<h1>Results</h1>
+
        <p>We have measured the EGFP fluorescence intensity with BioTek Synergy MX microplate reader.
−
<p>Here you can describe the results of your project and your future plans. </p>
+
All strains were pregrown overnight at 30°C in liquid –URA/2%Glc media, resuspended in fresh -URA/2%Glc media to OD600 ca. 1 and distributed to 96 well plate (clear flat bottom). 3 replicates from each strain were used. 100 µL of cells were added to each well. As a reference to OD600, fresh -URA/2%Glc media was used. After the 96 well plate was ready, the OD600 and fluorescence (excitation 485 nm, emission 528 nm, bandwidth 20, gain 80) were measured. The layout of the plate is described in table 3.</p>
−
</div>
+
        <p>Table 3. The 96 well plate layout for OD600 and Fluorescence measurements.</p>
 +
<table>
 +
<tr><td>&nbsp;</td><td>&nbsp;</td><td>&nbsp;</td><td>&nbsp;</td><td colspan="6">pRPL18B</td><td>&nbsp;</td></tr>
 +
<tr><td>&nbsp;</td><td>&nbsp;</td><td>Negative control</td><td>Positive control</td><td>Colony 1</td><td>Colony 2</td><td>Colony 3 </td><td>Colony 4</td><td>Colony 5</td><td>Colony 6</td><td>-Ura/2% Glc</td></tr>
 +
<tr><td>&nbsp;</td><td>&nbsp;</td><td>1</td><td>2</td><td>3</td><td>4</td><td>5</td><td>6</td><td>7</td><td>8</td><td>9</td></tr>
 +
<tr><td>Replicate 1</td><td>A</td><td>&nbsp;</td><td>&nbsp;</td><td>&nbsp;</td><td>&nbsp;</td><td>&nbsp;</td><td>&nbsp;</td><td>&nbsp;</td><td>&nbsp;</td><td>&nbsp;</td></tr>
 +
<tr><td>Replicate 2</td><td>B</td><td>&nbsp;</td><td>&nbsp;</td><td>&nbsp;</td><td>&nbsp;</td><td>&nbsp;</td><td>&nbsp;</td><td>&nbsp;</td><td>&nbsp;</td><td>&nbsp;</td></tr>
 +
<tr><td>Replicate 3</td><td>C</td><td>&nbsp;</td><td>&nbsp;</td><td>&nbsp;</td><td>&nbsp;</td><td>&nbsp;</td><td>&nbsp;</td><td>&nbsp;</td><td>&nbsp;</td><td>&nbsp;</td></tr>
 +
</table>
  
 +
 +
 +
        <p>As a fluorescence reference, the standard curve of fluorescence for fluorescein concentration was generated according with 2018 iGEM InterLab Study [2]. The only difference from the InterLab protocol was the concentration of the fluorescein used. In the InterLab study, the highest fluorescein concentration used was 10 µM. But due to the higher gain parameter used in our measurement experiment, we have used 0.313 µM to avoid out of range measurements. The layout of the 96 well plates is given in table 4.</p>
 +
        <p>Table 4. The layout of the plate for Fluorescein standard curve measurements.</p>
 +
<table>
 +
 +
<tr><td>Fluorescein µM</td><td></td><td>0,313</td><td>0,157</td><td>0,078</td><td>0,039</td><td>0,0196</td><td>0,0098</td><td>0,0049</td><td>0,0024</td><td>0</td></tr>
 +
<tr><td></td><td></td><td>1</td><td>2</td><td>3</td><td>4</td><td>5</td><td>6</td><td>7</td><td>8</td><td>9</td></tr>
 +
<tr><td>Replicate 1</td><td>A</td><td>79055</td><td>39438</td><td>20511</td><td>10034</td><td>5020</td><td>2731</td><td>1302</td><td>745</td><td>78</td></tr>
 +
<tr><td>Replicate 2</td><td>B</td><td>80361</td><td>40466</td><td>20735</td><td>10572</td><td>5367</td><td>2751</td><td>1371</td><td>784</td><td>86</td></tr>
 +
<tr><td>Replicate 3</td><td>C</td><td>76390</td><td>38680</td><td>19692</td><td>10112</td><td>5078</td><td>2719</td><td>1462</td><td>866</td><td>111</td></tr>
 +
<tr><td>Replicate 4</td><td>D</td><td>77470</td><td>38863</td><td>19833</td><td>10170</td><td>5235</td><td>2773</td><td>1344</td><td>765</td><td>121</td></tr>
 +
</table>
 +
        <h2>Results</h2>
 +
        <p> The results are presented in figure 1. As it turned out, the pRPL18B promoter is weaker than pTDH3 but still active. This corresponds to the data from the source article <a href="#ref-1">[1]</a>. </p>
 +
        <p>Figure 1<img src="results1.png"></p>
  
−
<div class="column third_size" >
+
        <h2>CONCLUSION</h2>
 +
        <p> We think the characterisation of the promoters is necessary to provide researchers with useful information and toolbox of promoters of different strength to better optimise synthetic pathways.</p>
 +
       
 +
       
 +
  <ol>
 +
        <li><a name="ref-1"></a>Denby, C. M., Li, R. A., Vu, V. T., Costello, Z., Lin, W., Chan, L. J. G., ... & Scheller, H. V. (2018). Industrial brewing yeast engineered for the production of primary flavor determinants in hopped beer. Nature communications, 9(1), 965.</li>
 +
        <li>https://static.igem.org/mediawiki/2018/0/09/2018_InterLab_Plate_Reader_Protocol.pdf, calibration 3</li>
 +
</ol>
 +
    </div>
 +
</section>
  
−
<h3>What should this page contain?</h3>
 
−
<ul>
 
−
<li> Clearly and objectively describe the results of your work.</li>
 
−
<li> Future plans for the project. </li>
 
−
<li> Considerations for replicating the experiments. </li>
 
−
</ul>
 
−
</div>
 
  
 +
</html>
  
  
−
 
+
{{Tartu_TUIT/footer}}
−
<div class="column two_thirds_size" >
+
−
<h3>Describe what your results mean </h3>
+
−
<ul>
+
−
<li> Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for. </li>
+
−
<li> Show data, but remember all measurement and characterization data must be on part pages in the Registry. </li>
+
−
<li> Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project. </li>
+
−
</ul>
+
−
</div>
+
−
 
+
−
 
+
−
<div class="clear extra_space"></div>
+
−
 
+
−
 
+
−
 
+
−
<div class="column two_thirds_size" >
+
−
<h3> Project Achievements </h3>
+
−
 
+
−
<p>You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.</p>
+
−
 
+
−
<ul>
+
−
<li>A list of linked bullet points of the successful results during your project</li>
+
−
<li>A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.</li>
+
−
</ul>
+
−
 
+
−
</div>
+
−
 
+
−
 
+
−
 
+
−
<div class="column third_size" >
+
−
<div class="highlight decoration_A_full">
+
−
<h3>Inspiration</h3>
+
−
<p>See how other teams presented their results.</p>
+
−
<ul>
+
−
<li><a href="https://2014.igem.org/Team:TU_Darmstadt/Results/Pathway">2014 TU Darmstadt </a></li>
+
−
<li><a href="https://2014.igem.org/Team:Imperial/Results">2014 Imperial </a></li>
+
−
<li><a href="https://2014.igem.org/Team:Paris_Bettencourt/Results">2014 Paris Bettencourt </a></li>
+
−
</ul>
+
−
</div>
+
−
</div>
+
−
 
+
−
 
+
−
 
+
−
 
+
−
 
+
−
</html>
+

Revision as of 00:26, 18 October 2018

Introduction

In order to determine the strength of pRPL18B promoter, we have conducted the following experiment. First of all, we have constructed 2 plasmids where EGFP is expressed under different constitutive promoters. We have chosen pTDH3 as a positive control due to its wide use in yeast research. The empty plasmid was used as a negative control.

MATERIAL AND METHOD

Plasmids used to conduct the experiment are listed in table 1.

Table 1. Plasmids created

 Insert 
numberPlasmid namePromoterGeneTerminatorbackbone
1pRS306 pTDH3-EGFP-tCYC1TDH3EGFPtCYC1pRS306
2pRS306 pRPL18B-EGFP-tCYC1pRPL18BEGFPtCYC1pRS306
3pRS306---pRS306

After construction of plasmids was finished, we have transformed S. cerevisiae CEN.PK-2-1C strain with plasmids listed above to create the strains (table 2).

Table 2. S. cerevisiae strains created.

 Strain nameGenotypePlasmid integrated
positive controlYST1CEN.PK-2-1C: MATa ura3-52 trp1-289 leu2-3,112 his3 ∆ SUC2pRS306 pTDH3-EGFP-tCYC1
Negative controlYST2CEN.PK-2-1C: MATa ura3-52 trp1-289 leu2-3,112 his3 ∆ SUC2pRS306
TestYST3CEN.PK-2-1C: MATa ura3-52 trp1-289 leu2-3,112 his3 ∆ SUC2pRS306 pRPL18B-EGFP-tCYC1

We have measured the EGFP fluorescence intensity with BioTek Synergy MX microplate reader. All strains were pregrown overnight at 30°C in liquid –URA/2%Glc media, resuspended in fresh -URA/2%Glc media to OD600 ca. 1 and distributed to 96 well plate (clear flat bottom). 3 replicates from each strain were used. 100 µL of cells were added to each well. As a reference to OD600, fresh -URA/2%Glc media was used. After the 96 well plate was ready, the OD600 and fluorescence (excitation 485 nm, emission 528 nm, bandwidth 20, gain 80) were measured. The layout of the plate is described in table 3.

Table 3. The 96 well plate layout for OD600 and Fluorescence measurements.

    pRPL18B 
  Negative controlPositive controlColony 1Colony 2Colony 3 Colony 4Colony 5Colony 6-Ura/2% Glc
  123456789
Replicate 1A         
Replicate 2B         
Replicate 3C         

As a fluorescence reference, the standard curve of fluorescence for fluorescein concentration was generated according with 2018 iGEM InterLab Study [2]. The only difference from the InterLab protocol was the concentration of the fluorescein used. In the InterLab study, the highest fluorescein concentration used was 10 µM. But due to the higher gain parameter used in our measurement experiment, we have used 0.313 µM to avoid out of range measurements. The layout of the 96 well plates is given in table 4.

Table 4. The layout of the plate for Fluorescein standard curve measurements.

Fluorescein µM0,3130,1570,0780,0390,01960,00980,00490,00240
123456789
Replicate 1A7905539438205111003450202731130274578
Replicate 2B8036140466207351057253672751137178486
Replicate 3C76390386801969210112507827191462866111
Replicate 4D77470388631983310170523527731344765121

Results

The results are presented in figure 1. As it turned out, the pRPL18B promoter is weaker than pTDH3 but still active. This corresponds to the data from the source article [1].

Figure 1

CONCLUSION

We think the characterisation of the promoters is necessary to provide researchers with useful information and toolbox of promoters of different strength to better optimise synthetic pathways.

  1. Denby, C. M., Li, R. A., Vu, V. T., Costello, Z., Lin, W., Chan, L. J. G., ... & Scheller, H. V. (2018). Industrial brewing yeast engineered for the production of primary flavor determinants in hopped beer. Nature communications, 9(1), 965.
  2. https://static.igem.org/mediawiki/2018/0/09/2018_InterLab_Plate_Reader_Protocol.pdf, calibration 3


SPONSORS