Difference between revisions of "Team:TokyoTech/InterLab"

 
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     <li style="font-size: 16px; text-indent:3em">- <a href="#overview" onclick="w3_close()" class="w3-bar-item w3-button w3-hover-white">1</a></li>
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<div class="w3-container" id="contact" style="margin-top:30px"><!-- ページタイトル -->
 
<div class="w3-container" id="contact" style="margin-top:30px"><!-- ページタイトル -->
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       <h1 class="w3-xxxlarge w3-text-red" style="padding-bottom: 10px;padding-top: 10px" align="center">Interlab</h2>
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       <h1 class="w3-xxxlarge w3-text-red" style="padding-bottom: 10px;padding-top: 10px" align="center">InterLab</h2>
 
     </div>
 
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−
     <p style="font-size: 16px; text-indent:1em">In iGEM 2017, the Interlab Study is one of the bronze medal criteria. We measured the GFP expression level of the six designated devices, positive control and negative control using the plate reader. It was the second time for our team to join this Interlab Study (The first time in iGEM 2015).  
+
     <p style="font-size: 16px; text-indent:1em">In iGEM 2017, the InterLab Study is one of the bronze medal criteria. We measured the GFP expression level of the six designated devices, positive control and negative control using the plate reader. It was the second time for our team to join this InterLab Study (The first time in iGEM 2015).  
 
     </p><br>
 
     </p><br>
  
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  <div class="w3-container" id="overview" style="margin-top:20px">
 
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     <h1 class="w3-xxxlarge w3-text-red" style="padding-bottom: 10px;padding-top: 10px"><b>Summary of the Experiment</b></h2>
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     <h1 class="w3-xxxlarge w3-text-red" style="padding-bottom: 10px;padding-top: 10px"><b>Summary</b></h2>
 
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     <p style="font-size: 16px; text-indent:1em">
 
     <p style="font-size: 16px; text-indent:1em">
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   Our purpose was to investigate the RBS intensity and promoter intensity effects on gene expression by obtaining the fluorescence data of samples and comparing them.
+
   Our purpose was to investigate the RBS intensity and promoter intensity effects on gene expression by obtaining the fluorescence data of samples and comparing them. We prepared Device1〜Device6, Positive Control and Negative Control as shown below.
 
     </p><br>
 
     </p><br>
  
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     <div class="w3-xxxlarge" style="padding-bottom: 10px;padding-top: 10px;text-align: center">
 
       <figure>
 
       <figure>
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       <img src="<!--画像URL-->" style="max-width:70%">
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       <img src="https://static.igem.org/mediawiki/parts/2/2f/T--TokyoTech--Interlab_Devices.png" style="max-width:70%">
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       <figcaption style="font-size: 16px">Fig. 画像タイトル</figcaption>
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       <figcaption style="font-size: 16px">Fig. 1 Designated devices</figcaption>
 
       </figure>
 
       </figure>
 
       </div><!-- 図終了 -->
 
       </div><!-- 図終了 -->
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     <p style="font-size: 16px; text-indent:1em">
 
     <p style="font-size: 16px; text-indent:1em">
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     1. </br>
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     1. We obtained LUDOX correction values.</p></br>
 
     <!-- 図開始 -->
 
     <!-- 図開始 -->
 
     <div class="w3-xxxlarge" style="padding-bottom: 10px;padding-top: 10px;text-align: center">
 
     <div class="w3-xxxlarge" style="padding-bottom: 10px;padding-top: 10px;text-align: center">
 
       <figure>
 
       <figure>
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       <img src="<https://2017.igem.org/File:T--TokyoTech--LUDOX_interlab.png>" style="max-width:60%">
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       <img src="https://static.igem.org/mediawiki/2017/4/4e/T--TokyoTech--LUDOX_interlab.png" style="max-width:30%">
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       <figcaption style="font-size: 16px">Fig. 1 画像タイトル</figcaption>
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       <figcaption style="font-size: 16px">Fig. 2 LUDOX correct value </figcaption>
 
       </figure>
 
       </figure>
 
       </div><!-- 図終了 -->
 
       </div><!-- 図終了 -->
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<p style="font-size: 16px; text-indent:1em">
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2. We obtained the two Fluorescein Standard Curve (normal and log scale).</br>
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2. We obtained the two Fluorescein Standard Curve (normal and log scale).</p></br>
 
  <!-- 図開始 -->
 
  <!-- 図開始 -->
 
     <div class="w3-xxxlarge" style="padding-bottom: 10px;padding-top: 10px;text-align: center">
 
     <div class="w3-xxxlarge" style="padding-bottom: 10px;padding-top: 10px;text-align: center">
 
       <figure>
 
       <figure>
−
       <img src="<https://static.igem.org/mediawiki/2017/8/82/T--TokyoTech--Fluorescein_Standard_Curve.png>" style="max-width:60%">
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       <img src="https://static.igem.org/mediawiki/2017/8/82/T--TokyoTech--Fluorescein_Standard_Curve.png" style="max-width:60%">
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       <figcaption style="font-size: 16px">Fig. 2 Fluorescein Standard Curve</figcaption>
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       <figcaption style="font-size: 16px">Fig. 3 Fluorescein Standard Curve</figcaption>
 
       </figure>
 
       </figure>
 
       </div><!-- 図終了 -->
 
       </div><!-- 図終了 -->
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     <div class="w3-xxxlarge" style="padding-bottom: 10px;padding-top: 10px;text-align: center">
 
       <figure>
 
       <figure>
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       <img src="<https://static.igem.org/mediawiki/2017/a/ad/T--TokyoTech--Fluorescein_Standard_Curve%28log_scale%29.png>" style="max-width:60%">
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       <img src="https://static.igem.org/mediawiki/2017/a/ad/T--TokyoTech--Fluorescein_Standard_Curve%28log_scale%29.png" style="max-width:60%">
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       <figcaption style="font-size: 16px">Fig. 3 Fluorescein Standard Curve (log scale)</figcaption>
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       <figcaption style="font-size: 16px">Fig. 4 Fluorescein Standard Curve (log scale)</figcaption>
 
       </figure>
 
       </figure>
 
       </div><!-- 図終了 -->
 
       </div><!-- 図終了 -->
−
 
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<p style="font-size: 16px; text-indent:1em">
 
3. We obtained the graph bellow. The RFU of GFP / Turbidity was in the following order, Device1>Device4>Device2>Positive Control>Device5>Device3>Device6>Negative Control.
 
3. We obtained the graph bellow. The RFU of GFP / Turbidity was in the following order, Device1>Device4>Device2>Positive Control>Device5>Device3>Device6>Negative Control.
 
     </p><br>
 
     </p><br>
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       <figure>
 
       <figure>
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       <img src="<https://2017.igem.org/File:T--TokyoTech--Fluo--OD600ave.png>" style="max-width:70%">
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       <img src="https://static.igem.org/mediawiki/2017/f/f3/T--TokyoTech--Fluo--OD600ave.png" style="max-width:70%">
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       <figcaption style="font-size: 16px">Fig. 4 Fluorescein (using AVERGE)</figcaption>
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       <figcaption style="font-size: 16px">Fig. 5 Fluorescein (using AVERGE)</figcaption>
 
       </figure>
 
       </figure>
 
       </div><!-- 図終了 -->
 
       </div><!-- 図終了 -->
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     <div class="w3-xxxlarge" style="padding-bottom: 10px;padding-top: 10px;text-align: center">
 
       <figure>
 
       <figure>
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       <img src="<https://static.igem.org/mediawiki/2017/a/a5/T--TokyoTech--Fluo--OD600geo.png>" style="max-width:70%">
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       <img src="https://static.igem.org/mediawiki/2017/a/a5/T--TokyoTech--Fluo--OD600geo.png" style="max-width:70%">
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       <figcaption style="font-size: 16px">Fig. 5 Fluorescein (using GEOMEAN)</figcaption>
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       <figcaption style="font-size: 16px">Fig. 6 Fluorescein (using GEOMEAN)</figcaption>
 
       </figure>
 
       </figure>
 
       </div><!-- 図終了 -->
 
       </div><!-- 図終了 -->
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   </p><br>
 
   </p><br>
  
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</div>
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<hr>
  
 
   <div class="w3-container" id="overview" style="margin-top:20px">
 
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     <hr style="width:50px;border:5px solid red" class="w3-round">
  
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     <p style="font-size: 16px">
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     <center>
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  Strain</br>
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    <object data="https://static.igem.org/mediawiki/2017/0/03/T--TokyoTech--interlab2.pdf" type="application/pdf" width="500px" height="500px"></object>
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All the plasmids were prepared in DH5a strain.</br></br>
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    </center>
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+
−
Plate reader</br>
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−
Tecan Infinite M200 PRO</br></br>
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−
Protocol</br>
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−
We followed the InterLab 2017 Plate Reader Protocol which iGEM offerd us.</br>
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−
リンクはる
+
−
1. OD600 Reference point</br>
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−
1) Add 100 μl LUDOX into wells A1, B1, C1, D1.</br>
+
−
 
+
−
2) Add 100 μl of H 2O into wells A2, B2, C2, D2. </br>
+
−
3) Measure absorbance 600 nm of all samples.</br> </br>
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−
 
+
−
2. Protocol fluorescein fluorescence standard curve </br>
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−
1) Spin down fluorescein stock tube to make sure pellet is at the bottom of tube. </br>
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−
2) Prepare 2x fluorescein stock solution (100 μM) by resuspending fluorescein in 1
+
−
mL of 1xPBS. </br>
+
−
 
+
−
3) Dilute the 2x fluorescein stock solution with 1xPBS to make a 1x fluorescein solution and resulting concentration of fluorescein stock solution 50 μM.</br>
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−
4) Add 100 μl of PBS into wells A2, B2, C2, D2....A12, B12, C12, D12.</br>
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−
5) Add 200 μl of fluorescein 1x stock solution into A1, B1, C1, D1.</br>
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−
6) Transfer 100 μl of fluorescein stock solution from A1 into A2. </br>
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7) Mix A2 by pipetting up and down 3x and transfer 100 μl into A3... </br>
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8) Mix A3 by pipetting up and down 3x and transfer 100 μl into A4... </br>
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9) Mix A4 by pipetting up and down 3x and transfer 100 μl into A5... </br>
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10) Mix A5 by pipetting up and down 3x and transfer 100 μl into A6... </br>
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11) Mix A6 by pipetting up and down 3x and transfer 100 μl into A7... </br>
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12) Mix A7 by pipetting up and down 3x and transfer 100 μl into A8... </br>
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13) Mix A8 by pipetting up and down 3x and transfer 100 μl into A9... </br>
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14) Mix A9 by pipetting up and down 3x and transfer 100 μl into A10... </br>
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15) Mix A10 by pipetting up and down 3x and transfer 100 μl into A11... </br>
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16) Mix A11 by pipetting up and down 3x and transfer 100 μl into liquid waste.</br>
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17)Repeat dilution series for rows B, C, D</br>
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18) Measure fluorescence of all samples at 490 nm as an excitation wavelength, 525 nm as an emission wavelength.</br></br>
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3. Cell measurement protocol</br>
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1) transform Escherichia coli DH5α with these following plasmids:</br>
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● Positive control</br>
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● Negative control</br>
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● Test Device 1: J23101+I13504</br>
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● Test Device 2: J23106+I13504</br>
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● Test Device 3: J23117+I13504</br>
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● Test Device 4: J23101.BCD2.E0040.B0015</br>
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● Test Device 5: J23106.BCD2.E0040.B0015</br>
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● Test Device 6: J23117.BCD2.E0040.B0015</br>
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2) Pick 2 colonies from each of plate and inoculate it on 10 mL LB medium + Chloramphenicol. Grow the cells overnight at 37˚C and 220 rpm.</br>
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3) Measure OD600 of the overnight cultures.</br>
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4) Dilute the cultures to a target OD 600 of 0.02 in 12 mL LB medium + Chloramphenicol in large test tube.</br>
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5) Incubate the cultures at 37°C and 220 rpm.</br>
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6) Take 500 μL samples of the cultures at 0, 2, 4, and 6 hours of incubation.</br>
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7) Place samples on ice.</br>
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8) At the end of sampling point, measure OD600 and RFU of GFP of all the samples at 490 nm as an excitation wavelength, 525 nm as an emission wavelength.</br>
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  2015 Tokyo_Tech Interlab
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    <a href=https://2015.igem.org/Team:Tokyo_Tech/Experiment/Interlab>2015 Tokyo_Tech InterLab</a>, accessed Augst 8, 2017
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Latest revision as of 00:17, 2 November 2017

<!DOCTYPE html> Coli Sapiens

iGEM Tokyo Tech

InterLab

Introduction

In iGEM 2017, the InterLab Study is one of the bronze medal criteria. We measured the GFP expression level of the six designated devices, positive control and negative control using the plate reader. It was the second time for our team to join this InterLab Study (The first time in iGEM 2015).


Summary

Our purpose was to investigate the RBS intensity and promoter intensity effects on gene expression by obtaining the fluorescence data of samples and comparing them. We prepared Device1〜Device6, Positive Control and Negative Control as shown below.


Fig. 1 Designated devices

Results

1. We obtained LUDOX correction values.


Fig. 2 LUDOX correct value

2. We obtained the two Fluorescein Standard Curve (normal and log scale).


Fig. 3 Fluorescein Standard Curve
Fig. 4 Fluorescein Standard Curve (log scale)

3. We obtained the graph bellow. The RFU of GFP / Turbidity was in the following order, Device1>Device4>Device2>Positive Control>Device5>Device3>Device6>Negative Control.


Fig. 5 Fluorescein (using AVERGE)
Fig. 6 Fluorescein (using GEOMEAN)

Discussion

Judging from the results, the gene expression is controlled by the intensity of promoter and the intensity of RBS. The promoter intensity of is supposed to be in the following order, J23101>J23106>J23117. The RBS intensity of I13504 is supposed to be higher than that of BCD2.E0040. Moreover, it is considered that the GFP expression level is dependent on individual to some extent.


Materials and Methods

Reference

2015 Tokyo_Tech InterLab, accessed Augst 8, 2017


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