Latest revision as of 00:13, 2 November 2017

<!DOCTYPE html> Coli Sapiens

iGEM Tokyo Tech

TraI Improvement Assay

Introduction

In the previous wiki page (Read TraI Assay page), we describe that the productivity of C8 in E. coli depends on the culture temperatures. However, to complete our co-culture system, the current 3OC8HSL (hereafter C8) productivity at 37℃ was not enough to transmit the AHL signal to human cells, because human cells are usually grown at 37℃. Therefore, we tried to mutate the traI gene and increase the productivity of C8 at 37℃.

Summary of experiment

TraI has not been characterized extensively, and thus, it is unclear what kind of mutations is appropriate for the above purpose. A preceding study describes that, in the case of LuxI, the amino acid substitution at the 34th and 63rd positions (both are substitutions from glutamate to glycine; E34G and E63G) increase the productivity of C6 (2). Since TraI has homology to LuxI over the entire amino acid sequences, we speculate that the same amino acid substitutions in TraI can increase the productivity of C8. Also, we here describe the modification of the culture conditions and the host strain choice for increased C8 production.

Results

When amino acid sequences of TraI and LuxI were aligned using the clustal W program (1), the E34 and E63 residues of LuxI were found to correspond to K34 and Q63 residues of TraR. According to this information, oligonucleotide primers to create TraI-K34G, TraI-Q63G, and TraI-K34G,Q63G mutants were designed. The primer sequences are shown in Fig. 1. The mutations were introduced to the pSB1C3-based traI plasmid using the inverse-PCR method, and successful introduction of the mutations were confirmed with Sanger sequencing.

Fig. 1 Sequences of the primers. Note that each primer set is divergent for inverse-PCR

The sequences of traI mutants and wild-type are shown in Fig. 2. The plasmids we used are shown in Fig. 3 and 4. The Sender and the Reporter strains were prepared in the same way as described in the previous wiki page (Read TraI Assay page).

Fig. 2 Sequence of traI wild type gene and mutant

Fig. 3 Structure of the plasmids used for creating the “Reporter” strain.

Fig. 4 Structure of the plasmids used for creating the “Sender” and “Mutated Sender” strains. One of these plasmids was used for preparing each transfromant

In the experiments shown in this page, one additional modification was made in experimental conditions; 1 microM of SAM (S-adenosylmethionine; structure is shown in Fig. 5) was added to the culture of the Sender. Since C8 is synthesized from SAM and ACP (acyl carrier protein) through the action of TraI in bacterial cells (3), we expected that the addition of SAM may increase the productivity.

Fig. 5 Chemical structure of SAM (S‐adenosylmethionine)

The result of C8 production using the TraI wild-type and the mutants is shown in Fig. 6. "W.T." means native traI.
The RFU value of the TraI(K34G)-expressing cells was about 3-fold higher than that of the TraI-expressing cells. E. coli introduced empty vector was used as Negative Control.
Other mutant did not show improvement of C8 production (data was not shown).
When these RFU values were converted to C8 concentrations using the calibration curve obtained in the reagent assay (Read TraI Assay page), they were calculated as 28 nM and 42 nM, respectively.

Fig. 6 Improvement of C8 production by the K34G mutant (37℃ culture)

Also, the other modification was made concerning the host strain of the Sender. The preceding iGEM study has shown that the amount of AHL produced by the luxI gene highly depends on the host strain; depending on the used strains as the Sender, there was approximatly 100-fold difference in cell number for obtaining the same activation level of the lux promoter (4). Therefore, we here used the other strain, MG1655hapB, as the Sender.
Note that, in the experiments of the previous wiki page (Read TraI Assay page ), only the DH5α strain was used as a host. The MG1655hapB strain is a mutant of MG1655 (the representative wild-type K12 strain) and has higher membrane permeability for hydrophobic compounds compared to the parent.
As a result (Fig. 7), we found that amount of C8 produced is dependent on strains. The RFU value in the MG1655hapB strain was about 3-fold higher than that in the DH5α strain. E. coli introduced empty vector was used as Negative Control.
When these RFU values were converted to C8 concentrations using the calibration curve obtained in the reagent assay (Read TraI Assay page), they were calculated as 28 and 37 nM, respectively.

Strain dependence of AHL production
We found that amount of C8 production is depend on E. coli’s strain. RFU is 2-fold higher than DH5α.
Calculated from the graph obtained in the reagent assay, C8 concentration of DH5α culture was 28 nM and MG1655hapB culture was 36 nM.

Fig. 7 Strain dependence of C8 production

Discussion

In the previous study, it was considered that the E34G mutation of LuxI most likely enhances the interactions between the enzyme and the ACP substrate. Therefore, we thought that K34G mutation of TraI also has the same effect.
It was also showed that the MG1655hapB strain produced more C8 than the DH5α strain. We speculate that the difference in permeability of hydrophobic compounds through the cell membrane is the main reason for this result.
Taken together, we conclude that increasing the productivity of C8 at 37℃ was successful. Notably, generation and functional identification the mutant traI (TraI(K34G)) meet the medal criteria of ”parts improvement”, because the wild-type traI parts was registered in iGEM parts collection earlier (Read BBa_K553001 page). However, further improvement of C8 production is necessary to transmit the signal from bacteria to human cells. Such improvement is possible through tuning the experimental conditions further.

Appendix: Material and Method

Reference

"> (1). http://www.genome.jp/tools-bin/clustalw
(2). Pavan Kumar Reddy Kambam, Daniel J. Sayut, Yan Niu, Dawn T. Eriksen, Lianhong Sun (2008) Directed evolution of LuxI for enhanced OHHL production. Biotechnology and Bioengineering Volume 101, Issue 2 1 October 2008 Pages 263-272
(3). MATTHEW R. PARSEK, DALE L. VAL, BRIAN L. HANZELKA, JOHN E. CRONAN, E. P. GREENBERG (1999) Acyl homoserine-lactone quorum-sensing signal generation. Proc. Natl. Acad. Sci. USA Vol. 96, pp. 4360-4365, April 1999 Biochemistry
(4). https://2007.igem.org/wiki/index.php/Chiba/Quorum_Sensing

@@ Line 3: / Line 3: @@
 <!DOCTYPE html>
 <html>
-<head>
 <title>Coli Sapiens</title>
 <meta charset="UTF-8">
 <meta name="viewport" content="width=device-width, initial-scale=1">
 <link rel="stylesheet" href="main.css">
-<link rel="stylesheet" href="https://fonts.googleapis.com/css?family=Poppins">
 <style>
-body,h1,h2,h3,h4,h5 {font-family: "Poppins", sans-serif}
+body,h1,h2,h3,h4,h5 {font-family: "Arial", sans-serif}
 body {font-size:16px;}
 .w3-half img{margin-bottom:-6px;margin-top:16px;opacity:0.8;cursor:pointer}
 .w3-half img:hover{opacity:1}
 </style>
-<head>
 <body>
+<div id="loader-bg">
+  <div id="loader">
+    <div class="photo-show">
+    <img src="https://static.igem.org/mediawiki/2017/f/f4/T--TokyoTech--load_1.png" width="600">
+    <img src="https://static.igem.org/mediawiki/2017/4/43/T--TokyoTech--load_2.png" width="600">
+    </div>
+  </div>
+</div>
 <!-- Sidebar/menu -->
 <nav class="w3-sidebar w3-red w3-collapse w3-top w3-large w3-padding" style="z-index:3;width:300px;font-weight:bold;" id="mySidebar"><br>
-   <a href="javascript:void(0)" onclick="w3_close()" class="w3-button w3-hide-large w3-display-topleft" style="width:100%;font-size:22px">Close Menu</a>
+   <a href="javascript:void(0)" onclick="w3_close()" class="w3-button w3-hide-large w3-display-topleft" style="width:100%;font-size:20px">Close Menu</a>
-   <div class="w3-container">
+   <div class="w3-container" style="margin-top: 20px;margin-bottom: 25px" id="wrap">
-     <img src="https://static.igem.org/mediawiki/2017/5/57/T--TokyoTech--logo2.png" style="width: 100%">
+     <img src="https://static.igem.org/mediawiki/2017/a/a8/T--TokyoTech--logo_white_bright_10211603.png" style="width: 100%">
    </div>
-   <div class="w3-bar-block" style="padding-top: 15px">
-     <ul id="normal" class="dropmenu">
+   <div style="padding-top: 15px; padding-left: 25px; id="wrap">
-     <li><a href="#overview" onclick="w3_close()" class="w3-bar-item w3-button w3-hover-white">Home</a>
+  <div id="contents" style="z-index: 10">
+    <ul class="click_menu_checkbox vertm_down">
+    <li style="margin-bottom: 10px; border: 5px double #fff; border-radius: 10px;">
+    <input type="checkbox" id="vmcb-a" />
+    <label for="vmcb-a"><a href="https://2017.igem.org/Team:TokyoTech">Home</a></label>
+    </li>
+    <li style="margin-bottom: 10px; border: 5px double #fff; border-radius: 10px">
+    <input type="checkbox" id="vmcb-b" />
+    <label for="vmcb-b"><a href="https://2017.igem.org/Team:TokyoTech/Medal">Achievements</a></label>
+    </li>
+    <li style="margin-bottom: 10px; border: 5px double #fff; border-radius: 10px">
+    <input type="checkbox" id="vmcb-c" />
+    <label for="vmcb-c"><a>Project</a></label>
+     <ul>
+      <li style="text-align: center;"><a href="https://2017.igem.org/Team:TokyoTech/Description" class="w3-bar-item w3-button w3-hover-white">Description</a></li>
+      <li style="text-align: center;"><a href="https://2017.igem.org/Team:TokyoTech/Project/Basic_Parts" onclick="w3_close()" class="w3-bar-item w3-button w3-hover-white">Basic Parts</a></li>
+      <li style="text-align: center;"><a href="https://2017.igem.org/Team:TokyoTech/Project/Composite_Parts" onclick="w3_close()" class="w3-bar-item w3-button w3-hover-white">Composite Parts</a></li>
+     </ul>
+    </li>
+    <li style="margin-bottom: 10px; border: 5px double #fff; border-radius: 10px">
+    <input type="checkbox" id="vmcb-d" />
+    <label for="vmcb-d"><a>Experiment</a></label>
+     <ul>
+        <li>
+        <input type="checkbox" id="vmcb-d1" />
+        <label for="vmcb-d1"><a href="https://2017.igem.org/Team:TokyoTech/Experiment/Overview" onclick="w3_close()" class="w3-bar-item w3-button w3-hover-white" style="text-align: center;">Overview</a></label>
+        </li>
+        <li style="padding-bottom: 10px; padding-top: 5px">
+        <input type="checkbox" id="vmcb-d2" />
+          <label for="vmcb-d2"><a style="text-align: center;">Bacteria to <br>Human Cells ▼</a></label>
+            <ul>
+              <li style="text-align: center;"><a href="https://2017.igem.org/Team:TokyoTech/Experiment/TraI_Assay" onclick="w3_close()" class="w3-bar-item w3-button w3-hover-white">TraI Assay</a></li>
+              <li style="text-align: center;"><a href="https://2017.igem.org/Team:TokyoTech/Experiment/TraI_Improvement" onclick="w3_close()" class="w3-bar-item w3-button w3-hover-white">TraI Improvement <br>Assay</a></li>
+              <li style="text-align: center;"><a href="https://2017.igem.org/Team:TokyoTech/Experiment/TraR_Reporter_Assay" onclick="w3_close()" class="w3-bar-item w3-button w3-hover-white" >TraR Reporter <br>Assay</a></li>
+              <li style="text-align: center;"><a href="https://2017.igem.org/Team:TokyoTech/Experiment/Transcriptome_Analysis" onclick="w3_close()" class="w3-bar-item w3-button w3-hover-white">Transcriptome <br>Analysis</a></li>
+              <li style="text-align: center;"><a href="https://2017.igem.org/Team:TokyoTech/Experiment/Chimeric_Transcription_Factor" onclick="w3_close()" class="w3-bar-item w3-button w3-hover-white">Chimeric <br> Transcription <br> Factor Assay</a></li>
+            </ul>
+      </li>
+      <li style="padding-bottom: 3px">
+      <input type="checkbox" id="vmcb-d3" />
+     <label for="vmcb-d3"><a style="text-align: center;">Human Cells to <br>Bacteria ▼</a></label>
+        <ul>
+          <li><a href="https://2017.igem.org/Team:TokyoTech/Experiment/AHK4_Assay" onclick="w3_close()" class="w3-bar-item w3-button w3-hover-white">AHK4 Assay</a></li>
+          </ul>
+        </li>
+        <li>
+        <input type="checkbox" id="vmcb-d4" />
+    <label for="vmcb-d4"><a href="https://2017.igem.org/Team:TokyoTech/InterLab" onclick="w3_close()" class="w3-bar-item w3-button w3-hover-white" style="text-align: center;">InterLab</a></label>
+        </li>
+    </ul>
+    </li>
+    <li style="margin-bottom: 10px; text-align: center; border: 5px double #fff; border-radius: 10px">
+    <input type="checkbox" id="vmcb-e" />
+    <label for="vmcb-e"><a href="https://2017.igem.org/Team:TokyoTech/Model">Modelling</a></label>
+    </li>
+    <li style="margin-bottom: 10px; text-align: center; border: 5px double #fff; border-radius: 10px">
+    <input type="checkbox" id="vmcb-f" />
+    <label for="vmcb-f"><a>Human Practices</a></label>
      <ul>
-     <li><a href="#overview" onclick="w3_close()" class="w3-bar-item w3-button w3-hover-white">1</a></li>
+     <li style="text-align: center;"><a href="https://2017.igem.org/Team:TokyoTech/HP" onclick="w3_close()" class="w3-bar-item w3-button w3-hover-white">Overview</a></li>
+    <li style="text-align: center;"><a href="https://2017.igem.org/Team:TokyoTech/HP/Silver" onclick="w3_close()" class="w3-bar-item w3-button w3-hover-white">Silver</a></li>
+    <li style="text-align: center;"><a href="https://2017.igem.org/Team:TokyoTech/HP/Gold_Integrated" onclick="w3_close()" class="w3-bar-item w3-button w3-hover-white">Gold (Integrated)</a></li>
+    <li style="text-align: center;"><a href="https://2017.igem.org/Team:TokyoTech/Demonstrate" onclick="w3_close()" class="w3-bar-item w3-button w3-hover-white">Demonstrate</a></li>
+    <li style="text-align: center;"><a href="https://2017.igem.org/Team:TokyoTech/Collaborations" onclick="w3_close()" class="w3-bar-item w3-button w3-hover-white">Collaborations</a></li>
      </ul>
      </li>
+    <li style="margin-bottom: 10px; text-align: center; border: 5px double #fff; border-radius: 10px">
+    <input type="checkbox" id="vmcb-g" />
+    <label for="vmcb-g"><a>About us</a></label>
+    <ul>
+    <li style="text-align: center;"><a href="https://2017.igem.org/Team:TokyoTech/Team" onclick="w3_close()" class="w3-bar-item w3-button w3-hover-white">Team</a></li>
+    <li style="text-align: center;"><a href="https://2017.igem.org/Team:TokyoTech/Attributions" onclick="w3_close()" class="w3-bar-item w3-button w3-hover-white">Attributions</a></li>
+    <li style="text-align: center;"><a href="https://2017.igem.org/Team:TokyoTech/Sponsors" onclick="w3_close()" class="w3-bar-item w3-button w3-hover-white">Sponsors</a></li>
      </ul>
-     <a href="#project" onclick="w3_close()" class="w3-bar-item w3-button w3-hover-white">Project</a>
+     </li>
-     <a href="#modeling" onclick="w3_close()" class="w3-bar-item w3-button w3-hover-white">Modeling</a>
+     </ul>
-     <a href="#hp" onclick="w3_close()" class="w3-bar-item w3-button w3-hover-white">Human Practices</a>
+     </div>
-    <a href="#notebook" onclick="w3_close()" class="w3-bar-item w3-button w3-hover-white">Notebook</a>
-    <a href="#team" onclick="w3_close()" class="w3-bar-item w3-button w3-hover-white">Team</a>
-    <a href="#sponsers" onclick="w3_close()" class="w3-bar-item w3-button w3-hover-white">Sponsers</a>
    </div>
 </nav>
@@ Line 63: / Line 146: @@
      <h1 class="w3-xxxlarge w3-text-red" style="padding-bottom: 10px;padding-top: 10px"><b>Introduction</b></h1><!-- 小見出し -->
      <hr style="width:50px;border:5px solid red" class="w3-round">
-     <p style="font-family: Poppins;font-size: 16px">
+     <p style="font-size: 16px; text-indent:1em">
 In the previous wiki page (Read <a href="https://2017.igem.org/Team:TokyoTech/Experiment/TraI_Assay">TraI Assay</a> page), we describe that the productivity of C8 in <span style="font-style: italic">E. coli</span> depends on the culture temperatures. However, to complete our co-culture system, the current 3OC8HSL (hereafter C8) productivity at 37℃ was not enough to transmit the AHL signal to human cells, because human cells are usually grown at 37℃. Therefore, we tried to mutate the <span style="font-style: italic">traI</span> gene and increase the productivity of C8 at 37℃.<br>
      </p>
@@ Line 74: / Line 157: @@
      <h1 class="w3-xxxlarge w3-text-red" style="padding-bottom: 10px;padding-top: 10px"><b>Summary of experiment</b></h1><!-- 小見出し -->
      <hr style="width:50px;border:5px solid red" class="w3-round">
-      <p style="font-family: Poppins;font-size: 16px">
+      <p style="font-size: 16px; text-indent:1em">
 TraI has not been characterized extensively, and thus, it is unclear what kind of mutations is appropriate for the above purpose. A preceding study describes that, in the case of LuxI, the amino acid substitution at the 34th and 63rd positions (both are substitutions from glutamate to glycine; E34G and E63G) increase the productivity of C6 (2). Since TraI has homology to LuxI over the entire amino acid sequences, we speculate that the same amino acid substitutions in TraI can increase the productivity of C8. Also, we here describe the modification of the culture conditions and the host strain choice for increased C8 production. <br>    </p>
 	</p>
@@ Line 84: / Line 167: @@
      <h1 class="w3-xxxlarge w3-text-red" style="padding-bottom: 10px;padding-top: 10px"><b>Results</b></h1><!-- 小見出し -->
      <hr style="width:50px;border:5px solid red" class="w3-round">
-	<p style="font-family: Poppins;font-size: 16px">
+	<p style="font-size: 16px; text-indent:1em">
 When amino acid sequences of TraI and LuxI were aligned using the clustal W program (1), the E34 and E63 residues of LuxI were found to correspond to K34 and Q63 residues of TraR. According to this information, oligonucleotide primers to create TraI-K34G, TraI-Q63G, and TraI-K34G,Q63G mutants were designed. The primer sequences are shown in Fig. 1. The mutations were introduced to the pSB1C3-based traI plasmid using the inverse-PCR method, and successful introduction of the mutations were confirmed with Sanger sequencing.<br>
 	<div class="w3-xxxlarge" style="padding-bottom: 10px;padding-top: 10px;text-align: center">
 	<figure>
-     	<img src="https://static.igem.org/mediawiki/2017/4/43/T--TokyoTech--TraIimprove100.jpg" style="max-width:50%">
+     	<img src="https://static.igem.org/mediawiki/2017/4/43/T--TokyoTech--TraIimprove100.jpg" style="max-width:80%">
-     	<figcaption style="font-family: Poppins;font-size: 16px">Fig. 1 Sequences of the primers. Note that each primer set is divergent for inverse-PCR</figcaption>
+     	<figcaption style="font-size: 16px">Fig. 1 Sequences of the primers. Note that each primer set is divergent for inverse-PCR</figcaption>
 	</figure>
 	</p>
-     <p style="font-family: Poppins;font-size: 16px">
+     <p style="font-size: 16px; text-indent:1em">
 The sequences of <span style="font-style: italic">traI</span> mutants and wild-type are shown in Fig. 2. The plasmids we used are shown in Fig. 3 and 4. The Sender and the Reporter strains were prepared in the same way as described in the previous wiki page (Read <a href="https://2017.igem.org/Team:TokyoTech/Experiment/TraI_Assay">TraI Assay</a> page).<br>
      	<figure>
-     	<img src="https://static.igem.org/mediawiki/2017/c/c9/T--TokyoTech--TraIimprove20.jpg" style="max-width:50%">
+     	<img src="https://static.igem.org/mediawiki/2017/c/c9/T--TokyoTech--TraIimprove20.jpg" style="max-width:80%">
-     	<figcaption style="font-family: Poppins;font-size: 16px">Fig. 2 Sequence of <span style="font-style: italic">traI</span> wild type gene and mutant</figcaption>
+     	<figcaption style="font-size: 16px">Fig. 2 Sequence of <span style="font-style: italic">traI</span> wild type gene and mutant</figcaption>
 	</figure>
 	<br>
 	<figure>
 	<img src="https://static.igem.org/mediawiki/2017/5/51/Fig2_structure_of_the_plasmid.png" style="max-width:75%">
-     	<figcaption style="font-family: Poppins;font-size: 16px">Fig. 3 Structure of the plasmids used for creating the “Reporter” strain. </figcaption>
+     	<figcaption style="font-size: 16px">Fig. 3 Structure of the plasmids used for creating the “Reporter” strain. </figcaption>
      	</figure>
 	</br>
 	<figure>
 	<img src="https://static.igem.org/mediawiki/2017/d/d7/TraI_Improvement_fig4.png" style="max-width:65%">
-     	<figcaption style="font-family: Poppins;font-size: 16px">Fig. 4 Structure of the plasmids used for creating the “Sender” and “Mutated Sender” strains. One of these plasmids was used for preparing each transfromant</figcaption>
+     	<figcaption style="font-size: 16px">Fig. 4 Structure of the plasmids used for creating the “Sender” and “Mutated Sender” strains. One of these plasmids was used for preparing each transfromant</figcaption>
      	</figure>
 	</p>
 	</div>
-	<p style="font-family: Poppins;font-size: 16px">
+	<p style="font-size: 16px; text-indent:1em">
-In the experiments shown in this page, one additional modification was made in experimental conditions; 1 μM of SAM (S-adenosylmethionine; structure is shown in Fig. 5) was added to the culture of the Sender. Since C8 is synthesized from SAM and ACP (acyl carrier protein) through the action of TraI in bacterial cells (3), we expected that the addition of SAM may increase the productivity.	</p>
+In the experiments shown in this page, one additional modification was made in experimental conditions; 1 microM of SAM (S-adenosylmethionine; structure is shown in Fig. 5) was added to the culture of the Sender. Since C8 is synthesized from SAM and ACP (acyl carrier protein) through the action of TraI in bacterial cells (3), we expected that the addition of SAM may increase the productivity.	</p>
 	<div class="w3-xxxlarge" style="padding-bottom: 10px;padding-top: 10px;text-align: center">
 	<figure>
@@ Line 117: / Line 200: @@
 </br>
 </br>
-     	<figcaption style="font-family: Poppins;font-size: 16px">Fig. 5 Chemical structure of SAM (S‐adenosylmethionine) </figcaption>
+     	<figcaption style="font-size: 16px">Fig. 5 Chemical structure of SAM (S‐adenosylmethionine) </figcaption>
      	</figure>
 	</p>
-	<p style="font-family: Poppins;font-size: 16px">
+	<p style="font-size: 16px; text-indent:1em">
 The result of C8 production using the TraI wild-type and the mutants is shown in Fig. 6. "W.T." means native <span style="font-style: italic">traI</span>.<br>
 The RFU value of the TraI(K34G)-expressing cells was about 3-fold higher than that of the TraI-expressing cells. <span style="font-style: italic">E. coli</span> introduced empty vector was used as Negative Control.<br>
@@ Line 127: / Line 210: @@
 	<figure>
 	<img src="https://static.igem.org/mediawiki/2017/3/32/T--TokyoTech--TraIimprove50.jpg" style="max-width:50%">
-     	<figcaption style="font-family: Poppins;font-size: 16px">Fig. 6 Improvement of C8 production by the K34G mutant (37℃ culture)</figcaption>
+     	<figcaption style="font-size: 16px">Fig. 6 Improvement of C8 production by the K34G mutant (37℃ culture)</figcaption>
      	</figure>
 	</p>
-	<p style="font-family: Poppins;font-size: 16px">
+	<p style="font-size: 16px; text-indent:1em">
 </br>
 Also, the other modification was made concerning the host strain of the Sender. The preceding iGEM study has shown that the amount of AHL produced by the <span style="font-style: italic">luxI</span> gene highly depends on the host strain; depending on the used strains as the Sender, there was approximatly 100-fold difference in cell number for obtaining the same activation level of the lux promoter (4). Therefore, we here used the other strain, MG1655hapB, as the Sender.<br>
@@ Line 138: / Line 221: @@
 When these RFU values were converted to C8 concentrations using the calibration curve obtained in the reagent assay (Read <a href="https://2017.igem.org/Team:TokyoTech/Experiment/TraI_Assay">TraI Assay</a> page), they were calculated as 28 and 37 nM, respectively.
 	</p>
-	<p style="font-family: Poppins;font-size: 16px">
+	<p style="font-size: 16px; text-indent:1em">
 Strain dependence of AHL production<br>
 We found that amount of C8 production is depend on <span style="font-style: italic">E. coli</span>’s strain. RFU is 2-fold higher than DH5α. <br>
@@ Line 144: / Line 227: @@
      	<figure>
      	<img src="https://static.igem.org/mediawiki/2017/f/f7/T--TokyoTech--TraIimprove3.jpg" style="max-width:50%">
-     	<figcaption style="font-family: Poppins;font-size: 16px">Fig. 7 Strain dependence of C8 production</figcaption>
+     	<figcaption style="font-size: 16px">Fig. 7 Strain dependence of C8 production</figcaption>
      	</figure>
 	</p>
@@ Line 154: / Line 237: @@
      <h1 class="w3-xxxlarge w3-text-red" style="padding-bottom: 10px;padding-top: 10px"><b>Discussion</b></h1>
      <hr style="width:50px;border:5px solid red" class="w3-round">
-    <p style="font-family: Poppins;font-size: 16px">
+   <p style="font-size: 16px; text-indent:1em">
 In the previous study, it was considered that the E34G mutation of LuxI most likely enhances the interactions between the enzyme and the ACP substrate. Therefore, we thought that K34G mutation of TraI also has the same effect.  <br>
 It was also showed that the MG1655hapB strain produced more C8 than the DH5α strain. We speculate that the difference in permeability of hydrophobic compounds through the cell membrane is the main reason for this result.<br>
@@ Line 176: / Line 259: @@
      <h1 class="w3-xxxlarge w3-text-red" style="padding-bottom: 10px;padding-top: 10px"><b>Reference</b></h1>
      <hr style="width:50px;border:5px solid red" class="w3-round">
-     <p style="font-family: Poppins;font-size: 16px">
+     <p style="font-size: 16px; text-indent:1em">">
 (1). http://www.genome.jp/tools-bin/clustalw<br>
 (2). Pavan Kumar Reddy Kambam, Daniel J. Sayut, Yan Niu, Dawn T. Eriksen, Lianhong Sun (2008) Directed evolution of LuxI for enhanced OHHL production. Biotechnology and Bioengineering Volume 101, Issue 2 1 October 2008 Pages 263-272<br>
@@ Line 187: / Line 270: @@
 </div>
-<div class="w3-light-grey w3-container w3-padding-32" style="margin-top:75px;padding-right:58px"><p class="w3-right">Hajime Fujita:  <a href="96haji.me" title="W3.CSS" target="_blank" class="w3-hover-opacity">All Rights Reserved</a></p></div>
+ <div class="w3-container" id="contact" style="margin-top:20px">
+ <p id="pageTop" style="text-align:right"><a href="#wrap"><img src="https://static.igem.org/mediawiki/2017/0/0d/T--TokyoTech--page_top_2.png" style="width:200px"></a></p>
+</div>
+<!-- W3.CSS Container -->
+<div class="w3-light-grey w3-container w3-padding-32" style="margin-top:75px;padding-right:58px"><p class="w3-right"><a href="http://96haji.me/" title="W3.CSS" target="_blank" class="w3-hover-opacity">Hajime Fujita with W3.CSS: All Rights Reserved</a></p></div>
+<script src="http://ajax.googleapis.com/ajax/libs/jquery/1.11.1/jquery.min.js"></script>
 <script>
 // Script to open and close sidebar
@@ Line 200: / Line 289: @@
      document.getElementById("myOverlay").style.display = "none";
 }
+</script>
+<script>
 // Modal Image Gallery
 function onClick(element) {
@@ Line 208: / Line 298: @@
    captionText.innerHTML = element.alt;
 }
+$(function() {
+  var h = $(window).height();
+  $('#wrap').css('display','none');
+  $('#loader-bg ,#loader').height(h).css('display','block');
+});
+$(window).load(function () { //全ての読み込みが完了したら実行
+  $('#loader-bg').delay(900).fadeOut(800);
+  $('#loader').delay(600).fadeOut(300);
+  $('#wrap').css('display', 'block');
+});
 </script>
+<script>
+//■page topボタン
+$(function(){
+var topBtn=$('#pageTop');
+topBtn.hide();
+//◇ボタンの表示設定
+$(window).scroll(function(){
+  if($(this).scrollTop()>80){
+    //---- 画面を80pxスクロールしたら、ボタンを表示する
+    topBtn.fadeIn();
+  }else{
+    //---- 画面が80pxより上なら、ボタンを表示しない
+    topBtn.fadeOut();
+  }
+});
+// ◇ボタンをクリックしたら、スクロールして上に戻る
+topBtn.click(function(){
+  $('body,html').animate({
+  scrollTop: 0},500);
+  return false;
+});
+});
+</script>
 </body>
 </html>

Difference between revisions of "Team:TokyoTech/Experiment/TraI Improvement"