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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

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          <input type="checkbox" id="vmcb-d1" />
-          <label for="vmcb-d1"><a style="text-align: center;">Bacteria <br>to Human Cells ▼</a></label>
+        <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>
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+          <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 <br> Impovement</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/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/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/C8_Toxicity" onclick="w3_close()" class="w3-bar-item w3-button w3-hover-white">C8 Toxicity <br> Assay</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>
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            <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>
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+     <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>
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-			<h1 class="w3-xxxlarge w3-text-red" style="padding-bottom: 10px;padding-top: 10px" align="center"><span style="font-style: italic">TraI</span> Improve Assay</h1>
+			<h1 class="w3-xxxlarge w3-text-red" style="padding-bottom: 10px;padding-top: 10px" align="center">
+TraI Improvement Assay</h1>
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      <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 previous study (<span style="font-style: italic">TraI</span> Assay), we found that the amount of C8 production heavily depend on culture temperature. But to construct co-culture system, current <span style="font-style: italic">TraI</span>’s C8 production in 37℃ is not enough to send AHL signal to mammalian cells. So, we mutate <span style="font-style: italic">TraI</span> gene and tried to improve the amount of C8 production in 37℃.<br>
+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>
-A report says LuxI’s C6 production got 72 folds compared to wildtype by mutating at 34th amino position and 63th amino position. We focused that LuxI gene and <span style="font-style: italic">TraI</span> gene have homology and mutated at 34th amino position and 63th amino position (1).<br>
-After experiment in various condition, we found that <span style="font-style: italic">TraI</span> gene mutated at 34th amino position shows 3 folds of RFU compared to wild type in LB medium with 1μM of SAM (S‐adenosylmethionine).<br>
-AHL is derived from SAM and <span style="font-style: italic">TraI</span> involved in a reaction of SAM and ACP (acyl carrier protein) to produce AHL (2).<br>
-At last we also found that C8 production is depend on strain.But experiment missed iGEM presentation<br>
      </p>
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      <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">
-At first, we designed primer to introduce mutation at 34th position and 63th position and mutate <span style="font-style: italic">TraI</span> gene. Primer sequence is shown Figure. 1.
+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>
-	<div class="w3-xxxlarge" style="padding-bottom: 10px;padding-top: 10px;text-align: center">
-	<figure>
-    	<img src="https://static.igem.org/mediawiki/2017/6/60/T--TokyoTech--TraIimprove1.jpg" style="max-width:50%">
-    	<figcaption style="font-family: Poppins;font-size: 16px">Figure. 1 Sequence of Primer</figcaption>
-	</figure>
-	</div>
-	<p style="font-family: Poppins;font-size: 16px">
-Then we added 1μM of SAM (structure is shown Figure.2) to sender <span style="font-style: italic">E. coli</span>’s culture because SAM is ingredients of AHL.<br>
 	</p>
-	<div class="w3-xxxlarge" style="padding-bottom: 10px;padding-top: 10px;text-align: center">
-	<figure>
-    	<img src="https://static.igem.org/mediawiki/2017/8/8e/T--TokyoTech--TraIimproveFig2.png" style="max-width:50%">
-    	<figcaption style="font-family: Poppins;font-size: 16px">Figure. 2 Chemical structure of SAM (S‐adenosylmethionine)</figcaption>
-	</figure>
-	</div>
-	<p style="font-family: Poppins;font-size: 16px">
-At last, we confirmed that <span style="font-style: italic">TraI</span> gene mutated at 34th amino position shows 3 folds of RFU compared to wild type in 37℃ and performed same experiment in 25℃.<br>
-The plasmids we used are shown in Fig.4~6. Same reporter <span style="font-style: italic">E. coli</span> as the <span style="font-style: italic">TraI</span> assay was used. We made E. coli with wild type <span style="font-style: italic">TraI</span> or mutated <span style="font-style: italic">TraI</span> at 34th amino position as Sender E. coli and confirmed the difference in C8 production. The sequence of <span style="font-style: italic">TraI</span> mutant and wild-type is shown in Fig. 3.
-	</p>
-	<div class="w3-xxxlarge" style="padding-bottom: 10px;padding-top: 10px;text-align: center">
-	<figure>
-    	<img src="https://static.igem.org/mediawiki/2017/8/8e/T--TokyoTech--TraIimproveFig2.png" style="max-width:50%">
-    	<figcaption style="font-family: Poppins;font-size: 16px">Figure. 3 Construction of LuxR gene and Plux-gfp gene</figcaption>
-	<img src="https://static.igem.org/mediawiki/2017/3/33/T--TokyoTech--TraI2.jpg" style="max-width:50%">
-    	<figcaption style="font-family: Poppins;font-size: 16px">Figure. 4 Sequence of <span style="font-style: italic">TraI</span> wild gene and mutant</figcaption>
-<img src="https://static.igem.org/mediawiki/2017/3/33/T--TokyoTech--TraI2.jpg" style="max-width:50%">
-    	<figcaption style="font-family: Poppins;font-size: 16px">Figure. 5 Construction of <span style="font-style: italic">TraI</span> gene (Wild Type)</figcaption>
-<img src="https://static.igem.org/mediawiki/2017/3/33/T--TokyoTech--TraI2.jpg" style="max-width:50%">
-    	<figcaption style="font-family: Poppins;font-size: 16px">Figure. 6 Construction of <span style="font-style: italic">TraI</span> gene (mutant)</figcaption>
-	</figure>
-	</div>
    </div>
 <hr>
@@ Line 189: / 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">
-C8 production of <span style="font-style: italic">TraI</span> wildtype and mutant is shown in Figure. 2.<br>
+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>
-RFU value of mutant is about 3 folds larger than wildtype.<br>
-Calculated from the graph obtained in the reagent assay,<br>
-OC8HSL concentration of <span style="font-style: italic">TraI</span> Wild type culture was nM and <span style="font-style: italic">TraI</span> mutant culuture was nM.<br>
-	</p>
 	<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:80%">
+    	<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-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/2017/6/60/T--TokyoTech--TraIimprove1.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">Figure. 7 Mutated <span style="font-style: italic">TraI</span> gene’s C8 production (37℃ culture)</figcaption>
+     	<figcaption style="font-size: 16px">Fig. 2 Sequence of <span style="font-style: italic">traI</span> wild type gene and mutant</figcaption>
 	</figure>
-	</div>
+	<br>
-	<p style="font-family: Poppins;font-size: 16px">
+	<figure>
-But mutant advantage is disappeared by lowering culture temperature to 25℃.<br>
+	<img src="https://static.igem.org/mediawiki/2017/5/51/Fig2_structure_of_the_plasmid.png" style="max-width:75%">
-The value of RFU exceeded the detection limit of the graph obtained in the reagent assay. Since the value of RFU peaks at more than 100 nM, both cultures are considered to synthesize more than 200nM of 3OC8HSL.<br>
+    	<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-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-size: 16px; text-indent:1em">
+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>
-	<img src="https://static.igem.org/mediawiki/2017/d/d8/T--TokyoTech--TraIimprove2.jpg" style="max-width:50%">
+	<img src="https://static.igem.org/mediawiki/2017/4/41/T--TokyoTech--TraIimprove200.jpeg" style="max-width:50%">
-     	<figcaption style="font-family: Poppins;font-size: 16px">Figure. 8 Mutated <span style="font-style: italic">TraI</span> gene’s C8 production (25℃ culture)</figcaption>
+</br>
+</br>
+     	<figcaption style="font-size: 16px">Fig. 5 Chemical structure of SAM (S‐adenosylmethionine) </figcaption>
      	</figure>
- 	</div>
+	</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>
+Other mutant did not show improvement of C8 production (data was not shown).<br>
+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 nM and 42 nM, respectively.<br>
+	<figure>
+	<img src="https://static.igem.org/mediawiki/2017/3/32/T--TokyoTech--TraIimprove50.jpg" style="max-width:50%">
+    	<figcaption style="font-size: 16px">Fig. 6 Improvement of C8 production by the K34G mutant (37℃ culture)</figcaption>
+    	</figure>
+	</p>
+	<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>
+Note that, in the experiments of the previous wiki page (Read <a href="https://2017.igem.org/Team:TokyoTech/Experiment/TraI_Assay">TraI Assay</a> 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.<br>
+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. <span style="font-style: italic">E. coli</span> introduced empty vector was used as Negative Control.<br>
+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-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 folds larger than DH5α. <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>
-Calculated from the graph obtained in the reagent assay, 3OC8HSL concentration of DH5α culture was nM and MG1655hapB culture was nM.
+Calculated from the graph obtained in the reagent assay, C8 concentration of DH5α culture was 28 nM and MG1655hapB culture was 36 nM.
-    	</p>
-	<div class="w3-xxxlarge" style="padding-bottom: 10px;padding-top: 10px;text-align: center">
      	<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">Figure. 9 Strain dependence of C8 production</figcaption>
+     	<figcaption style="font-size: 16px">Fig. 7 Strain dependence of C8 production</figcaption>
      	</figure>
-	</div>
+	</p>
    </div>
 <hr>
@@ Line 229: / 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 previous study, it is found that LuxI gene mutation at 34th amino position most likely enhances the interactions between the enzyme and the acyl ACP substrate. Therefore we thought that this <span style="font-style: italic">TraI</span> gene mutation at 34th amino position also enhances the interactions between the enzyme and the acyl ACP substrate. But in 25℃ of culture, the effect of interaction improvement is disappeared because it is thought that thermal motion of protein become calm and the acyl ACP substrate stably bind the enzyme in case of <span style="font-style: italic">TraI</span> wildtype. Consequently, we improved <span style="font-style: italic">TraI</span> gene’s C8 production in 37℃ condition same as temperature of human body.<br>
+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>
-We also found that MG1655hapb strain produce more C8 than DH5αstrain. <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>
-It is thought that strain dependence of C8 production resulted from permeability of <span style="font-style: italic">E. coli</span>’s cell<br>
+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 <span style="font-style: italic">traI</span> parts was registered in iGEM parts collection earlier (Read <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K553001">BBa_K553001</a> 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.<br>
-membrane because MG1655hapB strain has higher permeability compared to its wildtype MG1655.<br>
-We expect further improvement of C8 production to send a signal to mammalian cells. I hope the day in which human can talk with microorganism as a same living thing.<br>
      </p>
 </div>
 <hr>
+ <div id="mtt" class="w3-container" id="overview" style="margin-top:20px">
-<div class="w3-container" id="results" style="margin-top:20px">
+     <h1 class="w3-xxxlarge w3-text-red" style="padding-bottom: 10px;padding-top: 10px"><b> Appendix: Material and Method</b></h2>
-     <h1 class="w3-xxxlarge w3-text-red" style="padding-bottom: 10px;padding-top: 10px"><b>Materials and Methods</b></h1>
      <hr style="width:50px;border:5px solid red" class="w3-round">
-     <p style="font-family: Poppins;font-size: 16px">Supernatant assay<br>
+      <center>
-.Cultivate Sender <span style="font-style: italic">E. coli</span> in LB medium for about 15hours<br>
+     <object data="https://static.igem.org/mediawiki/2017/f/fb/TraI_improvement.pdf" type="application/pdf" style="width: 70%; height: 800px"></object>
-.Centrifuge the culture 16,000rpm and 5minutes<br>
+    </center>
-.Follow Reagent assay process (1~4) and Prepare Reporter culture.<br>
+    </div>
-.Mix 250μL of sender culture’s supernatant with Reporter culture in micro tube.<br>
-.Incubate the micro tube for 5 hours with Small shaking incubator in 37℃.<br>
-.Take 100μL of culture and Measure fluorescent (excitation wave length is 495nm, Measurement wavelength is 520nm gain is 45) and absorbance (Measurement wavelength is 600nm).<br>
-    </p>
-</div>
 <hr>
@@ Line 260: / 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">(1) 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>
+     <p style="font-size: 16px; text-indent:1em">">
-(2) 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<br>
+(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>
+(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<br>
+(4). https://2007.igem.org/wiki/index.php/Chiba/Quorum_Sensing<br>
      </p>
@@ Line 269: / 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 282: / Line 289: @@
      document.getElementById("myOverlay").style.display = "none";
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+</script>
+<script>
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 function onClick(element) {
@@ Line 290: / Line 298: @@
    captionText.innerHTML = element.alt;
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+$(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);
+  $('#wrap').css('display', 'block');
+});
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+<script>
+//■page topボタン
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+var topBtn=$('#pageTop');
+topBtn.hide();
+//◇ボタンの表示設定
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+  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();
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+});
+// ◇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);
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