Team:KIT-Kyoto/Modeling

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<h3>Project Concept</h3>
<h3>Project Concept</h3>
<p class="sentence">
<p class="sentence">
-
Two monoterpene synthetic systems were designed by combining two monoterpene synthetic enzyme genes which are already on the parts’ list, and gamma-terpinene synthetic enzyme gene we submitted as well as two protein devices. <br>
+
Two monoterpene synthetic systems were designed by combining two monoterpene synthetic enzyme genes which are already on the parts’ list, and &#947;-terpinene synthetic enzyme gene we submitted as well as two protein devices. <br>
-
One device is <em>E.coli</em> IPTG-inducing gamma-terpinene synthetic pathway, and the other is yeast-based gamma-terpinene synthetic pathway.
+
One device is <em>E. coli</em> IPTG-inducing &#947;-terpinene synthetic pathway, and the other is yeast-based &#947;-terpinene synthetic pathway.
<br>
<br>
</p>
</p>
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<div class="PDbox">
<div class="PDs1">
<div class="PDs1">
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<h3>Expression in Yeast</h3>
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<h3>Expression in <em>S. cerevisiae</em></h3>
<p class="sentence">
<p class="sentence">
-
The device in this yeast-based monoterpene synthetic pathway uses the S. cerevisiae cells which constantly express monoterpene synthetic enzyme by introducing each gene of monoterpene synthetic enzymes at the downstream of TEF promoter. GPP is constitutively synthesized in yeast cells and its volume is enough for intracellular monoterpene synthesis. Thus a certain monoterpene is easily synthesized by transferring this device into yeast cells.
+
The device in this yeast-based monoterpene synthetic pathway uses the <em>S. cerevisiae</em> cells which constantly express monoterpene synthetic enzyme by introducing each gene of monoterpene synthetic enzymes at the downstream of <em>TEF</em> promoter. GPP is constitutively synthesized in yeast cells and its volume is enough for intracellular monoterpene synthesis. Thus a certain monoterpene is easily synthesized by transferring this device into yeast cells.
</p>
</p>
</div>
</div>
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<div class="PDbox">
<div class="PDs2">
<div class="PDs2">
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<h3>Expression in <em>E.coli</em></h3>
+
<h3>Expression in <em>E. coli</em></h3>
<p class="sentence">
<p class="sentence">
-
The device expresses only when BL21(DE3) strain is used for genetic transfer. In this device, each monoperpene synthetic gene is under the control of tac promoter, so the expression of each monoterpene synthase gene is induced by adding IPTG. By changing the dose of IPTG and the timing of addition, the monoterpene volume changes and the intensity of scent also changes.  
+
The device expresses only when BL21(DE3) strain is used as the host. In this device, each monoperpene synthetic gene is under the control of <em>tac</em> promoter, so the expression of each monoterpene synthase gene is induced by adding IPTG. By changing the dose of IPTG and the timing of addition, the monoterpene volume changes and the intensity of scent also changes.  
-
<br>However, E.coli does not have enzyme to synthesize geranyl pyrophosphate (GPP), a basal material for all monoterpene synthesis. So one has to directly add GPP to the medium or transfer the DMAPP transferase gene (ERG20).
+
<br>However, <em>E. coli</em> does not have enzyme to synthesize geranyl pyrophosphate (GPP), a basal material for all monoterpene synthesis. So one has to directly add GPP to the medium or transfer the DMAPP transferase gene (ERG20).
</p>
</p>
</div>
</div>
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<div class="PDbox">
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<h3>How the Pathways Function</h3> <!--はてなマークつけないのが正しい-->
<p class="sentence">
<p class="sentence">
-
<h3>How the pathways function?</h3>
+
In the two pathways we designed, the behaviors of insects on the plate can be controlled via the scents that can be regulated by the types of monoterpene synthases, the volume and the timing of IPTG addition and the location of colonies on the plate. In our pathways, yeast and <em>E. coli</em> cells become a conductor to orchestrate insects.
-
<br>
+
-
In the two pathways we designed, the behaviors of insects on the plate can be controlled via the scents that can be regulated by the types of monoterpene synthases, the volume and the timing of IPTG addition and the location of colonies on the plate. In our pathways, yeast and E.coli cells become a conductor to orchestrate insects.
+
<br>
<br>
</p>
</p>
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<h2 id="bioassay">Biological Assay</h2>
<h2 id="bioassay">Biological Assay</h2>
<div class="scroll"></div>
<div class="scroll"></div>
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<h3>Purpose</h3>
<p class="sentence">
<p class="sentence">
 +
To evaluate the effect of limonene on the behavior of <em>Drosophila melanogaster</em>.
<br>
<br>
 +
</p>
 +
<h3>Materials</h3>
 +
<p class="sentence">
 +
The first-third instar larvae of <em>Drosophila melanogaster</em> Canton S (CS) as a wild type strain.
<br>
<br>
 +
</p>
 +
<div class="PDbox">
 +
<div class="PDs1">
 +
<h3>Method</h3>
 +
<p class="sentence">
 +
200 larvae were placed at the center of an agar plate (2.0%, radius: 8cm). A piece of filter paper was placed on each end of the plate (Figure 1). 2.5µl of limonene was dropped on one side of the filter paper (Limonene) and cover the plate with a lid. Three minutes later, the number of larvae within a 3cm radius from the filter paper was counted (A or B). The concentrations of limonene are diluted by 20% and 10% with DMSO. The control is DMSO.
 +
</p>
 +
<p class="sentence">
 +
The behavioral effects of incentives were measured by Response Index (RI). The RI is defined as (Ns-Nc)/(Ns+Nc), where Ns is the number of population attracted to limonene, and Nc is the number of population which moved to the control.
 +
</p>
 +
<p class="sentence">
 +
When all the population move into the radius with limonene, RI is 1 and when all the population move into the radius of control, RI is -1. Positive values mean positive chemotactic effect, and negative values mean negative chemotactic effect.<br>
 +
</p>
 +
</div>
 +
<div class="pdimg2">
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<img src="https://static.igem.org/mediawiki/2014/6/67/Kit_Bioassay.png" alt='bioassay' />
 +
</div>
 +
</div>
 +
<div class="clear"><hr /></div>
 +
<h3>Result</h3>
 +
<p class="sentence">
 +
<div class="bioassay">
 +
<strong>Table 1. With Limonene 100%</strong>
 +
<div class="m_table">
 +
<table class="materials">
 +
<tr>
 +
<th>Trial</th>
 +
<th>A<br>(Limonene)</th>
 +
<th>B<br>(Control)</th>
 +
<th>RI</th>
 +
</tr>
 +
<tr>
 +
<td>1st</td>
 +
<td>139</td>
 +
<td>11</td>
 +
<td>0.853</td>
 +
</tr>
 +
<tr>
 +
<td>2nd</td>
 +
<td>116</td>
 +
<td>52</td>
 +
<td>0.381</td>
 +
</tr>
 +
<tr>
 +
<td>3rd</td>
 +
<td>130</td>
 +
<td>11</td>
 +
<td>0.844</td>
 +
</tr>
 +
<tr>
 +
<td>Average</td>
 +
<td>128</td>
 +
<td>25</td>
 +
<td>0.678</td>
 +
</tr>
 +
</table>
 +
</div>
 +
<img src="/wiki/images/3/3b/Kit_Bioassay_fig2.png">
 +
</div>
<br>
<br>
 +
<div class="bioassay">
 +
<strong>Table 2. With Limonene 20%</strong>
 +
<div class="m_table">
 +
<table class="materials">
 +
<tr>
 +
<th>Trial</th>
 +
<th>A<br>(Limonene)</th>
 +
<th>B<br>(Control)</th>
 +
<th>RI</th>
 +
</tr>
 +
<tr>
 +
<td>1st</td>
 +
<td>115</td>
 +
<td>59</td>
 +
<td>0.322</td>
 +
</tr>
 +
<tr>
 +
<td>2nd</td>
 +
<td>96</td>
 +
<td>71</td>
 +
<td>0.149</td>
 +
</tr>
 +
<tr>
 +
<td>3rd</td>
 +
<td>133</td>
 +
<td>42</td>
 +
<td>0.520</td>
 +
</tr>
 +
<tr>
 +
<td>Average</td>
 +
<td>115</td>
 +
<td>57</td>
 +
<td>0.333</td>
 +
</tr>
 +
</table>
 +
</div>
 +
<img src="/wiki/images/7/79/Kit_Bioassay_fig3.png">
 +
</div>
<br>
<br>
 +
<div class="bioassay">
 +
<strong>Table 3. With Limonene 10%</strong>
 +
<div class="m_table">
 +
<table class="materials">
 +
<tr>
 +
<th>Trial</th>
 +
<th>A<br>(Limonene)</th>
 +
<th>B<br>(Control)</th>
 +
<th>RI</th>
 +
</tr>
 +
<tr>
 +
<td>1st</td>
 +
<td>89</td>
 +
<td>72</td>
 +
<td>0.106</td>
 +
</tr>
 +
<tr>
 +
<td>2nd</td>
 +
<td>98</td>
 +
<td>59</td>
 +
<td>0.248</td>
 +
</tr>
 +
<tr>
 +
<td>3rd</td>
 +
<td>92</td>
 +
<td>78</td>
 +
<td>0.082</td>
 +
</tr>
 +
<tr>
 +
<td>Average</td>
 +
<td>93</td>
 +
<td>70</td>
 +
<td>0.143</td>
 +
</tr>
 +
</table>
 +
</div>
 +
<img src="/wiki/images/1/13/Kit_Bioassay_fig4.png">
 +
</div>
<br>
<br>
-
<br>
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</p>
-
<br>
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<h3>Discussion</h3>
-
<br>
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<p class="sentence">
-
<br>
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Because RI was over 0 in all the trials, it was revealed that limonene attracts <em>Drosophila melanogaster</em>. Furthermore, it was suggested that the effects of incentives were increased relative to the concentration of limonene.<br>
-
<br>
+
</p>
-
<br>
+
<h3>Reference</h3>
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<br>
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<p class="sentence">
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<br>
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Shimoyama, N. (2014). TJB 200000764. <br>[online] Biol.tsukuba.ac.jp. Available at:<br> <a href="http://www.biol.tsukuba.ac.jp/tjb/Vol3No2/TJB200402200000764.html">http://www.biol.tsukuba.ac.jp/tjb/Vol3No2/TJB200402200000764.html</a> [Accessed 25 Aug. 2014].
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Latest revision as of 20:56, 17 October 2014

Modeling

Parts & Device

Project Concept

Two monoterpene synthetic systems were designed by combining two monoterpene synthetic enzyme genes which are already on the parts’ list, and γ-terpinene synthetic enzyme gene we submitted as well as two protein devices.
One device is E. coli IPTG-inducing γ-terpinene synthetic pathway, and the other is yeast-based γ-terpinene synthetic pathway.

fig1

Expression in S. cerevisiae

The device in this yeast-based monoterpene synthetic pathway uses the S. cerevisiae cells which constantly express monoterpene synthetic enzyme by introducing each gene of monoterpene synthetic enzymes at the downstream of TEF promoter. GPP is constitutively synthesized in yeast cells and its volume is enough for intracellular monoterpene synthesis. Thus a certain monoterpene is easily synthesized by transferring this device into yeast cells.

fig2

Expression in E. coli

The device expresses only when BL21(DE3) strain is used as the host. In this device, each monoperpene synthetic gene is under the control of tac promoter, so the expression of each monoterpene synthase gene is induced by adding IPTG. By changing the dose of IPTG and the timing of addition, the monoterpene volume changes and the intensity of scent also changes.
However, E. coli does not have enzyme to synthesize geranyl pyrophosphate (GPP), a basal material for all monoterpene synthesis. So one has to directly add GPP to the medium or transfer the DMAPP transferase gene (ERG20).

fig3

How the Pathways Function

In the two pathways we designed, the behaviors of insects on the plate can be controlled via the scents that can be regulated by the types of monoterpene synthases, the volume and the timing of IPTG addition and the location of colonies on the plate. In our pathways, yeast and E. coli cells become a conductor to orchestrate insects.

Biological Assay

Purpose

To evaluate the effect of limonene on the behavior of Drosophila melanogaster.

Materials

The first-third instar larvae of Drosophila melanogaster Canton S (CS) as a wild type strain.

Method

200 larvae were placed at the center of an agar plate (2.0%, radius: 8cm). A piece of filter paper was placed on each end of the plate (Figure 1). 2.5µl of limonene was dropped on one side of the filter paper (Limonene) and cover the plate with a lid. Three minutes later, the number of larvae within a 3cm radius from the filter paper was counted (A or B). The concentrations of limonene are diluted by 20% and 10% with DMSO. The control is DMSO.

The behavioral effects of incentives were measured by Response Index (RI). The RI is defined as (Ns-Nc)/(Ns+Nc), where Ns is the number of population attracted to limonene, and Nc is the number of population which moved to the control.

When all the population move into the radius with limonene, RI is 1 and when all the population move into the radius of control, RI is -1. Positive values mean positive chemotactic effect, and negative values mean negative chemotactic effect.

bioassay

Result

Table 1. With Limonene 100%
Trial A
(Limonene)		 B
(Control)		 RI
1st 139 11 0.853
2nd 116 52 0.381
3rd 130 11 0.844
Average 128 25 0.678

Table 2. With Limonene 20%
Trial A
(Limonene)		 B
(Control)		 RI
1st 115 59 0.322
2nd 96 71 0.149
3rd 133 42 0.520
Average 115 57 0.333

Table 3. With Limonene 10%
Trial A
(Limonene)		 B
(Control)		 RI
1st 89 72 0.106
2nd 98 59 0.248
3rd 92 78 0.082
Average 93 70 0.143

Discussion

Because RI was over 0 in all the trials, it was revealed that limonene attracts Drosophila melanogaster. Furthermore, it was suggested that the effects of incentives were increased relative to the concentration of limonene.

Reference

Shimoyama, N. (2014). TJB 200000764.
[online] Biol.tsukuba.ac.jp. Available at:
http://www.biol.tsukuba.ac.jp/tjb/Vol3No2/TJB200402200000764.html [Accessed 25 Aug. 2014].

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