Difference between revisions of "Team:BNU-CHINA/Results"

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{{BNU-CHINA/article | Project | Results |b/b1/Bnubgred}}
 
{{BNU-CHINA/article | Project | Results |b/b1/Bnubgred}}
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<html>
 +
<h2>l-/d-limonene synthase
 +
</h2>
 +
<h3>Agarose gel electrophoresis of l-/d- limonene synthase gene</h3>
 +
<figure><img src="#">
 +
    <figcaption>Fig.1 the agarose gel electrophoresis of l-/d- limonene synthase gene, the bands pointed by arrows represent l-limonene gene and d-limonene gene. Lane M, DNA marker DL2000; Fig1a, Lane 1 and 2, l-LS in pSB1C3 plasmids; Fig 1b, Lane1 and 2, d-LS in pSB1C3 plasmids; Plasmid are digested by EcoRI and PstI.
 +
    </figcaption>
 +
</figure>
 +
<p>In Fig.1 limonene synthase gene is inserted into the backbone pSB1C3. Then the plasmids are digested by Pst I and EcoR I. The length of the l-LS gene, d-LS gene and backbone are 1735bp, 1904bp and 2624bp,respectively. The target genes are marked with arrows, and the figure shows that limonene synthase gene was transferred into the <em>E.coli</em> BL21 cells successfully. 1.2 SDS-PAGE analysis of l-/d- limonene synthase
 +
</p>
 +
<h3>SDS-PAGE analysis of l-/d- limonene synthase
 +
</h3>
 +
<figure><img>
 +
    <figcaption>Fig.2 SDS-PAGE of l-limonenen synthase, d-limonene synthase and Gpps Lane M, molecular weight standards (kDa); Lane2 and 3, bacteria homogenate of d-limonene synthase; Lane 4 and 5, bacteria homogenate of l-limonene synthase; the lower bands pointed by arrows show GPPS and the upper bands pointer by arrows show limonene synthases.
 +
    </figcaption>
 +
</figure>
 +
<p>In Fig 2，we want to verify whether the limonene synthase and GPPS gene are expressed or not. The molecular weight of limonene and GPP synthase is 89 kDa (with GST on pGEX-4T-1 plasmid) and 49 kDa respectively. In Fig.2, the target proteins are marked with arrows, and the figure shows that synthases are expressed in the cell successfully.
 +
</p>
 +
<h3>Verification of attraction of limonene towards C. elegans
 +
</h3>
 +
<p>In order to know whether limonene attracts <em>C. elegans</em>, filter paper is dropped of 5 \(\mu\)L5% limonene (DMSO aq) and put on one side. The control is put on the other side with only 5 \(\mu\)L DMSO. To eliminate the effect of DMSO, two other control groups are made.
 +
</p>
 +
<h5>Table 1. The results of verification of limonene to attract C. elegans
 +
</h5>
 +
<table class="table table-condensed">
 +
    <tbody>
 +
        <tr>
 +
            <th></th>
 +
            <th></th>
 +
            <th>1</th>
 +
            <th>2</th>
 +
            <th>3</th>
 +
        </tr>
 +
        <tr>
 +
            <td>Control guoup 1</td>
 +
            <td>DMSO</td>
 +
            <td>117</td>
 +
            <td>113</td>
 +
            <td></td>
 +
        </tr>
 +
        <tr>
 +
            <td></td>
 +
            <td>DMSO</td>
 +
            <td>110</td>
 +
            <td>159</td>
 +
            <td></td>
 +
        </tr>
 +
        <tr>
 +
            <td>Control group 2</td>
 +
            <td>DMSO</td>
 +
            <td>180</td>
 +
            <td>86</td>
 +
            <td>30</td>
 +
        </tr>
 +
        <tr>
 +
            <td></td>
 +
            <td>M9</td>
 +
            <td>198</td>
 +
            <td>91</td>
 +
            <td>26</td>
 +
        </tr>
 +
    </tbody>
 +
</table>
 +
<p>a. The distribution of the C. elegans on the plates of control group Control group is used to eliminate the effect of DMSO, and some other experimental factors.
 +
</p>
 +
<table class="table table-condensed">
 +
    <tbody>
 +
        <tr>
 +
            <th></th>
 +
            <th>1</th>
 +
            <th>2</th>
 +
            <th>3</th>
 +
            <th>4</th>
 +
            <th>5</th>
 +
            <th>6</th>
 +
            <th>7</th>
 +
            <th>8</th>
 +
            <th>9</th>
 +
            <th>10</th>
 +
            <th>11</th>
 +
            <th>12</th>
 +
            <th>13</th>
 +
            <th>14</th>
 +
            <th>15</th>
 +
            <th>16</th>
 +
            <th>17</th>
 +
            <th>18</th>
 +
            <th>19</th>
 +
            <th>20</th>
 +
        </tr>
 +
        <tr>
 +
            <td>DMSO</td>
 +
            <td>110</td>
 +
            <td>8</td>
 +
            <td>129</td>
 +
            <td>74</td>
 +
            <td>90</td>
 +
            <td>19</td>
 +
            <td>51</td>
 +
            <td>172</td>
 +
            <td>144</td>
 +
            <td>89</td>
 +
            <td>125</td>
 +
            <td>49</td>
 +
            <td>114</td>
 +
            <td>28</td>
 +
            <td>63</td>
 +
            <td>109</td>
 +
            <td>346</td>
 +
            <td>54</td>
 +
            <td>70</td>
 +
        </tr>
 +
        <tr>
 +
            <td>Limonene+DMSO</td>
 +
            <td>149</td>
 +
            <td>64</td>
 +
            <td>37</td>
 +
            <td>185</td>
 +
            <td>128</td>
 +
            <td>100</td>
 +
            <td>94</td>
 +
            <td>150</td>
 +
            <td>202</td>
 +
            <td>190</td>
 +
            <td>114</td>
 +
            <td>124</td>
 +
            <td>43</td>
 +
            <td>129</td>
 +
            <td>57</td>
 +
            <td>79</td>
 +
        </tr>
 +
    </tbody>
 +
</table>
 +
<p>b. The distribution of the C. elegans on the plates of experimental group
 +
</p>
 +
<p>This group shows significantly biased movement by compareing l-limonene with DMSO.
 +
</p>
 +
<p>We find that nematodes of experimental group show significantly biased movement when we count the number of nematodes (Fig.1). However, nematodes of control group shows no significant difference about the distribution of nematodes on the plate.
 +
</p>
 +
<figure><img src="#">
 +
    <figcaption>Fig.1 The distribution of the C. elegans on the plates.
 +
    </figcaption>
 +
</figure>
 +
<p>C stands for the control group in which the filter paper is dropped with 5 \(\mu\)L DMSO; T stands for the experimental group in which the filter paper is dropped with 5 \(\mu\)L 5% l-limonene.
 +
</p>
 +
<p>According to the data we get, we did simple analysis which means we used the ratio of the number of nematodes distributed on two sides and drew a histogram(Fig.2). In the histogram, nematode distribution of experimental group shows significant difference that nematodes prefer limonene.
 +
</p>
 +
<figure><img src="#">
 +
    <figcaption>Fig2. Proportion of nematodes’ distribution
 +
    </figcaption>
 +
</figure>
 +
<p>During our verification of attraction of limonene, we analyzed 20 samples. In order to verify if there is any difference between test group and the control group at statistic level, we used paired t test to verify.
 +
</p>
 +
<h5>The methods are shown as the following:
 +
</h5>
 +
<p>The confidence of the following is 0.05, α=0.05. Firstly, we used the normality test. We selected the single sample K-S test to verify whether it had normality.
 +
</p>
 +
<ol>
 +
    <li>Judging standard of the single sample K-S test
 +
        <br/>If P > 0.05, it has normality, otherwise it doesn’t have normality.
 +
    </li>
 +
    <li>We used SPSS to derive the data and output the results
 +
    </li>
 +
</ol>
 +
<h5>Table.1 the output of the normality test
 +
</h5>
 +
<table>
 +
    <tbody>
 +
        <tr>
 +
            <th></th>
 +
            <th>Control</th>
 +
            <th>Test</th>
 +
        </tr>
 +
        <tr>
 +
            <td>Sample size</td>
 +
            <td>19</td>
 +
            <td>19</td>
 +
        </tr>
 +
        <tr>
 +
            <td>Mean</td>
 +
            <td>97.0526</td>
 +
            <td>143.3684</td>
 +
 +
        </tr>
 +
        <tr>
 +
            <td>Variance</td>
 +
            <td>74.46362</td>
 +
            <td>102.14108</td>
 +
        </tr>
 +
        <tr>
 +
            <td>K-S statistics</td>
 +
            <td>0.767</td>
 +
            <td>0.920</td>
 +
        </tr>
 +
        <tr>
 +
            <td>P value</td>
 +
            <td>0.598</td>
 +
            <td>0.366</td>
 +
        </tr>
 +
    </tbody>
 +
</table>
 +
<h2>Analysis</h2>
 +
<p>The p value of the control group is 0.598, while that of the test group is 0.366. Both of these two groups meet normality.
 +
</p>
 +
<p>After testing the normality, we did a significance test——paired t test to verify if there was any difference between these two groups.
 +
</p>
 +
<h3>1.  Set up a hypothesis</h3>
 +
<p>$$H_0: \mu1 = \mu2$$</p>
 +
<p>$$H1: \mu1
 +
    < \mu2$$</p>
 +
        <p>Among them, \(\mu_1\) is the mean of the control group, \(\mu_2\) is the mean of the test group.</p>
 +
        <h3>2. The judging standard of the paired t test.
 +
</h3>
 +
        <p>If p
 +
            < 0.05, we reject <em>H<sub>0</sub></em> and accept <em>H<sub>1</sub></em> and the limonene can attract the nematodes. And if p > = 0.05, we accept <em>H<sub>0</sub></em> and the limonene has no effect on the nematodes.
 +
        </p>
 +
<h3>3.  Output the results</h3>
 +
<h5>Table.2 paring sample test
 +
</h5>
 +
</html>

Revision as of 15:51, 17 September 2015

Team:BNU-CHINA - 2015.igem.org

Project Results

l-/d-limonene synthase

Agarose gel electrophoresis of l-/d- limonene synthase gene

Fig.1 the agarose gel electrophoresis of l-/d- limonene synthase gene, the bands pointed by arrows represent l-limonene gene and d-limonene gene. Lane M, DNA marker DL2000; Fig1a, Lane 1 and 2, l-LS in pSB1C3 plasmids; Fig 1b, Lane1 and 2, d-LS in pSB1C3 plasmids; Plasmid are digested by EcoRI and PstI.

In Fig.1 limonene synthase gene is inserted into the backbone pSB1C3. Then the plasmids are digested by Pst I and EcoR I. The length of the l-LS gene, d-LS gene and backbone are 1735bp, 1904bp and 2624bp,respectively. The target genes are marked with arrows, and the figure shows that limonene synthase gene was transferred into the E.coli BL21 cells successfully. 1.2 SDS-PAGE analysis of l-/d- limonene synthase

SDS-PAGE analysis of l-/d- limonene synthase

Fig.2 SDS-PAGE of l-limonenen synthase, d-limonene synthase and Gpps Lane M, molecular weight standards (kDa); Lane2 and 3, bacteria homogenate of d-limonene synthase; Lane 4 and 5, bacteria homogenate of l-limonene synthase; the lower bands pointed by arrows show GPPS and the upper bands pointer by arrows show limonene synthases.

In Fig 2，we want to verify whether the limonene synthase and GPPS gene are expressed or not. The molecular weight of limonene and GPP synthase is 89 kDa (with GST on pGEX-4T-1 plasmid) and 49 kDa respectively. In Fig.2, the target proteins are marked with arrows, and the figure shows that synthases are expressed in the cell successfully.

Verification of attraction of limonene towards C. elegans

In order to know whether limonene attracts C. elegans, filter paper is dropped of 5 \(\mu\)L5% limonene (DMSO aq) and put on one side. The control is put on the other side with only 5 \(\mu\)L DMSO. To eliminate the effect of DMSO, two other control groups are made.

Table 1. The results of verification of limonene to attract C. elegans
1 2 3
Control guoup 1 DMSO 117 113
DMSO 110 159
Control group 2 DMSO 180 86 30
M9 198 91 26

a. The distribution of the C. elegans on the plates of control group Control group is used to eliminate the effect of DMSO, and some other experimental factors.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
DMSO 110 8 129 74 90 19 51 172 144 89 125 49 114 28 63 109 346 54 70
Limonene+DMSO 149 64 37 185 128 100 94 150 202 190 114 124 43 129 57 79

b. The distribution of the C. elegans on the plates of experimental group

This group shows significantly biased movement by compareing l-limonene with DMSO.

We find that nematodes of experimental group show significantly biased movement when we count the number of nematodes (Fig.1). However, nematodes of control group shows no significant difference about the distribution of nematodes on the plate.

Fig.1 The distribution of the C. elegans on the plates.

C stands for the control group in which the filter paper is dropped with 5 \(\mu\)L DMSO; T stands for the experimental group in which the filter paper is dropped with 5 \(\mu\)L 5% l-limonene.

According to the data we get, we did simple analysis which means we used the ratio of the number of nematodes distributed on two sides and drew a histogram(Fig.2). In the histogram, nematode distribution of experimental group shows significant difference that nematodes prefer limonene.

Fig2. Proportion of nematodes’ distribution

During our verification of attraction of limonene, we analyzed 20 samples. In order to verify if there is any difference between test group and the control group at statistic level, we used paired t test to verify.

The methods are shown as the following:

The confidence of the following is 0.05, α=0.05. Firstly, we used the normality test. We selected the single sample K-S test to verify whether it had normality.

  1. Judging standard of the single sample K-S test
    If P > 0.05, it has normality, otherwise it doesn’t have normality.
  2. We used SPSS to derive the data and output the results
Table.1 the output of the normality test
Control Test
Sample size 19 19
Mean 97.0526 143.3684
Variance 74.46362 102.14108
K-S statistics 0.767 0.920
P value 0.598 0.366

Analysis

The p value of the control group is 0.598, while that of the test group is 0.366. Both of these two groups meet normality.

After testing the normality, we did a significance test——paired t test to verify if there was any difference between these two groups.

1. Set up a hypothesis

$$H_0: \mu1 = \mu2$$

$$H1: \mu1 < \mu2$$

Among them, \(\mu_1\) is the mean of the control group, \(\mu_2\) is the mean of the test group.

2. The judging standard of the paired t test.

If p < 0.05, we reject H0 and accept H1 and the limonene can attract the nematodes. And if p > = 0.05, we accept H0 and the limonene has no effect on the nematodes.

3. Output the results

Table.2 paring sample test