Team:SCU-China/Transmitter

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<ul class="nav nav-tabs nav-stacked" data-spy="affix" data-offset-top="125">
<ul class="nav nav-tabs nav-stacked" data-spy="affix" data-offset-top="125">
               <li ><a href="#Top">Back to top</a></li>
               <li ><a href="#Top">Back to top</a></li>
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<li ><a href="#1">Week 1 8.21-8.24</a></li>
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<li ><a href="#1">Week 1</a></li>
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<li ><a href="#2">Week 2 8.25-8.31</a></li>
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<li ><a href="#2">Week 2</a></li>
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<li ><a href="#3">Week 3</a></li>
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<li ><a href="#4">Week 4</a></li>
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<li ><a href="#5">Week 5</a></li>
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</ul> </div>
</ul> </div>
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<p>Finally we have to submit some biobricks that we made in our experiment. This notebook is focusing on constructing independent biobricks.There are about 30 biobricks that we plan to submit. In detail, we separate the into 4 groups:</p><p>
+
<p>Since our team is sorted into 4 groups, our team is focusing on the construction of the Transmitter Cell. The whole work that we should do is to build 2 gene lines which lie in the transmitter cells.</p><p>In detail, we use 9 biobricks supplied by IGEM Competition totally which are RBS (BBa_B0030), DT (BBa_B0015), pLux (BBa_R0062), LuxR (BBa_C0062), LasI (BBa_K081016 and BBa_K081009), CinR (BBa_K0077), RhlR (BBa_C0171), and RhlI (BBa_C0051).</p><h1 id="1" style="padding-top: 80px;
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Group one contains 8 different parts (Am4: Pcon+araC+pBAD; Am5: pRhl+RBS+mRFP+DT; Am6: pRHl/Las+RBS+GFP+DT; Am7: Pcon+RBS+LasR; Am8: RhlR+DT; Am9: RBS+amilCP+DT; Am10:RBS+amilCP+DT+Pcon+RBS; Am11: pRhl/Las+RBS); </p><p>
+
  margin-top: -45px;">Week 1 7.20-7.26</h1><p>1. We transformed the plasmids (BBa_B0030, BBa_B0015, BBa_R0062, BBa_K081016, BBa_K081009, BBa_K0077, BBa_C0171, BBa_C0051) into DH5&#945; E.Coli to amplify these plasmid</p><p>We extracted the plasmids (mentioned above) and used electrophoresis to test the concentration and quality of our plasmids.</p><p>Results:</p><table class="table table-striped"><thead><tr><td><p>Name</li>
-
Group two contains 8 different parts (Bm1: RBS+LasR+RhlR+DT; Bm2: Ptet+RBS+RhlR+DT; Bm3: Pcon+RBS_LasR+RhlR+DT; Bm4: pCin+CinR; Bm5: Pcon+RBS+Lac I; Bm6: pLac+RBS+luxI+DT; Bm7: RBS+CinI+DT; Bm8: pLux+RBS+LuxR+RBS+RhlI)</p>
+
 
-
<div id="1" style="padding-top: 80px;
+
</td><td><p>Concentration (ng/3 &#956;l)</p>
-
   margin-top: -45px;"><h1>Week 1 8.21-8.24</h1>
+
</td>
-
<p>1.We resuscitated the bacteria that contain Am4-Am11 parts.</p><p>
+
</tr></thead><tbody><tr><td><p>BBa_R0062</p>
-
2.We extracted all the plasmids and tested them by electrophoresis.</p><p>
+
</td><td><p>53.3/ 43.5</p>
-
3.All the plasmids were cleaved by EcoR I and Pst I and we tested their qualities by electrophoresis.</p><p>
+
</td>
-
4.We linked all the cleaved parts with pSB1C3 after we did the gel purification.</p><p>
+
</tr><tr><td><p>BBa_K081016</p>
-
5.We transformed all the parts into E.coli. and extracted the plasmids which were tested by double cleavage.</p><p>
+
</td><td><p>107.9/ 176.7</p>
-
6.We sent all the parts to sequence.</p></div>
+
</td>
 +
</tr><tr><td><p>BBa_B0030</p>
 +
</td><td><p>28.2,/8.96</p>
 +
</td>
 +
</tr><tr><td><p>BBa_C0171</p>
 +
</td><td><p>322.6/ 387.5</p>
 +
</td>
 +
</tr><tr><td><p>BBa_K0077</p>
 +
</td><td><p>193.4,/143.9</p>
 +
</td>
 +
</tr><tr><td><p>BBa_K081009</p>
 +
</td><td><p>64.4,/63.1</p>
 +
</td>
 +
</tr><tr><td><p>BBa_C0077</p>
 +
</td><td><p>152.7/ 156</p>
 +
</td>
 +
</tr><tr><td><p>BBa_C0062</p>
 +
</td><td><p>53.29/128.7</p>
 +
</td>
 +
</tr><tr><td><p>BBa_B0015</p>
 +
</td><td><p>116.2/19.5</p>
 +
</td>
 +
</tr></tbody>
 +
</table>
 +
<p>Note: The different concentrations are from different tubes.</p><h1 id="2" style="padding-top: 80px;
 +
  margin-top: -45px;">Week 2 7.27-8.03</h1><p>1. We transformed the BBa_C0062 and BBa_C0077 plasmids.</p><p>2. We extracted the BBa_J37019, BBa_I9026, BBa_K081005, BBa_I714075, BBa_K93600 and BBa_R0077 plasmids and determined the concentration of them by electrophoresis.</p><p>3. We cleaved the BBa_R0062, BBa_C0051, BBa_K0077 plasmids with EcoR I and Spe I enzyme and BBa_B0015 with Xba I and Pst I. Also, we prepared the pSB1A3 and pSB1K3 backbones with EcoR I and Pst I.</p><p>4. We cleaved the BBa_J37019 (Named E11) and BBa_R0077 (E21) with EcoR I and Spe I, BBa_I9026 (E12), BBa_K081016 (E22) and BBa_C0077 (E24) with Xba I and Pst I.</p><p>5. We linked the E11 and E12 with pSB1A3 (L11), the E21 and E22 with pSB1A3 (L21).</p><p>6. We transformed all the linked parts into E.coli.</p><p>7. We cleaved BBa_B0015 with Xba I and Pst I.</p><p>Results</p><table class="table table-striped"><caption>The concentration of plasmids</caption><thead><tr><td><p>Name</li>
 +
 
 +
</td><td><p>Concentration (ng/3&#956;l)</p>
 +
</td>
 +
</tr></thead><tbody><tr><td><p>BBa_J37019</p>
 +
</td><td><p>400/403</p>
 +
</td>
 +
</tr><tr><td><p>BBa_I9026</p>
 +
</td><td><p>247/140.6</p>
 +
</td>
 +
</tr><tr><td><p>BBa_R0077</p>
 +
</td><td><p>134/161.2</p>
 +
</td>
 +
</tr><tr><td><p>BBa_K081005</p>
 +
</td><td><p>51.2/27.6</p>
 +
</td>
 +
</tr><tr><td><p>BBa_I714075</p>
 +
</td><td><p>95.7/37.2</p>
 +
</td>
 +
</tr><tr><td><p>BBa_K93600</p>
 +
</td><td><p>41.6</p>
 +
</td>
 +
</tr><tr><td><p>L1</p>
 +
</td><td><p>92.3/54.2</p>
 +
</td>
 +
</tr><tr><td><p>L2</p>
 +
</td><td><p>88.4/60.4</p>
 +
</td>
 +
</tr></tbody>
 +
</table>
 +
<li>Note: The different concentrations are from different tubes.</p><p>2. The electrophoresis results are shown in Figure 1.</p>
 +
<img src="https://static.igem.org/mediawiki/2014/2/24/T1.png">
 +
<p>Figure 1. The cleaved plasmids</p><h1 id="3" style="padding-top: 80px;
 +
   margin-top: -45px;">Week 3 8.04-8.10</h1><p>1. We linked the L1 and BBa_B0015 with pSB1T3 (named F1), L2 and BBa_I9026 with pSB1T3 (F2). Then we transformed them into E.coli to amplify them and we extracted the plasmids to test their quality.</p><p>2. We linked E11 and E12 with pSB1T3 (named LA1), E21 and E22 with pSB1A3 (LA2).</p><p>3. We cleaved the BBa_J37019 (Named E11) and BBa_R0077 (E21) with EcoR I and Spe I, BBa_I9026 (E12), BBa_K081016 (E22) and BBa_C0077 (E24) with Xba I and Pst I again and tested their qualities by electrophoresis and then we did the gel purification.</p><p>4. We cleaved the BBa_R0077 with Spe I and Pst I (named EP21).</p><p>5. We linked EP21 and E22 with 3 different backbones (pSB1T3, pSB1A3, and pSB1C3) which are tested by electrophoresis.</p><p>Results:</p><table class="table table-striped"><caption>The concentration of plasmids and linkage product</caption><thead><tr><td><p>Name</li>
 +
 
 +
</td><td><p>Concentration (ng/3&#956;l)</p>
 +
</td>
 +
</tr></thead><tbody><tr><td><p>F11&#9;</p>
 +
</td><td><p>Failed</p>
 +
</td>
 +
</tr><tr><td><p>F21</p>
 +
</td><td><p>failed</p>
 +
</td>
 +
</tr><tr><td><p>E11</p>
 +
</td><td><p>8.53</p>
 +
</td>
 +
</tr><tr><td><p>E12</p>
 +
</td><td><p>Failed</p>
 +
</td>
 +
</tr><tr><td><p>E21</p>
 +
</td><td><p>24.9/7.5</p>
 +
</td>
 +
</tr><tr><td><p>E22</p>
 +
</td><td><p>14/5.3</p>
 +
</td>
 +
</tr><tr><td><p>E24</p>
 +
</td><td><p>18.1/17.7</p>
 +
</td>
 +
</tr></tbody>
 +
</table>
 +
<li>Note: The different concentrations are from different tubes.</p><p>2. The electrophoresis results are shown in Figure 2.</p><img src="https://static.igem.org/mediawiki/2014/4/49/T2.png"><p>Figure 2. A, C, and D the gel purification results and cleaved plasmids. B. the cleaved EP21.</p><h1 id="4" style="padding-top: 80px;
 +
  margin-top: -45px;">Week 4 8.11-8.17</h1><p>1. We did the liquid culture of BBa_R0077 and BBa_C0077.</p><p>2. We extracted the BBa_J37019, BBa_I9026 plasmids and then we cleaved both of them (BBa_J37019 with Spe I and Pst I; BBa_I9026 with Xba I and Pst I) and linked them with pSB1C3. Finally we tested the quality by transforming into E.coli.</p><p>3. We linked BBa_J37019 and BBa_I9026(LI1), BBa_R0077 and BBa_C0077 with pSB1C3.</p><p>4. We extracted BBa_I9026, BBa_J37019, BBa_C0077, and BBa_R0077.</p><p>5. We cleaved BBa_J37019 with E, S (D1, D2) or S, P (D3, D4) and BBa_I9026 plasmids with E, S, (D5, D6) and S, P (D7, D8) enzymes. Then we did the gel purification.</p><p>6. We linked the BBa_J37019 with BBa_I9026 with different types (named LI3).</p><p>7. We transformed LI3 into E.coli and extracted the plasmids to test the quality.</p><p>Results</p><table class="table table-striped"><caption>The concentration of plasmids</caption><thead><tr><td><p>Name</li>
 +
 
 +
</td><td><p>Concentration (ng/3&#956;l)</p>
 +
</td>
 +
</tr></thead><tbody><tr><td><p>BBa_I9026</p>
 +
</td><td><p>289.2/112.6</p>
 +
</td>
 +
</tr><tr><td><p>BBa_J37019</p>
 +
</td><td><p>193.2/40.7</p>
 +
</td>
 +
</tr><tr><td><p>BBa_C0077</p>
 +
</td><td><p>27.7/22.5/33.3</p>
 +
</td>
 +
</tr><tr><td><p>BBa_R0077</p>
 +
</td><td><p>177.8/179.8/200.3</p>
 +
</td>
 +
</tr></tbody>
 +
</table>
 +
<p>Note: The different concentrations are from different tubes.</p><p>2. The electrophoresis results are shown in Figure 3.</p><img src="https://static.igem.org/mediawiki/2014/thumb/a/a7/T3.png/471px-T3.png"><p>Figure A. the cleaved BBa_J37019 and BBa_I9026 plasmids. B. the cleaved linkage products. C and D is the gel purification of BBa_J37019 and BBa_I9026. E. the cleaved BBa_R0077 and BBa_C0077.</p><p>F. the linkage products.</p><h1 id="5" style="padding-top: 80px;
 +
  margin-top: -45px;">Week 5 8.18-8.24</h1><p>1. We linked the E11 and E12 with pSB1T3 by 3A assembly and E11 and E12 by traditional assembly.</p><p>2. We Amplified the LII3 which was tested by cleavage and electrophoresis.</p><p>3. We transformed the BBa_J37019, BBa_I9026, BBa_C0077, and L1T1.</p><p>4. We cultured the LIIF in liquid and transformed it.</p><p>5. We did the gel purification of BBa_J37019 and BBa_K747092.</p><p>Results</p><p>The electrophoresis results are shown in Figure 4.</p><img src="https://static.igem.org/mediawiki/2014/e/e7/T4.png"><p>Figure 4. A the gel purification results; B. the LII3 results.</p>
 +
 
-
<div id="2" style="padding-top: 80px;
 
-
  margin-top: -45px;"><h1>
 
-
Week 2 8.25-8.31</h1>
 
-
<p>1.We cleaved the Bm2, Bm6, and Bm7 plasmids to identify them.</p><p>
 
-
2.We did the liquid culture for the bacteria containing Am4-Am11 plasmids.</p><p>
 
-
3.We extracted the Am4-Am11 plasmids for storage.</p><p>
 
-
4.We did the gel purification and linked the Bm2, Bm6, and Bm7 parts with pSB1C3.</p><p>
 
-
5.We transformed our linkage products and extracted them.</p><p>
 
-
6.We identified them by double cleavage and sent them to sequence.</p><p>
 
-
</div>
 
</div></div>
</div></div>
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Latest revision as of 03:59, 18 October 2014

The Notebook of

Transmitter

Since our team is sorted into 4 groups, our team is focusing on the construction of the Transmitter Cell. The whole work that we should do is to build 2 gene lines which lie in the transmitter cells.

In detail, we use 9 biobricks supplied by IGEM Competition totally which are RBS (BBa_B0030), DT (BBa_B0015), pLux (BBa_R0062), LuxR (BBa_C0062), LasI (BBa_K081016 and BBa_K081009), CinR (BBa_K0077), RhlR (BBa_C0171), and RhlI (BBa_C0051).

Week 1 7.20-7.26

1. We transformed the plasmids (BBa_B0030, BBa_B0015, BBa_R0062, BBa_K081016, BBa_K081009, BBa_K0077, BBa_C0171, BBa_C0051) into DH5α E.Coli to amplify these plasmid

We extracted the plasmids (mentioned above) and used electrophoresis to test the concentration and quality of our plasmids.

Results:

Name

Concentration (ng/3 μl)

BBa_R0062

53.3/ 43.5

BBa_K081016

107.9/ 176.7

BBa_B0030

28.2,/8.96

BBa_C0171

322.6/ 387.5

BBa_K0077

193.4,/143.9

BBa_K081009

64.4,/63.1

BBa_C0077

152.7/ 156

BBa_C0062

53.29/128.7

BBa_B0015

116.2/19.5

Note: The different concentrations are from different tubes.

Week 2 7.27-8.03

1. We transformed the BBa_C0062 and BBa_C0077 plasmids.

2. We extracted the BBa_J37019, BBa_I9026, BBa_K081005, BBa_I714075, BBa_K93600 and BBa_R0077 plasmids and determined the concentration of them by electrophoresis.

3. We cleaved the BBa_R0062, BBa_C0051, BBa_K0077 plasmids with EcoR I and Spe I enzyme and BBa_B0015 with Xba I and Pst I. Also, we prepared the pSB1A3 and pSB1K3 backbones with EcoR I and Pst I.

4. We cleaved the BBa_J37019 (Named E11) and BBa_R0077 (E21) with EcoR I and Spe I, BBa_I9026 (E12), BBa_K081016 (E22) and BBa_C0077 (E24) with Xba I and Pst I.

5. We linked the E11 and E12 with pSB1A3 (L11), the E21 and E22 with pSB1A3 (L21).

6. We transformed all the linked parts into E.coli.

7. We cleaved BBa_B0015 with Xba I and Pst I.

Results

The concentration of plasmids

Name

Concentration (ng/3μl)

BBa_J37019

400/403

BBa_I9026

247/140.6

BBa_R0077

134/161.2

BBa_K081005

51.2/27.6

BBa_I714075

95.7/37.2

BBa_K93600

41.6

L1

92.3/54.2

L2

88.4/60.4
  • Note: The different concentrations are from different tubes.

    2. The electrophoresis results are shown in Figure 1.

    Figure 1. The cleaved plasmids

    Week 3 8.04-8.10

    1. We linked the L1 and BBa_B0015 with pSB1T3 (named F1), L2 and BBa_I9026 with pSB1T3 (F2). Then we transformed them into E.coli to amplify them and we extracted the plasmids to test their quality.

    2. We linked E11 and E12 with pSB1T3 (named LA1), E21 and E22 with pSB1A3 (LA2).

    3. We cleaved the BBa_J37019 (Named E11) and BBa_R0077 (E21) with EcoR I and Spe I, BBa_I9026 (E12), BBa_K081016 (E22) and BBa_C0077 (E24) with Xba I and Pst I again and tested their qualities by electrophoresis and then we did the gel purification.

    4. We cleaved the BBa_R0077 with Spe I and Pst I (named EP21).

    5. We linked EP21 and E22 with 3 different backbones (pSB1T3, pSB1A3, and pSB1C3) which are tested by electrophoresis.

    Results:

    The concentration of plasmids and linkage product

    Name

    Concentration (ng/3μl)

    F11

    Failed

    F21

    failed

    E11

    8.53

    E12

    Failed

    E21

    24.9/7.5

    E22

    14/5.3

    E24

    18.1/17.7
  • Note: The different concentrations are from different tubes.

    2. The electrophoresis results are shown in Figure 2.

    Figure 2. A, C, and D the gel purification results and cleaved plasmids. B. the cleaved EP21.

    Week 4 8.11-8.17

    1. We did the liquid culture of BBa_R0077 and BBa_C0077.

    2. We extracted the BBa_J37019, BBa_I9026 plasmids and then we cleaved both of them (BBa_J37019 with Spe I and Pst I; BBa_I9026 with Xba I and Pst I) and linked them with pSB1C3. Finally we tested the quality by transforming into E.coli.

    3. We linked BBa_J37019 and BBa_I9026(LI1), BBa_R0077 and BBa_C0077 with pSB1C3.

    4. We extracted BBa_I9026, BBa_J37019, BBa_C0077, and BBa_R0077.

    5. We cleaved BBa_J37019 with E, S (D1, D2) or S, P (D3, D4) and BBa_I9026 plasmids with E, S, (D5, D6) and S, P (D7, D8) enzymes. Then we did the gel purification.

    6. We linked the BBa_J37019 with BBa_I9026 with different types (named LI3).

    7. We transformed LI3 into E.coli and extracted the plasmids to test the quality.

    Results

    The concentration of plasmids

    Name

    Concentration (ng/3μl)

    BBa_I9026

    289.2/112.6

    BBa_J37019

    193.2/40.7

    BBa_C0077

    27.7/22.5/33.3

    BBa_R0077

    177.8/179.8/200.3

    Note: The different concentrations are from different tubes.

    2. The electrophoresis results are shown in Figure 3.

    Figure A. the cleaved BBa_J37019 and BBa_I9026 plasmids. B. the cleaved linkage products. C and D is the gel purification of BBa_J37019 and BBa_I9026. E. the cleaved BBa_R0077 and BBa_C0077.

    F. the linkage products.

    Week 5 8.18-8.24

    1. We linked the E11 and E12 with pSB1T3 by 3A assembly and E11 and E12 by traditional assembly.

    2. We Amplified the LII3 which was tested by cleavage and electrophoresis.

    3. We transformed the BBa_J37019, BBa_I9026, BBa_C0077, and L1T1.

    4. We cultured the LIIF in liquid and transformed it.

    5. We did the gel purification of BBa_J37019 and BBa_K747092.

    Results

    The electrophoresis results are shown in Figure 4.

    Figure 4. A the gel purification results; B. the LII3 results.

    • Sichuan university

    Retrieved from "http://2014.igem.org/Team:SCU-China/Transmitter"