Team:UANL Mty-Mexico/Notebook

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<html>
<html>
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<h2>Notebook</h2>
+
<p><h2>Notebook</h2></p>
<p><b>Reference Data</b></p>
<p><b>Reference Data</b></p>
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<th><b>Part Name</b></th>
<th><b>Part Name</b></th>
<th><b>Part Short name</b></th>
<th><b>Part Short name</b></th>
-
<th><b>In pSB1A3 (bp)</b></th>
+
<th><b>In pSB 1A3 (bp)</b></th>
-
<th><b>In pSB1C3 (bp)</b></th>
+
<th><b>In pSB 1C3 (bp)</b></th>
-
<th><b>In PUC57 (bp)</b></th>
+
<th><b>In pUC57 (bp)</b></th>
<th><b>Part size (bp)</b></th>
<th><b>Part size (bp)</b></th>
</tr>
</tr>
<!-- Favorite-Name-Type-Description-Designer-Length-->
<!-- Favorite-Name-Type-Description-Designer-Length-->
<tr>
<tr>
-
<td> pLambdaCI + TetR </td>
+
<td> RBS + TetR </td>
<td>TetR</td>
<td>TetR</td>
-
<td>2925</td>
+
<td>2,925</td>
-
<td>2802 </td>
+
<td>2,802 </td>
-
<td>3478</td>
+
<td>3,478</td>
-
<td>732</td>
+
<td>   732</td>
</tr>
</tr>
<tr>
<tr>
<td>pLac + GFP</td>
<td>pLac + GFP</td>
<td> GFP </td>
<td> GFP </td>
-
<td>3162</td>
+
<td>3,162</td>
-
<td>3034</td>
+
<td>3,034</td>
-
<td>3715</td>
+
<td>3,715</td>
-
<td>964</td>
+
<td>   964</td>
</tr>
</tr>
<tr>
<tr>
<td>pCons LacI</td>
<td>pCons LacI</td>
<td> LacI </td>
<td> LacI </td>
-
<td>3483</td>
+
<td>3,483</td>
-
<td>3352</td>
+
<td>3,352</td>
-
<td>4036</td>
+
<td>4,036</td>
<td>1,282</td>
<td>1,282</td>
</tr>  
</tr>  
<tr>
<tr>
-
<td>pTetR+mCherry</td>
+
<td>pTetR + mCherry</td>
<td>mCherry</td>
<td>mCherry</td>
-
<td>3107</td>
+
<td>3,107</td>
-
<td>2957</td>
+
<td>2,957</td>
-
<td>3660</td>
+
<td>3,660</td>
-
<td>887</td>
+
<td>   887</td>
</tr>
</tr>
<tr>
<tr>
<td>BBa_K1140005</td>
<td>BBa_K1140005</td>
<td> 3-E1 </td>
<td> 3-E1 </td>
-
<td>2811</td>
+
<td>2,811</td>
<td>---</td>
<td>---</td>
<td>---</td>
<td>---</td>
-
<td>935</td>
+
<td> 935</td>
</tr>
</tr>
</table>
</table>
-
<p>This table works as a reference of the parts that were used in the laboratory and in the diary. We have a short name for each part so for example, if a diary entry says "TetR" we mean the complete part "pLambdaCI + TetR". Another important aspect is that we were working with the plasmids pSB1C3, pSB1A3 and PUC57 so here there is the information about the length of the part and the length in each plasmid. </p>
+
<p align="justify">This table works as a reference of the parts that were used in the laboratory and in the diary. We have a short name for each part so for example, if a diary entry says "TetR" we mean the complete part "RBS + TetR". Another important aspect is that we were working with the plasmids pSB 1C3, pSB 1A3 and pUC57 so here there is the information about the length of the part and the length in each plasmid. </p>
<br>
<br>
 +
<p><b>August 4th, 2013</b></p>
 +
<p align=”justify”>Today we prepared solutions in order to start working on the lab the following day. The solutions will be required for the MiniPrep, Transformation. We also prepared aliquots, mQ water with RNAse, etc.  </p>
 +
<br>
 +
<p><b>August 5th, 2013</b></p>
 +
<p align=”justify”>Today we started with the transformations of the synthetic DNAs that just arrived.</p>
 +
<br>
 +
<p><b>August 6th, 2013</b></p>
 +
<p align=”justify”>We inoculated the colonies obtained from the transformations.</p>
 +
<br>
 +
<p><b>August 7th, 2013</b></p>
<p><b>August 7th, 2013</b></p>
-
<p>This day we did Minipreparation of DNA. The parts that were obtained were the following: pCos R Lac1, pLac R GFP, pTetR R mCherry, pTetR, 3-1E</p>
+
<p align="justify">This day we did Minipreparation of DNA. The parts that were obtained were the following: LacI, GFP, mCherry, TetR and 3-1E</p>
<br>
<br>
<p><b>August 8th, 2013</b></p>
<p><b>August 8th, 2013</b></p>
-
<p><u>Test of mCherry with Thermo-mixer</u>-<u>Cualitative experiment</u></p>
+
<p>Test of mCherry with Thermo-mixer-Cualitative experiment</p>
-
<p>20 Colonies of the dish with pTet-R-mCherry were planted in 0.5 mL of Agar with Kanamycin in order to observe the red color after several hours at 42ᵒ C</p>
+
<p align="justify">20 Colonies of the dish with pTet-R-mCherry were planted in 0.5 mL of Agar with Kanamycin in order to observe the red colour after several hours at 42ᵒC. The experiment was performed following these parameters. </p>
<table class="table table-striped">
<table class="table table-striped">
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</tr>
</tr>
</table>
</table>
-
<p>Most tubes shown development seeing them on the light.</p>
+
<p>Most tubes shown development seeing them on the light but there was no color shown.</p>
<br>
<br>
<p><b>August 10th, 2013</b></p>
<p><b>August 10th, 2013</b></p>
-
<p>20 tubes with mCherry were put in the shaker with 2 controls with CDS (with oxygen everyone). </p>
+
20 tubes with mCherry were put in the shaker with 2 controls with CDS (with oxygen everyone), following these parameters:
-
<p>Temperature: 37ᵒ</p>
+
-
<p>Revolutions: 900 rpm</p>
+
-
<p>Start: 11:35 am ->  9:00 am (Time 21: 35 hours)</p>
+
-
<p>The tubes show mCherry expression, some more intense than others.The experiment was repeated at 30°C OVERNIGHT with a  
+
<li><ul>Temperature: 37ᵒC </ul></li>
-
Volume of 500ul at 30°C with tubes of 1.5ml. They were left at 4°C meanwhile they were incubated.
+
<li><ul>Revolutions: 900 rpm</ul></li>
-
</p>
+
<li><ul>Start: 11:35 am ->  9:00 am (Time 21:35 hours)</ul></li>
 +
 
 +
<p align="justify">The tubes show mCherry expression, some more intense than others. The experiment was repeated at 30°C overnight with a volume of 500 µL at 30°C with tubes of 1.5 ml. They were left at 4°C meanwhile they were incubated.</p>
<table class="table table-striped">
<table class="table table-striped">
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<td> Black Set</td>
<td> Black Set</td>
<td>42ºC</td>
<td>42ºC</td>
-
<td>900rpm</td>
+
<td>900 rpm</td>
<td>15 hours</td>
<td>15 hours</td>
<td> :( </td>
<td> :( </td>
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<td>Blue Set</td>
<td>Blue Set</td>
<td> 37ºC </td>
<td> 37ºC </td>
-
<td>900rpm</td>
+
<td>900 rpm</td>
<td>21 hours</td>
<td>21 hours</td>
<td> :) </td>
<td> :) </td>
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<td>Red Set</td>
<td>Red Set</td>
<td> 32ºC</td>
<td> 32ºC</td>
-
<td>900rpm</td>
+
<td>900 rpm</td>
<td></td>
<td></td>
<td>:)</td>
<td>:)</td>
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<br>
<br>
<p><b>August 12th, 2013</b></p>
<p><b>August 12th, 2013</b></p>
-
<p>The experiment was repeated at 42°C with oxygen. It began at 2:15 pm and ended at 1:20 pm</p>
+
<p align="justify">The experiment was repeated at 42°C allowing the oxygen in to the tube. It began at 2:15 pm and ended at 1:20 pm of the next day.</p>
-
<center><img src="https://static.igem.org/mediawiki/2013/a/a9/42oC_clonas_1-10_Control_37oC.jpg" width="500px" height="400px" class="img-rounded"></center>
+
<center>
-
<center><font size=2>Image 1. Tubes with the mCherry pellet that were incubated at 42ºC</font></center>
+
<figure>
-
 
+
<img src="https://static.igem.org/mediawiki/2013/9/99/37Cunal2013.jpg" width=400px style="border:1px solid black" >
 +
<figcaption><span class="text-muted"><br>Figure 1. Tubes with the mCherry pellet that were incubated at 42ºC.</span> <br>
 +
</figcaption>
 +
</figure>
 +
</center>
<br>
<br>
<p><b>August 16th, 2013</b></p>
<p><b>August 16th, 2013</b></p>
<p>Electrophoresis gel of the digestions from 15/08/13</p>
<p>Electrophoresis gel of the digestions from 15/08/13</p>
-
 
+
<center>
 +
<figure>
 +
<img src="https://static.igem.org/mediawiki/2013/2/2b/Fig2_657.png" width=300px style="border:1px solid black" >
 +
<figcaption><span class="text-muted"><br>Figure 2. Electrophoresis gel of the digestions from 15/08/13</span> <br>
 +
</figcaption>
 +
</figure>
 +
</center>
 +
<br>
<table class="table table-striped">
<table class="table table-striped">
<tr>
<tr>
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<!-- Favorite-Name-Type-Description-Designer-Length-->
<!-- Favorite-Name-Type-Description-Designer-Length-->
<tr>
<tr>
-
<td> DNA</td>
+
<td>DNA</td>
-
<td>2uL</td>
+
<td>2 µL</td>
<td></td>
<td></td>
</tr>
</tr>
<tr>
<tr>
-
<td>EcoRI</td>
+
<td><i>Eco</i>RI</td>
-
<td>0.3uL </td>
+
<td>0.3 µL </td>
-
<td>1.5uL</td>
+
<td>1.5 µL</td>
</tr>
</tr>
<tr>
<tr>
-
<td>PstI</td>
+
<td><i>Pst</i>I</td>
-
<td>0.3uL </td>
+
<td>0.3 µL </td>
-
<td>1.5uL</td>
+
<td>1.5 µL</td>
</tr>  
</tr>  
<tr>
<tr>
-
<td>Buffer O</td>
+
<td>Buffer O (Fermentas)</td>
-
<td>1uL</td>
+
<td>1 µL</td>
-
<td>5uL</td>
+
<td>5 µL</td>
</tr>
</tr>
<tr>
<tr>
<td></td>
<td></td>
-
<td>10uL</td>
+
<td>10 µL</td>
-
<td>50uL</td>
+
<td>50 µL</td>
</tr>
</tr>
</table>
</table>
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<br>
<br>
<p><b>August 18th, 2013</b></p>
<p><b>August 18th, 2013</b></p>
-
<p>Ligation with a 1:1 ratio DNA:Vector</p>
+
<p>There where no colonies present from the previous day.</p>
 +
<p>Ligation with a 1:1 ratio DNA:Vector. Is was performed with the following parameters:</p>
<table class="table table-striped">
<table class="table table-striped">
<tr>
<tr>
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</tr>
</tr>
<tr>
<tr>
-
<td>pConsLac1</td>
+
<td>pCons-LacI</td>
<td> 1326</td>
<td> 1326</td>
<td>1.6</td>
<td>1.6</td>
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</tr>
</tr>
<tr>
<tr>
-
<td>pTetRmCherry</td>
+
<td>pTetR-mCherry</td>
<td> 960</td>
<td> 960</td>
<td>2.2</td>
<td>2.2</td>
<td>0.45/1</td>
<td>0.45/1</td>
</tr><tr>
</tr><tr>
-
<td>pLac1GFP</td>
+
<td>pLacI-GFP</td>
<td> 1005</td>
<td> 1005</td>
<td>2.1</td>
<td>2.1</td>
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</tr>
</tr>
</table>
</table>
-
<p>This ligation was not sucessful in the transformation.</p>
 
-
</html>
 
<br>
<br>
<p><b>August 19th, 2013</b></p>
<p><b>August 19th, 2013</b></p>
-
<p>Dishes of:PTetmCherry, PConsLac, TetR, PLacGFP, 3-1E were planted again.</p>
+
<p>This ligation was not successful in the transformation.</p>
 +
<p>Dishes of:pTet-mCherry, pCons-LacI, pTetR, pLacI-GFP, and the 3-1E DNA were planted again.</p>
<br>
<br>
<p><b>August 26th, 2013</b></p>
<p><b>August 26th, 2013</b></p>
-
<p>All the dishes were succesfully transformed but only the PLacGFP grew in the test tubes with medium and was succesful in the MiniPrep.</p>
+
<p align="justify">All the dishes were transformed but only the pLacGFP grew successfully.</p>
 +
<br>
 +
<p><b>August 28th, 2013</b></p>
 +
<p align="justify">Today we picked up colonies from the dishes from the previous day. We also prepared new competent cells.</p>
<br>
<br>
<p><b>August 29th, 2013</b></p>
<p><b>August 29th, 2013</b></p>
-
<p>All the dishes were succesfully transformed, we got colonies of all the parts, they grew in the test tubes and the miniPrep was sucessfully done for all. </p>
+
<p align="justify">All the dishes were successfully transformed and we planted in petri dishes. We got colonies of all the parts, they grew in the test tubes and miniPrep was done. </p>
<br>
<br>
<p><b>August 30th, 2013</b></p>
<p><b>August 30th, 2013</b></p>
-
<p>Digestions with EcorI</p>
+
<p align="justify">The MiniPrep was successful and we did digestions with <i>Eco</i>RI. Transformations (one tube each one): TetR, pConLac1, TetRmCherry, 3-1E, pLacGFP. *They were planted in Ampicilin dish.</p>
-
<p>Transformations (one tube each one): TetR, pConLac1, TetRmCherry, 3-1E, pLacGFP</p>
+
<center>
-
<p>*They were planted with Ampicilin.</p>
+
<figure>
 +
<img src="https://static.igem.org/mediawiki/2013/6/6e/MinPreps30Aug.png" width=300px style="border:1px solid black" >
 +
<figcaption><span class="text-muted"><br>Figure 3. Electrophoresis gel from the August 30 miniPreps</span> <br>
 +
</figcaption>
 +
</figure>
 +
</center>
<br>
<br>
<p><b>August 31th, 2013</b></p>
<p><b>August 31th, 2013</b></p>
<p>MiniPrep and digestions from the ones of 08/30/2013. We didn't obtained any construction.</p>
<p>MiniPrep and digestions from the ones of 08/30/2013. We didn't obtained any construction.</p>
<p>Transformations  (one tube each one): TetR, pConLac1, TetRmCherry, 3-1E, pLacGFP</p>
<p>Transformations  (one tube each one): TetR, pConLac1, TetRmCherry, 3-1E, pLacGFP</p>
-
<p>*They were planted with Ampicilin.</p>
+
<p>*They were planted in Ampicilin dish.</p>
<br>
<br>
-
<p><b>September 2nd, 2013</b></p>
+
 
-
<p>Today we inoculated colonies for TetR, pConLac1, TetRmCherry, 3-1E, pLacGFP. We alsos prepared a digestion with EcoRI and PstI.</p>
+
<p><b>September 11th, 2013</b></p>
 +
<p align="justify">Digestions with <i>Eco</i>RI and <i>Pst</i>I and Ligations</p>
<br>
<br>
-
<p><b>September 3rd, 2013</b></p>
+
<p><b>September 12th, 2013</b></p>
-
<p>Digestion of the colonies from 09/02/2013. The ones from the gel (see image 2) were saved. The others are in a green box. We also planted mCherry and pBB1A3 vector.</p>
+
<p align="justify">We didn't obtained any part so we started again transforming them one more time.</p>
<br>
<br>
 +
<p><b>September 13th, 2013</b></p>
 +
<p align="justify">There were no results presented so we transformed again.</p>
 +
<br>
 +
<p><b>September 14th, 2013</b></p>
 +
<p align="justify">We got no results in the construction desired.</p>
 +
<br>
 +
<p><b>September 19th, 2013</b></p>
 +
<p align="justify">Quantification with the Nanodrop of the previous samples</p>
 +
<br>
 +
<table class="table table-striped">
 +
<tr>
 +
<th><b></b></th>
 +
<th><b>ng/µL</b></th>
 +
<th><b>260/280</b></th>
 +
</tr>
 +
<!-- Favorite-Name-Type-Description-Designer-Length-->
 +
<tr>
 +
<td>pSB1C3</td>
 +
<td>1293.4</td>
 +
<td>1.99</td>
 +
</tr>
 +
<tr>
 +
<td>GFP</td>
 +
<td>133.5</td>
 +
<td>1.84</td>
 +
</tr>
 +
<tr>
 +
<td>TetR</td>
 +
<td>119.6</td>
 +
<td>1.78</td>
 +
</tr>
 +
<tr>
 +
<td>tRFP</td>
 +
<td>104.3</td>
 +
<td>1.74</td>
 +
</tr>
 +
<tr>
 +
<td>pConsLac</td>
 +
<td>130.5</td>
 +
<td>1.59</td>
 +
</tr>
 +
</table>
 +
 +
<p><b>September 20th, 2013</b></p>
 +
<p align="justify">We prepared new calcium competent cells, <i>E</i>. <i>coli</i> strain Top10. </p>
 +
<br>
 +
<p><b>September 23th, 2013</b></p>
 +
<p align="justify">We did the transformations again for all the parts.</p>
 +
<br>
 +
<p><b>September 24th, 2013</b></p>
 +
<p align="justify">Again there where no results.</p>
 +
<br>
 +
 +
<p align="justify"><b>Note:</b> The constructions with the pSB1A3 and pSB1C3 showed no results but the controls with pUC vector were fine and did work in our experiments.</p>
 +
<br>
 +
 +
<p><b>October 9th, 2013</b></p>
 +
<p align="justify">New Digestions with <i>Eco</i>RI and <i>Pst</i>I.</p>
 +
<br>
 +
 +
<p><b>October 10th, 2013</b></p>
 +
<p align="justify">New ligations were done</p>
 +
<br>
 +
<p><b>October 11th, 2013</b></p>
 +
<p align="justify">New Petri dishes were made, and use for transformation today.</p>
 +
<br>
 +
<p><b>October 13th, 2013</b></p>
 +
<p align="justify">We inoculated the colonies obtained from the transformations.</p>
 +
<br>
 +
<p><b>October 14th, 2013</b></p>
 +
<p align="justify">We did minipreparation of DNA. After, the DNA was digested with <i>Eco</i>RI, <i>Pst</i>I and <i>Hin</i>fI for an enzymatic analysis (our Gel #700!!!). After hours, we did a electrophoresis and we realised that 2 new parts were now in the pSB 1C3 vector.</p>
 +
<br>
 +
<figure>
 +
<center>
 +
<img src="https://static.igem.org/mediawiki/2013/7/76/700Zorrab.png" width=300px style="border:1px solid black" >
 +
<figcaption><span class="text-muted"><br>Figure 4. Electrophoresis gel from the August 30 miniPreps cut with <i>Hin</i>fI, Lambda-<i>Pst</i>I as marker.</span> <br>
 +
</figure>
 +
</center>
 +
</p>
 +
<br>
 +
<p><b>October 17th, 2013</b></p>
 +
<p align="justify">We started a new test in order to measure number of plasmids per cell in M1, M2 and M12.</p>
 +
<br>
 +
<p><b>October 18th, 2013</b></p>
 +
<p align="justify">First results for number of plasmids.</p>
 +
<br>
 +
<p><b>October 21th, 2013</b></p>
 +
<p align="justify">More results for number of plasmids.</p>
 +
<br>
 +
<p><b>October 22th, 2013</b></p>
 +
<p align="justify">A test for measuring expression of GFP (in pUC57 and pSB1C3) was prepared in microcentrifuge tubes with LB medium and IPTG 1mM for 17 hours at 37 °C (in bacteria <i>E. coli</i> Top10) in thermomixer.</p>
 +
<p>We still have problems with expression of mCherry, so we did new cultures with stored (red) M1, M2 and M12 in order.</p>
 +
<br>
 +
<p><b>October 23th, 2013</b></p>
 +
<p align="justify">We used fluorometer to measure expression of GFP, but any fluorescence was detected, so new microcentrifuge tubes with GFP construction were prepared at same conditions of last day.</p>
 +
<p>The cultures of M1, M2, M11 and M12 were red and presented fluorescence, so we did minipreparation and culture again them in new tubes. With the DNA that was obtained, a digestion with <i>Eco</i>RI and <i>Pst</i>I was done and a new ligation.
 +
</p>
 +
<br>
 +
<p><b>October 24th, 2013</b></p>
 +
<p align="justify">Again, any fluorescence was detected for GFP cultures. New microcentrifuge tubes with GFP construction were prepared with IPTG 0.5 mM.</p>
 +
<p>A new transformation was done, using new mCherry and latest TetR and LacI ligations.</p>
 +
<p>Cultures of M11 and M12 grew, but any with M1 and M2. We did minipreparation and the DNA was measured in nanodrop. 
 +
</p>
 +
<br>
 +
<p><b>October 25th, 2013</b></p>
 +
<p align="justify">Residual fluorescence was detected for GFP cultures.</p>
 +
<p>We inoculated the colonies obtained from the transformations.
 +
</p>
 +
<br>
 +
<p><b>October 26th, 2013</b></p>
 +
<p align="justify">Cultures of mCherry grew, but the others did not. We did minipreparation of that tubes and digested them with <i>Eco</i>RI, <i>Pst</i>I and <i>Hin</i>fI. The results can be seen on gel # 709 (<i>Eco</i>RI/<i>Pst</i>I) and 710 (<i>Hin</i>fI). Probably, we have multiple clones with mCherry in pSB1C3.</p>
 +
<p>New ligations of TetR, LacI and GFP with pSB1C3 were done and a transformation was made in a new competent stock, Top10 strain.
 +
</p>
 +
<br>
 +
<figure>
 +
<center>
 +
<img src="https://static.igem.org/mediawiki/2013/2/2d/Gel709a.jpg" width=250px style="border:1px solid black" >
 +
<figcaption><span class="text-muted"><br>Figure 5. Electrophoresis gel (709) with the <i>Eco</i>RI/<i>Pst</i>I clones digestion, Lambda-<i>Pst</i>I as marker.</span> <br>
 +
</figure>
 +
</center>
 +
</p>
 +
<br>
 +
<br>
 +
<figure>
 +
<center>
 +
<img src="https://static.igem.org/mediawiki/2013/5/59/Gel710a.jpg" width=500px style="border:1px solid black" >
 +
<figcaption><span class="text-muted"><br>Figure 6. Electrophoresis gel (710) with the <i>Hin</i>fI clones digestion, Lambda-<i>Pst</i>I as marker.</span> <br>
 +
</figure>
 +
</center>
 +
</p>
 +
<br>
 +
<p><b>October 28th, 2013</b></p>
 +
<p align="justify">We did minipreparation and digested them with <i>Hin</i>fI to identify more positive clones. As a result we get at the most two positive clones, one for pSB-mCherry and one for pSB-GFP
 +
</p>
 +
<br>
 +
<figure>
 +
<center>
 +
<img src="https://static.igem.org/mediawiki/2013/0/0a/713.jpg" width=500px style="border:1px solid black" >
 +
<figcaption><span class="text-muted"><br>Figure 7. Electrophoresis gel with the <i>Hin</i>fI clones digestion for mCherry, GFP, LacI TetR with pSB1C3 vector as control, Lambda-<i>Pst</i>I as marker.</span> <br>
 +
</figure>
 +
</center>
 +
</p>
 +
<br>
 +
<p><b>Stability</b></p>
<p><b>Stability</b></p>
-
<p>Digestion of the colonies from 09/02/2013. The ones from the gel (see image 2) were saved. The others are in a green box. We also planted mCherry and pBB1A3 vector.</p>
+
<p align="justify">For the <b>stability</b> experiments the tests were performed from September 14 to 20. The protocols are in the section of Safety. <a href="https://2013.igem.org/Team:UANL_Mty-Mexico/Safety/laboratory" target="2">Click here</a> </p>
 +
<br>
 +
<p align="justify">For the <b>fluorescence</b> experiments the tests were performed from September 10 to 27. The protocols are in the section of Wetlab. <a href="https://2013.igem.org/Team:UANL_Mty-Mexico/Wetlab#mCherry" target="2">Click here</a>  </p>
<br>
<br>
 +
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Notebook

Reference Data

Part Name Part Short name In pSB 1A3 (bp) In pSB 1C3 (bp) In pUC57 (bp) Part size (bp)
RBS + TetR TetR 2,925 2,802 3,478 732
pLac + GFP GFP 3,162 3,034 3,715 964
pCons LacI LacI 3,483 3,352 4,036 1,282
pTetR + mCherry mCherry 3,107 2,957 3,660 887
BBa_K1140005 3-E1 2,811 --- --- 935

This table works as a reference of the parts that were used in the laboratory and in the diary. We have a short name for each part so for example, if a diary entry says "TetR" we mean the complete part "RBS + TetR". Another important aspect is that we were working with the plasmids pSB 1C3, pSB 1A3 and pUC57 so here there is the information about the length of the part and the length in each plasmid.


August 4th, 2013

Today we prepared solutions in order to start working on the lab the following day. The solutions will be required for the MiniPrep, Transformation. We also prepared aliquots, mQ water with RNAse, etc.


August 5th, 2013

Today we started with the transformations of the synthetic DNAs that just arrived.


August 6th, 2013

We inoculated the colonies obtained from the transformations.


August 7th, 2013

This day we did Minipreparation of DNA. The parts that were obtained were the following: LacI, GFP, mCherry, TetR and 3-1E


August 8th, 2013

Test of mCherry with Thermo-mixer-Cualitative experiment

20 Colonies of the dish with pTet-R-mCherry were planted in 0.5 mL of Agar with Kanamycin in order to observe the red colour after several hours at 42ᵒC. The experiment was performed following these parameters.

Start End Hours
37ºC 2:50 pm 7:02 pm 4 hr 12 min
42ºC 7:05 pm 10:35 am 15 hr 40 min

Most tubes shown development seeing them on the light but there was no color shown.


August 10th, 2013

20 tubes with mCherry were put in the shaker with 2 controls with CDS (with oxygen everyone), following these parameters:
    • Temperature: 37ᵒC
    • Revolutions: 900 rpm
    • Start: 11:35 am -> 9:00 am (Time 21:35 hours)

    • The tubes show mCherry expression, some more intense than others. The experiment was repeated at 30°C overnight with a volume of 500 µL at 30°C with tubes of 1.5 ml. They were left at 4°C meanwhile they were incubated.

    Set Temperature Velocity Time Live
    Black Set 42ºC 900 rpm 15 hours :(
    Blue Set 37ºC 900 rpm 21 hours :)
    Red Set 32ºC 900 rpm :)

    Time 10:25 – 9:15 = 22:50 hrs


    August 12th, 2013

    The experiment was repeated at 42°C allowing the oxygen in to the tube. It began at 2:15 pm and ended at 1:20 pm of the next day.


    Figure 1. Tubes with the mCherry pellet that were incubated at 42ºC.

    August 16th, 2013

    Electrophoresis gel of the digestions from 15/08/13


    Figure 2. Electrophoresis gel of the digestions from 15/08/13

    DNA 1x 5x
    DNA 2 µL
    EcoRI 0.3 µL 1.5 µL
    PstI 0.3 µL 1.5 µL
    Buffer O (Fermentas) 1 µL 5 µL
    10 µL 50 µL

    August 18th, 2013

    There where no colonies present from the previous day.

    Ligation with a 1:1 ratio DNA:Vector. Is was performed with the following parameters:

    Size (bp) Vector DNA:Vector
    pTetR 768 2.8 0.33/1
    pCons-LacI 1326 1.6 0.62/1
    pTetR-mCherry 960 2.2 0.45/1
    pLacI-GFP 1005 2.1 0.47/1

    August 19th, 2013

    This ligation was not successful in the transformation.

    Dishes of:pTet-mCherry, pCons-LacI, pTetR, pLacI-GFP, and the 3-1E DNA were planted again.


    August 26th, 2013

    All the dishes were transformed but only the pLacGFP grew successfully.


    August 28th, 2013

    Today we picked up colonies from the dishes from the previous day. We also prepared new competent cells.


    August 29th, 2013

    All the dishes were successfully transformed and we planted in petri dishes. We got colonies of all the parts, they grew in the test tubes and miniPrep was done.


    August 30th, 2013

    The MiniPrep was successful and we did digestions with EcoRI. Transformations (one tube each one): TetR, pConLac1, TetRmCherry, 3-1E, pLacGFP. *They were planted in Ampicilin dish.


    Figure 3. Electrophoresis gel from the August 30 miniPreps

    August 31th, 2013

    MiniPrep and digestions from the ones of 08/30/2013. We didn't obtained any construction.

    Transformations (one tube each one): TetR, pConLac1, TetRmCherry, 3-1E, pLacGFP

    *They were planted in Ampicilin dish.


    September 11th, 2013

    Digestions with EcoRI and PstI and Ligations


    September 12th, 2013

    We didn't obtained any part so we started again transforming them one more time.


    September 13th, 2013

    There were no results presented so we transformed again.


    September 14th, 2013

    We got no results in the construction desired.


    September 19th, 2013

    Quantification with the Nanodrop of the previous samples


    ng/µL 260/280
    pSB1C3 1293.4 1.99
    GFP 133.5 1.84
    TetR 119.6 1.78
    tRFP 104.3 1.74
    pConsLac 130.5 1.59

    September 20th, 2013

    We prepared new calcium competent cells, E. coli strain Top10.


    September 23th, 2013

    We did the transformations again for all the parts.


    September 24th, 2013

    Again there where no results.


    Note: The constructions with the pSB1A3 and pSB1C3 showed no results but the controls with pUC vector were fine and did work in our experiments.


    October 9th, 2013

    New Digestions with EcoRI and PstI.


    October 10th, 2013

    New ligations were done


    October 11th, 2013

    New Petri dishes were made, and use for transformation today.


    October 13th, 2013

    We inoculated the colonies obtained from the transformations.


    October 14th, 2013

    We did minipreparation of DNA. After, the DNA was digested with EcoRI, PstI and HinfI for an enzymatic analysis (our Gel #700!!!). After hours, we did a electrophoresis and we realised that 2 new parts were now in the pSB 1C3 vector.



    Figure 4. Electrophoresis gel from the August 30 miniPreps cut with HinfI, Lambda-PstI as marker.


    October 17th, 2013

    We started a new test in order to measure number of plasmids per cell in M1, M2 and M12.


    October 18th, 2013

    First results for number of plasmids.


    October 21th, 2013

    More results for number of plasmids.


    October 22th, 2013

    A test for measuring expression of GFP (in pUC57 and pSB1C3) was prepared in microcentrifuge tubes with LB medium and IPTG 1mM for 17 hours at 37 °C (in bacteria E. coli Top10) in thermomixer.

    We still have problems with expression of mCherry, so we did new cultures with stored (red) M1, M2 and M12 in order.


    October 23th, 2013

    We used fluorometer to measure expression of GFP, but any fluorescence was detected, so new microcentrifuge tubes with GFP construction were prepared at same conditions of last day.

    The cultures of M1, M2, M11 and M12 were red and presented fluorescence, so we did minipreparation and culture again them in new tubes. With the DNA that was obtained, a digestion with EcoRI and PstI was done and a new ligation.


    October 24th, 2013

    Again, any fluorescence was detected for GFP cultures. New microcentrifuge tubes with GFP construction were prepared with IPTG 0.5 mM.

    A new transformation was done, using new mCherry and latest TetR and LacI ligations.

    Cultures of M11 and M12 grew, but any with M1 and M2. We did minipreparation and the DNA was measured in nanodrop.


    October 25th, 2013

    Residual fluorescence was detected for GFP cultures.

    We inoculated the colonies obtained from the transformations.


    October 26th, 2013

    Cultures of mCherry grew, but the others did not. We did minipreparation of that tubes and digested them with EcoRI, PstI and HinfI. The results can be seen on gel # 709 (EcoRI/PstI) and 710 (HinfI). Probably, we have multiple clones with mCherry in pSB1C3.

    New ligations of TetR, LacI and GFP with pSB1C3 were done and a transformation was made in a new competent stock, Top10 strain.



    Figure 5. Electrophoresis gel (709) with the EcoRI/PstI clones digestion, Lambda-PstI as marker.




    Figure 6. Electrophoresis gel (710) with the HinfI clones digestion, Lambda-PstI as marker.


    October 28th, 2013

    We did minipreparation and digested them with HinfI to identify more positive clones. As a result we get at the most two positive clones, one for pSB-mCherry and one for pSB-GFP



    Figure 7. Electrophoresis gel with the HinfI clones digestion for mCherry, GFP, LacI TetR with pSB1C3 vector as control, Lambda-PstI as marker.


    Stability

    For the stability experiments the tests were performed from September 14 to 20. The protocols are in the section of Safety. Click here


    For the fluorescence experiments the tests were performed from September 10 to 27. The protocols are in the section of Wetlab. Click here


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