Team:DTU-Denmark-2/Notebook/Lab notes

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<a href="https://2011.igem.org/Team:DTU-Denmark-2/Team/Notebook/Lab_notes#June"><font size="4"> June</font></a><br><br>
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<br>
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<a href="https://2011.igem.org/Team:DTU-Denmark-2/Team/Notebook/Lab_notes#July"><font size="4">July</font></a><br><br>
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<b>Lab notes</b><br> <br> <br>
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&nbsp; &nbsp;<a href="https://2011.igem.org/Team:DTU-Denmark-2/Team/Notebook/Lab_notes#Tuesday 19.07.2011"> Tuesday 19.07.2011</a><br><br>
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&nbsp; &nbsp;<a href="https://2011.igem.org/Team:DTU-Denmark-2/Team/Notebook/Lab_notes#Thursday 21.07.2011">Thursday 21.07.2011</a><br><br>
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&nbsp; &nbsp; <a href="https://2011.igem.org/Team:DTU-Denmark-2/Team/Notebook/Lab_notes#Friday 22.07.2011">Friday 22.07.2011</a><br><br>
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&nbsp; &nbsp; <a href="https://2011.igem.org/Team:DTU-Denmark-2/Team/Notebook/Lab_notes#Monday 25.07.2011">Monday 25.07.2011</a><br><br>
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&nbsp; &nbsp;<a href="https://2011.igem.org/Team:DTU-Denmark-2/Team/Notebook/Lab_notes#Tuesday 26.07.2011">Tuesday 26.07.2011</a><br><br>
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&nbsp; &nbsp;<a href="https://2011.igem.org/Team:DTU-Denmark-2/Team/Notebook/Lab_notes#Wednesday 27.07.2011">Wednesday 27.07.2011</a><br><br>
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<a href="https://2011.igem.org/Team:DTU-Denmark-2/Team/Notebook/Lab_notes#August"><font size="4">August</font></a><br><br>
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&nbsp; &nbsp;<a href="https://2011.igem.org/Team:DTU-Denmark-2/Team/Notebook/Lab_notes#Thursday 04.08.2011">Thursday 04.08.2011</a><br><br>
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&nbsp; &nbsp;<a href="https://2011.igem.org/Team:DTU-Denmark-2/Team/Notebook/Lab_notes#Friday 05.08.2011">Friday 05.08.2011</a><br><br>
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&nbsp; &nbsp;<a href="https://2011.igem.org/Team:DTU-Denmark-2/Team/Notebook/Lab_notes#Monday 08.08.2011">Monday 08.08.2011</a><br><br>
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&nbsp; &nbsp;<a href="https://2011.igem.org/Team:DTU-Denmark-2/Team/Notebook/Lab_notes#Wednesday 10.08.2011">Wednesday 10.08.2011</a><br><br>
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&nbsp; &nbsp;<a href="https://2011.igem.org/Team:DTU-Denmark-2/Team/Notebook/Lab_notes#Friday 12.08.2011">Friday 12.08.2011</a><br><br>
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&nbsp; &nbsp;<a href="https://2011.igem.org/Team:DTU-Denmark-2/Team/Notebook/Lab_notes#Monday 15.08.2011">Monday 15.08.2011</a><br><br>
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&nbsp; &nbsp;<a href="https://2011.igem.org/Team:DTU-Denmark-2/Team/Notebook/Lab_notes#Tuesday 16.08.2011">Tuesday 16.08.2011</a><br><br>
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<a href="https://2011.igem.org/Team:DTU-Denmark-2/Team/Notebook/Lab_notes#August"><font size="4">September</font></a><br><br>
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<a href="https://2011.igem.org/Team:DTU-Denmark-2/Team/Notebook/Lab_notes#August"><font size="4">October</font></a><br><br>
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<a href="https://2011.igem.org/Team:DTU-Denmark-2/Team/Notebook/Lab_notes#August"><font size="4">November</font></a><br><br>
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<!--<span id="test">test</span>-->
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<a href="https://2011.igem.org/Team:DTU-Denmark-2/Notebook/Lab_notes#July" class="h1">July</a><br>
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<a href="https://2011.igem.org/Team:DTU-Denmark-2/Notebook/Lab_notes#Week 1"class="h2">Week 1</a><br>
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<a href="https://2011.igem.org/Team:DTU-Denmark-2/Notebook/Lab_notes#Week 2 "class="h2"> Week 2</a><br>
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<a href="https://2011.igem.org/Team:DTU-Denmark-2/Notebook/Lab_notes#Week 3"class="h2"> Week 3</a><br>
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<a href="https://2011.igem.org/Team:DTU-Denmark-2/Notebook/Lab_notes#Week 4"class="h2"> Week 4</a><br><br>
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<a href="https://2011.igem.org/Team:DTU-Denmark-2/Notebook/Lab_notes#August"class="h1">August</a><br>
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<a href="https://2011.igem.org/Team:DTU-Denmark-2/Notebook/Lab_notes#Week 5"class="h2"> Week 5</a><br>
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<a href="https://2011.igem.org/Team:DTU-Denmark-2/Notebook/Lab_notes#Week 6"class="h2"> Week 6</a><br>
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<a href="https://2011.igem.org/Team:DTU-Denmark-2/Notebook/Lab_notes#Week 7"class="h2"> Week 7</a><br>
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<a href="https://2011.igem.org/Team:DTU-Denmark-2/Notebook/Lab_notes#Week 8"class="h2"> Week 8</a><br><br>
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<a href="https://2011.igem.org/Team:DTU-Denmark-2/Notebook/Lab_notes#September"class="h1">September</a><br>
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<a href="https://2011.igem.org/Team:DTU-Denmark-2/Notebook/Lab_notes#Week 9"class="h2"> Week 9</a><br>
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<a href="https://2011.igem.org/Team:DTU-Denmark-2/Notebook/Lab_notes#Week 10"class="h2"> Week 10</a><br><br>
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<a href="https://2011.igem.org/Team:DTU-Denmark-2/Notebook/Lab_notes#October"class="h1">October</a><br>
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<a href="https://2011.igem.org/Team:DTU-Denmark-2/Notebook/Lab_notes#November"class="h1">November</a><br>
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<a name="June"></a><h2>June</h2>
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<br>
<br>
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<br>
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<a name="July"></a><h2><b>July</b></h2>
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<br>
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<br>
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<a name="Week 1"></a><h3><b>Week 1: 04.07.2011 - 10.07.2011</b></h3>
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<a name="Tuesday 19.07.2011"></a><h3>Tuesday 19.07.2011</h3>
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First week! Time was spent on designing the Plug 'n' Play assembly system, figuring out what 48 biobricks to construct, and searching for appropriate DNA templates.
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First day in the lab and 48 biobricks to go, how exciting!
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<br>
<br>
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Of the 48 biobricks we will have when the project is finished we had only established 17 of them and ordered primers. We started the day with 11 PCR reactions of the first bash of 17 biobricks, we still need some DNA templates to due all 17. 6 PCR were correct and was purified with a purification Kit (GE Healthcare). So only 42 biobricks to go!
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<br>
 +
<a name="Week 2"></a><h3><b>Week 2: 11.07.2011 - 17.07.2011</b></h3>
 +
<a name="Wednesday 13.07.2011"></a><h4>Wednesday</h4>
 +
The first batch of primers have been ordered, so we are waiting in excitement for them to arrive, so we can start our work in the lab.
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<a name="Week 3"></a><h3><b>Week 3: 18.07.2011 - 24.07.2011</b></h3>
 +
<a name="Tuesday 19.07.2011"></a><h4>Tuesday</h4>
 +
First day in the lab and 48 biobricks to go. This is exciting!
 +
<br>
 +
We plan to construct 48 new BioBricks. We have now established 17 of them and ordered primers for them with USER tails. We started the day with 11 PCR reactions. 6 of them were correct, and they were purified with a purification Kit (GE Healthcare). Only 42 biobricks to go!
<br>
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<br>
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Line 74: Line 100:
<br>
<br>
<ul>
<ul>
-
<li>pALC</li>
+
<li>pAlc (<a href="http://partsregistry.org/Part:BBa_K678001">BBa_K678001</a>)</li>
-
<li>TtrpC</li>
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<li>TtrpC (<a href="http://partsregistry.org/Part:BBa_K678036 ">BBa_K678036</a>)</li>
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<li>Terminator 1</li>
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<li>Terminator 1 (<a href="http://partsregistry.org/Part:BBa_678037">BBa_K678037</a>)</li>
-
<li>Terminator 2</li>
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<li>Terminator 2 (<a href="http://partsregistry.org/Part:BBa_K678038">BBa_K678038</a>)</li>
-
<li>Terminator 3</li>
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<li>Terminator 3 (<a href="http://partsregistry.org/Part:BBa_K678039">BBa_K678039</a>)</li>
-
<li>argB</li>
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<li>argB (<a href="http://partsregistry.org/Part:BBa_K678043">BBa_K678043</a>)</li>
-
<ul>
+
</ul>
 +
 
 +
<img src="https://static.igem.org/mediawiki/2011/a/a7/20.07.2011.png" height="270px" alt="""/> </img>
<br>
<br>
<br>
<br>
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<a name="Thursday 21.07.2011"></a><h3>Thursday 21.07.2011</h3>
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<a name="Thursday 21.07.2011"></a><h4>Thursday</h4>
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Some of the PCR reactions that didn't work Tuesday was repeated with a different annealing temperature and amplification of new biobricks.
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The failed PCR reactions from tuesday were repeated. This time we used a different annealing temperature, and....IT WORKED!
<br>
<br>
<br>
<br>
<b>New biobricks in the bag:</b>
<b>New biobricks in the bag:</b>
 +
<ul>
<ul>
 +
<li>PGK (<a href="http://partsregistry.org/Part:BBa_K678004">BBa_K678004</a>)</li>
 +
<li>BGHpA (<a href="http://partsregistry.org/Part:BBa_K678019">BBa_K678019</a>)</li>
 +
<li>eGFP+k_GOI (<a href="http://partsregistry.org/Part:BBa_K678007">BBa_K678007</a>)</li>
 +
</ul>
 +
 +
<img src="https://static.igem.org/mediawiki/2011/e/ee/21.07.2011.png" height="270px" alt="""/> </img>
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<li>PGK</li>
 
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<li>BGHpA</li>
 
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<li>eGFP+k_GOI</li>
 
<br>
<br>
<br>
<br>
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The fragments will be frozen and purified later.
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The fragments will be frozen and later purified.
<br>
<br>
<br>
<br>
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We still have not succeed to make biobricks pyrG and pyrG-DR (use as a marker in fungi), despite two PCR attempts with a high annealing (59°) and low (56°) temperature.
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We still have not succeed to make BioBricks pyrG and pyrG-DR (used as a marker in fungi), despite different PCR attempts with a high annealing (59°) and low (56°) temperature.
<br>
<br>
 +
<br>
 +
<b>Work for iGEM Copenhagen team</b>
 +
<br>
 +
We have established collaboration with the iGEM team from Copenhagen University. We will costumize a number of their BioBricks to use with our Plug 'n' Play system. This is a great way of showing how easy it is to costumize the Plug 'n' Play system for any application, and the Copenhagen team will save a lot of time.
 +
Today we transformed competent E.coli cells with some of the standard biological parts from iGEM,<a href="http://partsregistry.org/Part:BBa_R0010"> BBa_R0010</a>,<a href="http://partsregistry.org/Part:BBa_R0015"> Bba_B0015</a> and <a href="http://partsregistry.org/Part:BBa_J52034">BBa_J52034</a>, and later we will make them fit the Plug 'n' Play with DNA system.
 +
<br>
 +
<br>
 +
<a name="Friday 22.07.2011"></a><h4>Friday</h4>
 +
We made a PCR of a troublesome BioBrick again - but this time with a small modification. We made a PCR mix with and without MgCl<sub>2</sub> 50mM in the reactions. And...IT WORKED with MgCl<sub>2</sub> 50mM!
 +
<br>
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<br>
 +
<b>Biobrick obtained:</b>
 +
<ul>
 +
<li>CMV (<a href="http://partsregistry.org/Part:BBa_J52034">BBa_J52034</a>)</li>
 +
<li>hygR (<a href="http://partsregistry.org/Part:BBa_K678042">BBa_K678042</a>)</li>
 +
</ul>
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<a name="USER cloning"></a><h2>USER cloning</h2>
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<img src="https://static.igem.org/mediawiki/2011/c/ce/22.07.2011_1.png" height="270px" alt="""/> </img>
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<a name="Tranformation in E.coli"></a><h3>Tranformation in E.coli</h3>
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<a name="Purification of plasmids"></a><h3>Purification of plasmids</h3>
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<img src="https://static.igem.org/mediawiki/2011/2/28/22.07.2011_2.png" height="270px" alt="""/> </img>
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<a name="Fungi"></a><h2>Fungi</h2>
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<a name="Transformation in fungi"></a><h3>Transformation in fungi</h3>
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<br>
 +
<br>
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The fragments will be frozen and later purified .
<br>
<br>
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<b>Genetic Transformation of filamentous fungi – protocol from Center for Microbial Biotechnology (CMB) at DTU, Author Nielsen, J. B.</b>
 
<br>
<br>
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<a name="Week 4"></a><h3><b>Week 4: 25.07.2011 - 31.07.2011</b></h3>
<br>
<br>
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<b>Media:</b>
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<a name="Monday 25.07.2011"></a><h4>Monday</h4>
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<li>Minimal medium (MM) (1L) -50 mL D-glucose 20% w/V, 20 mL 50x mineral mix, 10 mL 1 M sodium nitrate, 20 g ager.</li>
 
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<li>Transformation media(TM)(1L)- 342.3 g Sucrose, 20 mL 50x mineral mix, 20 g agar</li>
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We got a new delivery of primers from IDT today. They are to be diluted and stocked, before use.
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<li>Mineral Mix (1L)- 26g KCL, 26g MgSO4·7H2O, 76g KH2PO4, 50 mL Trace element solution, MIlli-Q water to volume 1000 mL</li>
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We have had some difficulty with obtaining 5 of our new biobricks; SV40+ori, SV40 pA, LacZ, pyrG and pyrG-DR. We ran a touch PCR program, where the annealing temperature ran from 55° to 65° for each sample in each cycle.
 +
<br>
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<br>
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<b>The PCR reactions were successful, and following biobricks are ready for Plug'n'Play:  </b>
 +
 +
<ul>
 +
<li>SV40 +ori (<a href="http://partsregistry.org/Part:BBa_K678005">BBa_K678005</a>)</li>
 +
<li>SV40 pA (<a href="http://partsregistry.org/Part:BBa_K678012">BBa_K678012</a>)</li>
 +
<li>LacZ (<a href="http://partsregistry.org/Part:BBa_K678026">BBa_K678026</a>)</li>
 +
</ul>
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<li>D-glucose 20% (0.5 L) - 100g D-glucose and MilliQ water up to 500 ml</li>
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<img src="https://static.igem.org/mediawiki/2011/7/76/26.07.2011_1.png" height="270px" alt="""/> </img>
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<li>Aspergillus protoplastationbuffer (APB)- Final conc. 1.1 M MgSO4 and 10 mM Na-phosphate buffer. pH is adjusted with 2 N NaOH to 5.8.</li>
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 +
<br>
 +
<br>
 +
<a name="Tuesday 26.07.2011"></a><h4>Tuesday</h4>
 +
The primers recieved yesterday will be used for amplification of many many many new biobricks today.
 +
<br>
 +
<br>
 +
<b>Out of 15 PCR reactions 5 succeeded:  </b>
-
<li>Aspergillus transformation buffer (ATB)- Final conc: 1.2 M Sorbitol; 50 mM CaCl2·2 H2O; 20 mM Tris; and 0.6 M KCl. pH is adjusted with 2 N HCl to 7.2.</li>
+
<br>
 +
<ul>
 +
<li>GFP_GOI (<a href="http://partsregistry.org/Part:BBa_K678028">BBa_K678028</a>) </li>
 +
<li>GFP_modul (<a href="http://partsregistry.org/Part:BBa_K678027">BBa_K678027</a>)</li>
 +
<li>GFP_TS (<a href="http://partsregistry.org/Part:BBa_K678029">BBa_K678029</a>)</li>
 +
<li>eGFP_TS (<a href="http://partsregistry.org/Part:BBa_K678007">BBa_K678007</a>)</li>
 +
<li>eGFP_module (<a href="http://partsregistry.org/Part:BBa_K678009">BBa_K678009</a>)</li>
 +
</ul>
-
<li>PCT (200ml) - Final conc: 50% w/vol PEG 8000; 50 mM CaCl2; 20 mM Tris; and 0.6 M KCl. pH is adjusted with 2 N HCl to 7.5. Store at 4 °C</li>
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<img src="https://static.igem.org/mediawiki/2011/b/bf/26.07.2011_2.png" height="270px" alt="""/> </img>
 +
 
 +
<img src="https://static.igem.org/mediawiki/2011/a/ab/26.07.2011_3.png" height="270px" alt="""/> </img>
 +
 
 +
 +
<br>
 +
<br>
 +
<a name="Wednesday 27.07.2011"></a><h4>Wednesday</h4>
 +
 
 +
Today we have run 8 reactions, 3 standard and 5 touch PCR reactions on some of the biobricks that we still have difficulties obtaining.  
 +
<br>
 +
<br>
 +
<b>One succeeded:</b>
 +
<ul>
 +
<li>hgH-polyA (<a href="http://partsregistry.org/Part:BBa_K678018">BBa_K678018</a>) </li>
</ul>
</ul>
 +
 +
<img src="https://static.igem.org/mediawiki/2011/e/e5/27.07.2011.png" height="270px" alt="""/> </img>
 +
<br>
<br>
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<b>Initiation:</b> The host strain is grown as three-point stabs on Minimal medium plates with the require suppliants added. MM will for convenience throughout the protocol refer to MM with the supplements included.
+
<a name="August"></a><h2><b>August</b></h2>
<br>
<br>
 +
<a name="Week 5"></a><h3><b>Week 5: 01.08.2011 - 07.08.2011</b></h3>
 +
<a name="Monday 01.08.2011"></a><h4>Monday</h4>
 +
New week, new energy, and lots of team spirit!
 +
Today 15 PCR reactions were conducted, including parts for iGEM Copenhagen team. 14 succeeded. The annealing temperature was increased to 63°, and it worked like magic!
<br>
<br>
-
<b>Inoculation:</b> The conidia are harvest by adding 5 ml of MM and firmly rub with a sterile Drigalsky spatula. The conidial suspension is pipette to a sterile 500 ml shake flask containing 100 ml MM. The cultures are incubated at 30 °C with 150 rpm of shaking over night (14-20 hours)
 
<br>
<br>
 +
<b>New biobricks in the bag:</b>
 +
<ul>
 +
<li>RFP_GOI (<a href="http://partsregistry.org/Part:BBa_K678031">BBa_K678031</a>) </li>
 +
<li>RFP_modul (<a href="http://partsregistry.org/Part:BBa_K678030">BBa_K678030</a>)</li>
 +
<li>RFP_TS (<a href="http://partsregistry.org/Part:BBa_K678032">BBa_K678032</a>)</li>
 +
<li>bleR (<a href="http://partsregistry.org/Part:BBa_K678044">BBa_K678044</a>)</li>
 +
<li>pgpdA (<a href="http://partsregistry.org/Part:BBa_K678024">BBa_K678024</a>)</li>
 +
<li>yA (<a href="http://partsregistry.org/Part:BBa_K678035">BBa_K678035</a>)</li>
 +
<li>Plasmid_fun (<a href="http://partsregistry.org/Part:BBa_K678046">BBa_K678046</a>)</li>
 +
<li>Plasmid_mam (<a href="http://partsregistry.org/Part:BBa_K678023">BBa_K678023</a>)</li>
 +
<li>Amp cas  (<a href="http://partsregistry.org/Part:BBa_K678047">BBa_K678047</a>)</li>
 +
<li>Hygromycin (<a href="http://partsregistry.org/Part:BBa_K678021">BBa_K678021</a>)</li>
 +
<li>CYP79A2 (Copenhagen part) (<a href="http://partsregistry.org/Part:BBa_K527001">BBa_K527001</a>)</li>
 +
<li>Terminator (Copenhagen part) (<a href="http://partsregistry.org/Part:BBa_B0015">BBa_B0015</a>)</li>
 +
<li>pIPTG (Copenhagen part) (<a href="http://partsregistry.org/Part:BBa_R0010">BBa_R0010</a>)</li>
 +
<li>CYP79B2(Copenhagen part) (<a href="http://partsregistry.org/Part:BBa_K527002">BBa_K527002</a>)</li>
 +
</ul>
<br>
<br>
-
<b>Mycelial harvest:</b> A funnel with a sterile Mira cloth (filter) is used to harvest mycelia. To remove residual glucose from mycelia the biomass are wash with Aspergillus protoplastationbuffer (APB). The filtered biomass is transferred to a new Falcon tube with a sterile spoon.
+
 
 +
<img src="https://static.igem.org/mediawiki/2011/e/e2/1.08.2011_1.png" height="270px" alt="""/> </img>
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 +
<img src="https://static.igem.org/mediawiki/2011/4/4d/01.08.2011_2.png" height="270px" alt="""/> </img>
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<img src="https://static.igem.org/mediawiki/2011/9/97/01.08.2011_3.png" height="270px" alt="""/> </img>
 +
 
 +
 
 +
<a name=" Wednesday 03.08.2011"></a><h4> Wednesday </h4>
 +
There are still 2 BioBricks that we haven't been able to obtain with PCR: pyrG and pyrG-DR.  
<br>
<br>
 +
Today we tried again with gradient PCR to find the optimal melting temperature, but no magic happened. 
<br>
<br>
-
<b>Protoplastation:</b> Mycellium is resuspenden in 10 ml filter-sterillized(0.45μm filters) APB containing 40 mg Glucanex/ml. The Glucanex is dissolved in APB with gentle magnetic stirring less than 100/min. Mycelia with dissolved Glucanex are mixed at 30 °C with 150 rpm of shaking for 2-3 hours.<br>
 
-
Portoplast solutions are diluted in APB adding up to 40 ml mark. An overlay of max. 5ml Aspergillus transformation buffer (ATB) diluted to ½x with sterile MilliQ-water is carefully placed on top of the APB. Centrifuged at 13 min 3000 RCF in Sorvall centrifuge. Protoplates should be observed as a halo of with slurry in the interphase of the two liquids. Withdraw of the protoplasts are done with pipette and placed in a Falcon tube. ATB is added up to 40 ml mark. Centrifuge at 300 RCF in 13 min and supernatanted are discarded.<br>
 
-
The protoplastes are resuspended in 1 ml ATB with a 5 ml pipette.
 
<br>
<br>
 +
<a name="Thursday 04.08.2011"></a><h4>Thursday</h4>
 +
22 BioBricks, which have been obtained with PCR during the last week of laboratory work, was purified today. The purification of DNA was done by using the Illustra GFX PCR DNA and Gel Band Purification Kit (GE Healthcare). The procedure was carried out according to the manufactures protocol for purification of DNA from TAE and TBE agarose gels, see protocol section for more information.<br><br>
 +
Despite several attempts we have not been able to obtain BioBrick pyrG and pyrG-DR. Therefore we made a test with well-known primers without USER-tails (sometimes these USER-tails can be tricky). This was done to verify if something was wrong with the DNA template. The gel electrophoresis did not show any bands, and thereby it revealed that the DNA template was the source of error. Problem solved!
<br>
<br>
-
<b>Genetic transformation:</b> Aliquotes of 50 μl are transferred to a 1.5 ml Eppendorf tube containgen 10 μl of DNA for transformation. Protoplast and DNA are incubated at room temperature for at least 30 min.<br>
+
<br>
-
Protoplat and DNA suspension are added to 1 ml PCT in a 15 ml tube and shake gently. Incubated for 1-5 min at room temperature. Diluted in 3 ml ATB. The tube is filled with molten transformation medium (TM) agar (temperature of 40-45°C) to apporeimately 12 ml. The tube is mix rapidly by inverting the tube twice. Poured directly on pre-made TM plates and incubated at 37°C for 3-8 days.
+
 
 +
We also started cultivation of the mammalian cell line, U2OS, which is derived from a patient with osteosarcoma (bone cancer). They were defrosted and cultivated in DMEM medium containing serum, penicillin and streptomycin. The cell line was then transferred to a 25 cm2 culture flask, see protocol section.
<br>
<br>
<br>
<br>
-
<a name="Mammalian cells"></a><h2>Mammalian cells</h2>
+
<a name="Week 6"></a><h3><b>Week 5: 08.08.2011 - 14.08.2011</b></h3>
<br>
<br>
-
<a name="Cell culture and reagents"></a><h3>Cell culture and reagents</h3>
+
<a name="Monday 08.08.2011"></a><h4>Monday</h4>
-
HeLa and U2OS cells were kindly provided by The Danish Cancer Society. HeLa cells are a spontaneously immortilized cell line derived from cervical cancer cells taken from Henrietta Lacks, a patient that died of cancer in 1951. The HeLa cell line is the oldest and most commonly used human cell line, e.g. the cells were used to test the first polio vaccine in the 1950s, and since then they have been used for research in cancer, AIDS, etc. U2OS cell line is derived from the bone tissue of a patient suffering from osteosarcoma. U2OS cells show epithelial adherent morphology.  
+
 
 +
Today we made the first USER cloning, we are very excited to see if the system works the way, we want it to! <br>
 +
 
 +
All our biobricks are divided into devices, see protocol for USER MIX. <br>
 +
Device 1,2,3,5 and 6 was not registrated later.
<br>
<br>
-
The cells are cultivated in Dulbecco’s Modified Eagle Medium (DMEM), supplemented with Penicillin, Streptomycin, and 10 % heat-inactivated foetal calf serum (FCS). Penicillin and Streptomycin are added to prevent any microbial growth. FCS is added to supply essential non-defined components, such as serum proteins and lipids. Supplemented DMEM medium is referred to as complete DMEM throughout the report. The cells are adherent and are kept in 75 cm2 culture flask until 80-100% confluency, where they are passed on to new culture flasks or cover slips.
+
<b>The following devices were cloned today:</b>
<br>
<br>
 +
 +
<ul>
 +
<li><b>Device 1</b>:<br> plasmid_mam (<a href="http://partsregistry.org/Part:BBa_K678023">BBa_K678023</a>) + CMV (<a href="http://partsregistry.org/Part:BBa_J52034">BBa_J52034</a>) + eGFP (<a href="http://partsregistry.org/Part:BBa_K678006">BBa_K678006</a>) + hGH pA (<a href="http://partsregistry.org/Part:BBa_K678018">BBa_K678018</a>) + Hygromycin (<a href="http://partsregistry.org/Part:BBa_K678021">BBa_K678021</a>)</li>
 +
 +
<li><b>Device 2</b>: <br>plasmid_mam (<a href="http://partsregistry.org/Part:BBa_K678023">BBa_K678023</a>) + SV40+ori (<a href="http://partsregistry.org/Part:BBa_K678005">BBa_K678005</a>) + eGFP (<a href="http://partsregistry.org/Part:BBa_K678006">BBa_K678006</a>) + SV40 pA (<a href="http://partsregistry.org/Part:BBa_K678012">BBa_K678012</a>) + Hygromycin (<a href="http://partsregistry.org/Part:BBa_K678021">BBa_K678021</a>)</li>
 +
 +
<li><b>Device 3</b>: <br>plasmid_mam (<a href="http://partsregistry.org/Part:BBa_K678023">BBa_K678023</a>) + PGK (<a href="http://partsregistry.org/Part:BBa_K678004">BBa_K678004</a>) + eGFP_module (<a href="http://partsregistry.org/Part:BBa_K678006">BBa_K678006</a>) + BGH pA (<a href="http://partsregistry.org/Part:BBa_K678019">BBa_K678019</a>) + Neomycin (<a href="http://partsregistry.org/Part:BBa_K678022">BBa_K678022</a>)</li>
 +
 +
<li><b>Device <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K678058">BBa_K678058</a></b>:<br>plasmid_fun (<a href="http://partsregistry.org/Part:BBa_K678046">BBa_K678046</a>) + pAlc (<a href="http://partsregistry.org/Part:BBa_K678001">BBa_K678001</a>) + LacZ (<a href="http://partsregistry.org/Part:BBa_K678026">BBa_K678026</a>) + T1 (<a href="http://partsregistry.org/Part:BBa_K678037">aBBa_K678037</a>) + argB (<a href="http://partsregistry.org/Part:BBa_K678043">BBa_K678043</a>)</li>
 +
 +
<li><b>Device 5</b>:<br> plasmid_fun (<a href="http://partsregistry.org/Part:BBa_K678046">BBa_K678046</a>) + gpdA (<a href="http://partsregistry.org/Part:BBa_K678024">BBa_K678024</a>) + GFP_module (<a href="http://partsregistry.org/Part:BBa_K678027">BBa_K678027</a>) + trpC (<a href="http://partsregistry.org/Part:BBa_K678036">BBa_K678036</a>) + hygR (<a href="http://partsregistry.org/Part:BBa_K678042">BBa_K678042</a>)</li>
 +
 +
<li><b>Device 6</b>: <br>plasmid_fun (<a href="http://partsregistry.org/Part:BBa_K678046">BBa_K678046</a>) + gpdA (<a href="http://partsregistry.org/Part:BBa_K678024">BBa_K678024</a>) + RFP_module (<a href="http://partsregistry.org/Part:BBa_K678030">BBa_K678030</a>) + trpC (<a href="http://partsregistry.org/Part:BBa_K678036">BBa_K678036</a>) + hygR (<a href="http://partsregistry.org/Part:BBa_K678042">BBa_K678042</a>)</li>
 +
 +
<li>Device 7 (Copenhagen): <br>plasmid_mam (<a href="http://partsregistry.org/Part:BBa_K678023">BBa_K678023</a>) + pIPTG (<a href="http://partsregistry.org/Part:BBa_R0010">BBa_R0010</a>) + CYP79B2 (<a href="http://partsregistry.org/Part:BBa_K527002">BBa_K527002</a>) + Terminator (<a href="http://partsregistry.org/Part:BBa_B0015">BBa_B0015</a>) + amp (<a href="http://partsregistry.org/Part:BBa_K678047">BBa_K678047</a>)</li>
 +
 +
<li>Device 8 (Copenhagen): <br>plasmid_mam (<a href="http://partsregistry.org/Part:BBa_K678023">BBa_K678023</a>) + pIPTG (<a href="http://partsregistry.org/Part:BBa_R0010">BBa_R0010</a>) + CYP79A2 (<a href="http://partsregistry.org/Part:BBa_K527001">BBa_K527001</a>) + Terminator (<a href="http://partsregistry.org/Part:BBa_B0015">BBa_B0015</a>) + amp (<a href="http://partsregistry.org/Part:BBa_K678047">BBa_K678047</a>)</li>
 +
 +
</ul>
<br>
<br>
-
<a name="Medium"></a><h3>Medium</h3>
+
 
 +
 
 +
<b>PCR reactions done today:</b>
 +
<br>
 +
 
<ul>
<ul>
-
<li>500 ml DMEM</li>
+
<li>eGFP+k_GOI (<a href="http://partsregistry.org/Part:BBa_K678007">BBa_K678007</a>)</li>
-
<li>50 ml Fetal Calf Serum (FCS)</li>  
+
<li>eGFP_TS (<a href="http://partsregistry.org/Part:BBa_K678007">BBa_K678007</a>)</li>
-
<li>5 ml Penicillin</li>  
+
<li>yA (<a href="http://partsregistry.org/Part:BBa_K678035">BBa_K678035</a>)</li>
-
<li>5 ml Streptomycin </li>  
+
<li>GFP_TS (<a href="http://partsregistry.org/Part:BBa_K678029">BBa_K678029</a>)</li>
 +
<li>RFP_TS (<a href="http://partsregistry.org/Part:BBa_K678032">BBa_K678032</a>)</li>
 +
<li>RFP_GOI (<a href="http://partsregistry.org/Part:BBa_K678031">BBa_K678031</a>) </li>
 +
<li>MTS (<a href="http://partsregistry.org/Part:BBa_K678025">BBa_K678025</a>) </li>
 +
<li>GFP_PTS1_fun (<a href="http://partsregistry.org/Part:BBa_K678033">BBa_K678033</a>) </li>
 +
<li>GFP_PTS1_mam (<a href="http://partsregistry.org/Part:BBa_K678017">BBa_K678017</a> )</li>
 +
<li>RFP_NLS_module (<a href="http://partsregistry.org/Part:BBa_K678034">BBa_K678034</a>)  </li>
 +
<li>pyrG (<a href="http://partsregistry.org/Part:BBa_K678040">BBa_K678040</a> ) </li>
 +
<li>pyrG-DR (<a href="http://partsregistry.org/Part:BBa_K678041">BBa_K678041</a>)  </li>
</ul>
</ul>
 +
 +
<img src="https://static.igem.org/mediawiki/2011/c/c5/08.08.2011_1.png" height="270px" alt="""/>
 +
 +
<br>
<br>
-
Add FCS, penicillin, and streptomycin to a new flask of DMEM. Store at 5°C.
 
<br>
<br>
 +
After recieving the new DNA template we finally succeeded in getting biobricks pyrG and pyrG-DR. 
<br>
<br>
-
<a name="Cultivation of the cells"></a><h3>Cultivation of the cells</h3>
+
The U2OS cell line was splitted and passed on to new culture flasks. They looked really lovely in the microscope, which bodes well for future confocal microscopy. They seem to thrive in the mammalian cell lab, and they are passed on every second or third day, depending on the initial cell concentration.
 +
<br>
 +
<br>
 +
<a name="Tuesday 09.08.2011"></a><h4>Tuesday</h4>
 +
 
 +
<i>E.coli </i> was inoculated with the plasmides done by USER cloning yesterday.
 +
<br>
 +
<br>
 +
<b>PCR done today:</b>
-
25 cm2 culture flask <br>
 
-
Complete DMEM <br>
 
<ul>
<ul>
-
<li>Place the appropriate amount of EDTA-trypsin and complete DMEM in the incubator (37°C), before handling the cells (about 1-2 hours before).</li>  
+
<li> RFP_TS (<a href="http://partsregistry.org/Part:BBa_K678032">BBa_K678032</a>)</li>
 +
<li>RFP_GOI (<a href="http://partsregistry.org/Part:BBa_K678031">BBa_K678031</a>)</li>
 +
<li>RFP_NLS_Module (<a href="http://partsregistry.org/Part:BBa_K678034">BBa_K678034</a>)</li>
-
<li>The HeLA and the U2OS cells are kept at -80°C until defrosted gently at 37°.</li>
+
</ul>
-
<li>Exactly when defrosted, they are mixed with 12 ml complete DMEM in the pipette. </li>  
+
<br>
-
<li>The cell suspension is transferred to a 15 ml vial and centrifuged at 280 g for 10 minutes. The supernatant is discarded. </li>  
+
<img src="https://static.igem.org/mediawiki/2011/5/52/09.08.2011_1.png" height="270px" alt="""/>
 +
<img src="https://static.igem.org/mediawiki/2011/b/bc/09.08.2011_2.png" height="270px" alt="""/>
-
<li>The cell pellet is resuspended in 5 ml complete DMEM and transferred to a 25 cm2 culture flask.</li> 
 
-
<li>The cells are kept at 37°C in a  5% CO2 incubator until the following day, where they are passed on to larger culture flasks.</li>  
+
 
 +
 
 +
<br>
 +
<br>
 +
<b>Purification with GFX kit of the following biobricks:</b>
 +
<br>
 +
 
 +
<ul>
 +
<li>eGFP+k_GOI (<a href="http://partsregistry.org/Part:BBa_K678007">BBa_K678007</a>)</li>
 +
<li>eGFP_TS (<a href="http://partsregistry.org/Part:BBa_K678007">BBa_K678007</a>)</li>
 +
<li>yA (<a href="http://partsregistry.org/Part:BBa_K678035">BBa_K678035</a>)</li>
 +
<li>GFP_TS (<a href="http://partsregistry.org/Part:BBa_K678029">BBa_K678029</a>)</li>
 +
<li>RFP_TS (<a href="http://partsregistry.org/Part:BBa_K678032">BBa_K678032</a>)</li>
 +
<li>RFP_GOI (<a href="http://partsregistry.org/Part:BBa_K678031">BBa_K678031</a>) </li>
 +
<li>MTS (<a href="http://partsregistry.org/Part:BBa_K678025">BBa_K678025</a>) </li>
 +
<li>GFP_PTS1_fun (<a href="http://partsregistry.org/Part:BBa_K678033">BBa_K678033</a>) </li>
 +
<li>GFP_PTS1_mam (<a href="http://partsregistry.org/Part:BBa_K678017">BBa_K678017</a>)</li>
 +
<li>RFP_NLS_module (<a href="http://partsregistry.org/Part:BBa_K678034">BBa_K678034</a>) </li>
 +
<li>pyrG (<a href="http://partsregistry.org/Part:BBa_K678040">BBa_K678040</a>) </li>
 +
<li>pyrG-DR (<a href="http://partsregistry.org/Part:BBa_K678041">BBa_K678041</a>)  </li>
</ul>
</ul>
<br>
<br>
-
<a name="Passing and maintenance of HeLa and U2OS cells"></a><h3>Passing and maintenance of HeLa and U2OS cells</h3>
+
<b>USER cloning done today:</b>
-
Procedure for cells in 75 cm2 culture flask:<br>
+
-
75 cm2 culture flask <br>
+
-
50 ml vial<br>
+
-
1, 2, 5, 10, 25 ml pipettes <br>
+
-
Complete DMEM medium<br>
+
-
0.05 % EDTA-trypsin<br>
+
<br>
<br>
<ul>
<ul>
-
<li>The appropriate amount of EDTA-trypsin and complete DMEM in the incubator (37°C), before handling the cells.</li>   
+
<li><b>Device <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K678065">BBa_K678065</a> </b>: <br>plasmid_fun (<a href="http://partsregistry.org/Part:BBa_K678046">BBa_K678046</a>) + gpdA (<a href="http://partsregistry.org/Part:BBa_K678024">BBa_K678024</a>) + MTS (<a href="http://partsregistry.org/Part:BBa_K678025">BBa_K678025</a>) + GFP_TS (<a href="http://partsregistry.org/Part:BBa_K678029">BBa_K678029</a>) + TtrpC (<a href="http://partsregistry.org/Part:BBa_K678036">BBa_K678036</a>) + hygR (<a href="http://partsregistry.org/Part:BBa_K678042">BBa_K678042</a>)</li>
-
<li>The filter of a 1 ml pipette is broken off, and the pipette is attached to the vacuum suction maschine. The medium is removed, and the pipette is put in a 50 ml vial for later use.</li>  
+
<li><b>Device 10</b>: <br>plasmid_fun (<a href="http://partsregistry.org/Part:BBa_K678046">BBa_K678046</a>) + gpdA (<a href="http://partsregistry.org/Part:BBa_K678024">BBa_K678024</a>) + GFP_PTS1_module_fun (<a href="http://partsregistry.org/Part:BBa_K678033">BBa_K678033</a>) + TtrpC (<a href="http://partsregistry.org/Part:BBa_K678036">BBa_K678036</a>) + hygR (<a href="http://partsregistry.org/Part:BBa_K678042">BBa_K678042</a>)</li>
-
<li>1 ml 0,05% EDTA-trypsin/PBS is added to wash the cells. Tilt the flask quickly, so the EDTA-trypsin covers the cells. Remove the liquid quickly. </li>  
+
<li><b>Device 11</b>:<br> plasmid_fun (<a href="http://partsregistry.org/Part:BBa_K678046">BBa_K678046</a>) + gpdA (<a href="http://partsregistry.org/Part:BBa_K678024">BBa_K678024</a>) + RFP_NLS_module (<a href="http://partsregistry.org/Part:BBa_K678034">BBa_K678034</a>) + TtrpC (<a href="http://partsregistry.org/Part:BBa_K678036">BBa_K678036</a>) + hygR (<a href="http://partsregistry.org/Part:BBa_K678042">BBa_K678042</a>)</li>
-
<li>1 ml 0.05% trypsin-EDTA/PBS is added and the flask is incubated for 3 min at 37°C and 5 % CO2.</li>
+
<li><b>Device <a href="http://partsregistry.org/Part:BBa_K678050">BBa_K678050</a></b>:<br> plasmid_mam (<a href="http://partsregistry.org/Part:BBa_K678023">BBa_K678023</a>) + CMV (<a href="http://partsregistry.org/Part:BBa_J52034">BBa_J52034</a>) + GFP_PST1_module_mam (<a href="http://partsregistry.org/Part:BBa_K678017">BBa_K678017</a>) + BGH pA (<a href="http://partsregistry.org/Part:BBa_K678019">BBa_K678019</a>) + Hygromycin (<a href="http://partsregistry.org/Part:BBa_K678021">BBa_K678021</a>)</li>
-
<li>9 ml complete medium is added to inactivate the EDTA-trypsin and to wash the cells of the surface. Make sure the cells are well resuspended, before you transfer them. </li>  
+
<li><b>Device <a href="http://partsregistry.org/Part:BBa_K678049">BBa_K678049</a></b>:<br> plasmid_mam (<a href="http://partsregistry.org/Part:BBa_K678023">BBa_K678023</a>) + CMV (<a href="http://partsregistry.org/Part:BBa_J52034">BBa_J52034</a>) + eGFP_module (<a href="http://partsregistry.org/Part:BBa_K678027">BBa_K678027</a>) + BGH pA (<a href="http://partsregistry.org/Part:BBa_K678019">BBa_K678019</a>) + Hygromycin (<a href="http://partsregistry.org/Part:BBa_K678021">BBa_K678021</a>)</li>
 +
</ul>
-
<li>2,5 ml (depends on the concentration of the cells) cell suspension is transferred to a new 75 cm2 culture flask. </li>
+
<br>
 +
<a name="Wednesday 10.08.2011"></a><h4>Wednesday</h4>
 +
The U2OS cells was investigated in the microscope. They had a confluency of about 90%, an therefore ready to pass on to new culture flasks. This was done according to the protocol (see protocol for passing and maintenance of mammalian cells")
-
<li>The cells are kept at 37°C in a 5% CO2 incubator until they are 80-100% confluent. This takes about 2-3 days. Then they are passed on to a new flask or plate.</li>
 
-
</ul>
 
<br>
<br>
-
<a name="Transferring the cells to coverslips"></a><h3>Transferring the cells to coverslips</h3>
+
 
 +
<a name="Friday 12.08.2011"></a><h4>Friday</h4>
 +
 
 +
Today 13 PCR reaction were carried out, some parts were for characterisation of two promoters: pAlc and DMKP-P6, and the rest were parts for the Copenhagen iGEM team.
 +
<br>
 +
<br>
<ul>
<ul>
-
<li>The filter of a 1 ml pipette is broken off, and the pipette is attached to the vacuum suction maschine. The medium is removed, and the pipette is put in a 50 ml vial for later use.
+
<li>GFP_GOI (<a href="http://partsregistry.org/Part:BBa_K678028">BBa_K678028</a>) </li>
 +
<li>GFP_PTS_fun (<a href="http://partsregistry.org/Part:BBa_K678033">BBa_K678033</a>)</li>
 +
<li>GFP_PTS_mam (<a href="http://partsregistry.org/Part:BBa_K678017">BBa_K678017</a>)</li>
 +
<li>RFP_NLS (<a href="http://partsregistry.org/Part:BBa_K678034">BBa_K678034</a>)</li>
 +
<li>DMKP_P6 (<a href="http://partsregistry.org/Part:BBa_K678000">BBa_K678000</a>)</li>
 +
<li>ptrA (<a href="http://partsregistry.org/Part:BBa_K678045">BBa_K678045</a>)</li>
 +
<li>1A2 (<b>C7</b>)</li>
 +
<li>2C9 (<b>C8</b>)</li>
 +
<li>pAlc-L (<a href="http://partsregistry.org/Part:BBa_K678001">BBa_K678001</a>)</li>
 +
<li>DMKP_P6-L (<a href="http://partsregistry.org/Part:BBa_K678000">BBa_K678000</a>)</li>
 +
<li>RFP_TS (<a href="http://partsregistry.org/Part:BBa_K678032">BBa_K678032</a>)</li>
 +
<li>RFP_GOI (<a href="http://partsregistry.org/Part:BBa_K678031">BBa_K678031</a>)</li>
 +
</ul> 
 +
<br>
 +
<br>
-
<li>1,5 ml 0,05% EDTA-trypsin/PBS is added to wash the cells. Tilt the flask quickly, so the EDTA-trypsin covers the cells. Remove the liquid quickly.</li>
+
<img src="https://static.igem.org/mediawiki/2011/b/b0/12.08.2011.png"height="250px" alt="""/>
-
<li>1,5 ml 0.05% trypsin-EDTA/PBS is added and the flask is incubated for 3 min at 37°C and 5 % CO2. </li>  
+
<br>
 +
<br>
 +
The U2OS cells were splitted and passed on to two new 75 cm2 culture flasks. Hopefully, they will be ready to pass on to cover slips on monday.
 +
<br>
 +
<a name="Week 7"></a><h3><b>Week 7: 15.08.2011 - 21.08.2011</b></h3>
 +
<br>
 +
<a name="Monday 15.08.2011"></a><h4>Monday</h4>
 +
The U2OS cells were splitted and passed on to a new 75 cm2 culture flask and to coverslips placed in a 6-well plate. They were placed in the incubator overnight - and will be ready for transfection with plasmides tomorrow.
 +
<br>
-
<li>9 ml complete medium is added to inactivate the EDTA-trypsin and to wash the cells of the surface. Make sure the cells are well resuspended, before you transfer them. </li>  
+
In preparation for characterization of two promoters with β-galactosidase assay the plasmid p68 cut with restriction enzyme.
 +
<br>
 +
<br>
 +
Purification of PCR products with GFX kit.  
 +
<br>
 +
<br> 
 +
<b>9 PCR reactions were done today</b>
 +
<br>
 +
<ul>
 +
<li> eYFP_module (<a href="http://partsregistry.org/Part:BBa_K678009">BBa_K678009</a>) </li>
 +
<li>eYFP_GOI (<a href="http://partsregistry.org/Part:BBa_K678010">BBa_K678010</a>)</li>
 +
<li>eYFP_TS (<a href="http://partsregistry.org/Part:BBa_K678011">BBa_K678011</a>)</li>
 +
<li>eCFP_module (<a href="http://partsregistry.org/Part:BBa_K678015">BBa_K678015</a>)</li>
 +
<li>eCFP_GOI (<a href="http://partsregistry.org/Part:BBa_K678048">BBa_K678048</a>)</li>
 +
<li>eCFP_TS (<a href="http://partsregistry.org/Part:BBa_K678016">BBa_K678016</a>)</li>
 +
<li>mCheryy_module (<a href="http://partsregistry.org/Part:BBa_K678020">BBa_K678020</a>)</li>
 +
<li>mCherry_GOI (<a href="http://partsregistry.org/Part:BBa_K678013">BBa_K678013</a>)</li>
 +
<li>mCHerry_TS (<a href="http://partsregistry.org/Part:BBa_K678014">BBa_K678014</a>)</li>
 +
</ul> 
 +
<br>
 +
<br>
-
<li>Calculate the concentration of cells you want in the wells</li>  
+
<img src="https://static.igem.org/mediawiki/2011/2/2d/15.08.2011.png" height="270px" alt="""/>
-
<li>Cover slips are placed in the bottom of a 6-well plate (2 cover slips/well). 2 ml cell suspension is added gently to each well.</li>  
+
<img src="https://static.igem.org/mediawiki/2011/9/9a/15.08.2011_23.png" height="270px" alt="""/>
-
<li>The plate is placed in the incubator at 37C and 5% CO2 O/N.</li>
 
-
</ul>
 
-
</td>
 
-
</th>
 
-
</table>
 
-
</body>
 
-
</html>
 
 +
<br>
 +
<br>
 +
<b>USER cloning and transformation in <i>E.coli </i> today: </b>
 +
<br>
 +
<ul>
 +
<li><b>Device <a href="http://partsregistry.org/Part:BBa_K678059">BBa_K678059</a></b>:<br> plasmid_fun (<a href="http://partsregistry.org/Part:BBa_K678046">BBa_K678046</a>) + PAlc (<a href="http://partsregistry.org/Part:BBa_K678001">BBa_K678001</a>) + LacZ (<a href="http://partsregistry.org/Part:BBa_K678026">BBa_K678026</a>) + T1 (<a href="http://partsregistry.org/Part:BBa_K678037">BBa_K678037</a>) + ptrA (<a href="http://partsregistry.org/Part:BBa_K678045">BBa_K678045</a>)</li>
 +
<li><b>Device <a href="http://partsregistry.org/Part:BBa_K678060">BBa_K678060</a></b>:</br> plasmid_fun (<a href="http://partsregistry.org/Part:BBa_K678046">BBa_K678046</a>) + gpdA (<a href="http://partsregistry.org/Part:BBa_K6780242">BBa_K6780242</a>) + GFP_module (<a href="http://partsregistry.org/Part:BBa_K678027">BBa_K678027</a>) + TtrpC (<a href="http://partsregistry.org/Part:BBa_K678036">BBa_K678036</a>) + pyrG (<a href="http://partsregistry.org/Part:BBa_K678040">BBa_K678040</a>)</li>
 +
<li><b>Device <a href="http://partsregistry.org/Part:BBa_K678061">BBa_K678061</a></b>:<br> plasmid_fun (<a href="http://partsregistry.org/Part:BBa_K678046">BBa_K678046</a>) + gpdA (<a href="http://partsregistry.org/Part:BBa_K6780242">BBa_K6780242</a>) + GFP_PTS1_module_fun (<a href="http://partsregistry.org/Part:BBa_K678033">BBa_K678033</a>) + TtrpC (<a href="http://partsregistry.org/Part:BBa_K678036">BBa_K678036</a>) + pyrG (<a href="http://partsregistry.org/Part:BBa_K678040">BBa_K678040</a>)</li>
 +
<li><b>Device <a href="http://partsregistry.org/Part:BBa_K678062">BBa_K678062</a></b>:<br> plasmid_fun (<a href="http://partsregistry.org/Part:BBa_K678046">BBa_K678046</a>) + gpdA (<a href="http://partsregistry.org/Part:BBa_K6780242">BBa_K6780242</a>) + RFP_module (<a href="http://partsregistry.org/Part:BBa_K678030">BBa_K678030</a>) + TtrpC (<a href="http://partsregistry.org/Part:BBa_K678036">BBa_K678036</a>) + pyrG (<a href="http://partsregistry.org/Part:BBa_K678040">BBa_K678040</a>)</li>
 +
<li><b>Device <a href="http://partsregistry.org/Part:BBa_K678063">BBa_K678063</a></b>:<br> plasmid_fun (<a href="://partsregistry.org/Part:BBa_K678046">BBa_K678046</a>) + gpdA (<a href="http://partsregistry.org/Part:BBa_K6780242">BBa_K6780242</a>) + RFP_NLS_module (<a href="http://partsregistry.org/Part:BBa_K678034">BBa_K678034</a>) + TtrpC (<a href="http://partsregistry.org/Part:BBa_K678036">BBa_K678036</a>) + pyrG (<a href="http://partsregistry.org/Part:BBa_K678040">BBa_K678040</a>)</li>
 +
<li><b>Device <a href="http://partsregistry.org/Part:BBa_K678064">BBa_K678064</a></b>:<br> plasmid_fun (<a href="http://partsregistry.org/Part:BBa_K678046">BBa_K678046</a>) + gpdA (<a href="http://partsregistry.org/Part:BBa_K6780242">BBa_K6780242</a>) + MTS (<a href="http://partsregistry.org/Part:BBa_K678025">BBa_K678025</a>) + GFP_TS (<a href="http://partsregistry.org/Part:BBa_K678029">BBa_K678029</a>) + TtrpC (<a href="http://partsregistry.org/Part:BBa_K678036">BBa_K678036</a>) + pyrG (<a href="http://partsregistry.org/Part:BBa_K678040">BBa_K678040</a>)</li>
 +
<li><b>Device <a href="http://partsregistry.org/Part:BBa_K678066">BBa_K678066</a></b>:<br> plasmid_fun (<a href="http://partsregistry.org/Part:BBa_K678046">BBa_K678046</a>) + DMKP_P6 (<a href="http://partsregistry.org/Part:BBa_K678000">BBa_K678000</a>) + GFP_GOI (<a href="http://partsregistry.org/Part:BBa_K678028">BBa_K678028</a>) + RFP_TS (<a href="http://partsregistry.org/Part:BBa_K678032">BBa_K678032</a>) + T2 (<a href="http://partsregistry.org/Part:BBa_K678038">BBa_K678038</a>) + bleR (<a href="http://partsregistry.org/Part:BBa_K678044">BBa_K678044</a>)</li>
-
= Lab notes =
+
<li><b>Device <a href="http://partsregistry.org/Part:BBa_K678067">BBa_K678067</a></b>:<br> plasmid_fun (<a href="http://partsregistry.org/Part:BBa_K678046">BBa_K678046</a>) + pAlc (<a href="http://partsregistry.org/Part:BBa_K678001">BBa_K678001</a>) + RFP_GOI (<a href="http://partsregistry.org/Part:BBa_K678031">BBa_K678031</a>) + RFP_TS (<a href="http://partsregistry.org/Part:BBa_K678032">BBa_K678032</a>) + T3 (<a href="http://partsregistry.org/Part:BBa_K678039">BBa_K678039</a>) + pyrG_DR (<a href="http://partsregistry.org/Part:BBa_K678041">BBa_K678041</a>)</li>
-
=='''June'''==
+
<li><b>Device <a href="http://partsregistry.org/Part:BBa_K678049">BBa_K678049</a></b>:<br> plasmid_mam (<a href="http://partsregistry.org/Part:BBa_K678023">BBa_K678023</a>) + CMV (<a href="http://partsregistry.org/Part:BBa_J52034">BBa_J52034</a>) + eGFP_Module (<a href="http://partsregistry.org/Part:BBa_K678006">BBa_K678006</a>) + BGH pA (<a href="http://partsregistry.org/Part:BBa_K678019">BBa_K678019</b>) + Hygromycin (<a href="http://partsregistry.org/Part:BBa_K678021">BBa_K678021</a>)</li>
-
<br />
+
 
-
=='''July'''==
+
<li><b>Device <a href="http://partsregistry.org/Part:BBa_K678050">BBa_K678050</a></b>:<br> plasmid_mam (<a href="http://partsregistry.org/Part:BBa_K678023">BBa_K678023</a>) + CMV (<a href="http://partsregistry.org/Part:BBa_J52034">BBa_J52034</a>) + GFP_PTS1_module_mam (<a href="http://partsregistry.org/Part:BBa_K678017">BBa_K678017</a>) + BGH pA (<a href="http://partsregistry.org/Part:BBa_K678019">BBa_K678019</a>) + Hygromycin (<a href="http://partsregistry.org/Part:BBa_K678021">BBa_K678021</a>)</li>
-
===Tuesday 19.07.2011===
+
 
-
First day in the lab and 48 biobricks to go, how exciting!
+
<li><b>Device <a href="http://partsregistry.org/Part:BBa_K678054">BBa_K678054</a></b>:<br> plasmid_mam (<a href="http://partsregistry.org/Part:BBa_K678023">BBa_K678023</a>) + PGK (<a href="http://partsregistry.org/Part:BBa_K678004">BBa_K678004</a>) + eGFP+K_GOI (<a href="http://partsregistry.org/Part:BBa_K678007">BBa_K678007</a>) + eGFP_TS (<a href="http://partsregistry.org/Part:BBa_K678008">BBa_K678008</a>) + hGH_polyA (<a href="http://partsregistry.org/Part:BBa_K678018">BBa_K678018</a>) + Neomycin (<a href="http://partsregistry.org/Part:BBa_K678022">BBa_K678022</a>)</li>
 +
 
 +
</ul>
<br>
<br>
 +
 +
 +
 +
<a name="Tuesday 16.08.2011"></a><h4>Tuesday</h4>
 +
We transfected U2OS cells with plasmides containing GFP and placed them in the incubator overnight.
<br>
<br>
 +
<br>
 +
<b>USER cloning and transformation in <i>E.coli </i> today: </b>
 +
<br>
 +
<ul>
 +
<li><b>Device <a href="http://partsregistry.org/Part:BBa_K678051">BBa_K678051</a></b>:<br> plasmid_mam (<a href="http://partsregistry.org/Part:BBa_K678023">BBa_K678023</a>) + CMV (<ahref="http://partsregistry.org/Part:BBa_J52034">BBa_J52034</a>) + eYFP_module (<a href="http://partsregistry.org/Part:BBa_K678009">BBa_K678009</a>) + BGH pA (<a href="http://partsregistry.org/Part:BBa_K678019">BBa_K678019</a>) + Hygromysin (<a href="http://partsregistry.org/Part:BBa_K678021">BBa_K678021</a>)</li>
-
Of the 48 biobricks we will have when the project is finished we had only established 17 of them and ordered primers. We started the day with 11 PCR reactions of the first bash of 17 biobricks, we still need some DNA templates to due all 17. 6 PCR were correct and was purified with a purification Kit (GE Healthcare). So only 42 biobricks to go!
+
<li><b>Device <a href="http://partsregistry.org/Part:BBa_K678052">BBa_K678052</a></b>:<br> plasmid_mam (<a href="http://partsregistry.org/Part:BBa_K678023">BBa_K678023</a>) + CMV (<a href="http://partsregistry.org/Part:BBa_J52034">BBa_J52034</a>) + mCherry_module (<a href="http://partsregistry.org/Part:BBa_K678020">BBa_K678020</a>) + BGH pA (<a href="http://partsregistry.org/Part:BBa_K678019">BBa_K678019</a>) + Hygromysin (<a href="http://partsregistry.org/Part:BBa_K678021">BBa_K678021</a>)</li>
-
'''Biobricks in the bag:'''
+
<li><b>Device <a href="http://partsregistry.org/Part:BBa_K678053">BBa_K678053</a></b>:  <br>plasmid_mam (<a href="http://partsregistry.org/Part:BBa_K678023">BBa_K678023</a>) + CMV (<a href="http://partsregistry.org/Part:BBa_J52034">BBa_J52034</a>) + eCFP_module (<a href="http://partsregistry.org/Part:BBa_K678015">BBa_K678015</a>) + BGH pA (<a href="http://partsregistry.org/Part:BBa_K678019">BBa_K678019</a>) + Hygromysin (<a href="http://partsregistry.org/Part:BBa_K678021">BBa_K678021</a>)</li>
-
*pALC
+
<li><b>Device <a href="http://partsregistry.org/Part:BBa_K678055">BBa_K678055</a></b>:<br>  plasmid_mam (<a href="http://partsregistry.org/Part:BBa_K678023">BBa_K678023</a>) + PGK (<a href="http://partsregistry.org/Part:BBa_K678004">BBa_K678004</a>) + eYFP_GOI (<a href="http://partsregistry.org/Part:BBa_K678010">BBa_K678010</a>) + eYFP_TS (<a href="http://partsregistry.org/Part:BBa_K678011">BBa_K678011</a>) + hGH_polyA (<a href="http://partsregistry.org/Part:BBa_K678018">BBa_K678018</a>) + Neomycin (<a href="http://partsregistry.org/Part:BBa_K678022">BBa_K678022</a>)</li>
-
*TtrpC
+
-
*Terminator 1
+
-
*Terminator 2
+
-
*Terminator 3
+
-
*argB
+
 +
<li><b>Device <a href="http://partsregistry.org/Part:BBa_K678056">BBa_K678056</a></b>:<br>  plasmid_mam (<a href="http://partsregistry.org/Part:BBa_K678023">BBa_K678023</a>) + SV40+ori (<a href="http://partsregistry.org/Part:BBa_K678005">BBa_K678005</a>) + mCherry_GOI (<a href="http://partsregistry.org/Part:BBa_K678013">BBa_K678013</a>) + mCherry_TS (<a href="http://partsregistry.org/Part:BBa_K678014">BBa_K678014</a>) + SV40 pA (<a href="http://partsregistry.org/Part:BBa_K678012">BBa_K678012</a>) + Neomycin (<a href="http://partsregistry.org/Part:BBa_K678022">BBa_K678022</a>)</li>
-
***Insert GEL Photo
+
<li><b>Device <a href="http://partsregistry.org/Part:BBa_K678057">BBa_K678057</a></b>:<br>  plasmid_mam (<a href="http://partsregistry.org/Part:BBa_K678023">BBa_K678023</a>) + Sv40+ori (<a href="http://partsregistry.org/Part:BBa_K678005">BBa_K678005</a>) + eCFP_GOI (<a href="http://partsregistry.org/Part:BBa_K678048">BBa_K678048</a>) + eCFP_TS (<a href="http://partsregistry.org/Part:BBa_K678016">BBa_K678016</a>) + SV40 pA (<a href="http://partsregistry.org/Part:BBa_K678012">BBa_K678012</a>) + Neomycin (<a href="http://partsregistry.org/Part:BBa_K678022">BBa_K678022</a>)</li>
 +
 
 +
<li><b>Device <a href="http://partsregistry.org/Part:BBa_K678059">BBa_K678059</a></b>:<br>  plasmid_fun (<a href="http://partsregistry.org/Part:BBa_K678046">BBa_K678046</a>) + Palc (<a href="http://partsregistry.org/Part:BBa_K678001">BBa_K678001</a>) + LacZ (<a href="http://partsregistry.org/Part:BBa_K678026">BBa_K678026</a>) + T1 (<a href="http://partsregistry.org/Part:BBa_K678037">BBa_K678037</a>) + ptrA (<a hrf="http://partsregistry.org/Part:BBa_K678045">BBa_K678045</a>)</li>
 +
 
 +
</ul>
 +
<br>
 +
<a name="Wednesday 17.08.2011"></a><h4>Wednesday</h4>
 +
<br>
 +
Inoculations of <i>E.coli </i> transformants with USER devices cloned yesterday.
 +
<br>
 +
<br>
 +
Restriction analysis of device 4, 7, 8, and 27.
<br>
<br>
-
===Thursday 21.07.2011===
+
<br>
 +
<b>USER cloning and transformation in <i>E.coli </i> today: </b>
-
Some of the PCR reactions that didn't work Tuesday was repeated with a different annealing temperature and amplification of new biobricks.
+
<ul>
-
+
<li><b>Device <a href="http://partsregistry.org/Part:BBa_K678058">BBa_K678058</a></b>:<br> plasmid_fun (<a href="http://partsregistry.org/Part:BBa_K678046">BBa_K678046</a>) + pAlc (<a href="http://partsregistry.org/Part:BBa_K678001">BBa_K678001</a>) + LacZ (<a href="http://partsregistry.org/Part:BBa_K678026">BBa_K678026</a>) + T1 (<a href="http://partsregistry.org/Part:BBa_K678037">aBBa_K678037</a>) + argB (<a href="http://partsregistry.org/Part:BBa_K678043">BBa_K678043</a>)</li>
 +
<li><b>Device <a href="http://partsregistry.org/Part:BBa_K678059">BBa_K678059</a></b>:<br> plasmid_fun (<a href="http://partsregistry.org/Part:BBa_K678046">BBa_K678046</a>) + PAlc (<a href="http://partsregistry.org/Part:BBa_K678001">BBa_K678001</a>) + LacZ (<a href="http://partsregistry.org/Part:BBa_K678026">BBa_K678026</a>) + T1 (<a href="http://partsregistry.org/Part:BBa_K678037">BBa_K678037</a>) + ptrA (<a href="http://partsregistry.org/Part:BBa_K678045">BBa_K678045</a></li>
-
'''New biobricks in the bag:'''
+
<li><b>Device <a href="http://partsregistry.org/Part:BBa_K678060">BBa_K678060</a></b>: <br>plasmid_fun (<a href="http://partsregistry.org/Part:BBa_K678046">BBa_K678046</a>) + gpdA (<a href="http://partsregistry.org/Part:BBa_K6780242">BBa_K6780242</a>) + GFP_module (<a href="http://partsregistry.org/Part:BBa_K678027">BBa_K678027</a>) + TtrpC (<a href="http://partsregistry.org/Part:BBa_K678036">BBa_K678036</a>) + pyrG (<a href="http://partsregistry.org/Part:BBa_K678040">BBa_K678040</a>)</li>
-
** Insert of gel photo
+
<li><b>Device <a href="http://partsregistry.org/Part:BBa_K678061">BBa_K678061</a></b>:<br> plasmid_fun (<a href="http://partsregistry.org/Part:BBa_K678046">BBa_K678046</a>) + gpdA (<a href="http://partsregistry.org/Part:BBa_K6780242">BBa_K6780242</a>) + GFP_PTS1_module_fun (<a href="http://partsregistry.org/Part:BBa_K678033">BBa_K678033</a>) + TtrpC (<a href="http://partsregistry.org/Part:BBa_K678036">BBa_K678036</a>) + pyrG (<a href="http://partsregistry.org/Part:BBa_K678040">BBa_K678040</a>)</li>
-
*PGK
+
<li><b>Device <a href="http://partsregistry.org/Part:BBa_K678062">BBa_K678062</a></b>: <br>plasmid_fun (<a href="http://partsregistry.org/Part:BBa_K678046">BBa_K678046</a>) + gpdA (<a href="http://partsregistry.org/Part:BBa_K6780242">BBa_K6780242</a>) + RFP_module (<a href="http://partsregistry.org/Part:BBa_K678030">BBa_K678030</a>) + TtrpC (<a href="http://partsregistry.org/Part:BBa_K678036">BBa_K678036</a>) + pyrG (<a href="http://partsregistry.org/Part:BBa_K678040">BBa_K678040</a>)</li>
-
*BGHpA
+
-
*eGFP+k_GOI
+
-
The fragments will be frozen and purified later.  
+
<li><b>Device <a href="http://partsregistry.org/Part:BBa_K678063">BBa_K678063</a></b>:<br> plasmid_fun (<a href="http://partsregistry.org/Part:BBa_K678046">BBa_K678046</a>) + gpdA (<a href="http://partsregistry.org/Part:BBa_K6780242">BBa_K6780242</a>) + RFP_NLS_module (<a href="http://partsregistry.org/Part:BBa_K678034">BBa_K678034</a>) + TtrpC (<a href="http://partsregistry.org/Part:BBa_K678036">BBa_K678036</a>) + pyrG (<a href="http://partsregistry.org/Part:BBa_K678040">BBa_K678040</a>)</li>
 +
<li><b>Device <a href="http://partsregistry.org/Part:BBa_K678064">BBa_K678064</a></b>: <br>plasmid_fun (<a href="http://partsregistry.org/Part:BBa_K678046">BBa_K678046</a>) + gpdA (<a href="http://partsregistry.org/Part:BBa_K6780242">BBa_K6780242</a>) + MTS (<a href="http://partsregistry.org/Part:BBa_K678025">BBa_K678025</a>) + GFP_TS (<a href="http://partsregistry.org/Part:BBa_K678029">BBa_K678029</a>) + TtrpC (<a href="http://partsregistry.org/Part:BBa_K678036">BBa_K678036</a>) + pyrG (<a href="http://partsregistry.org/Part:BBa_K678040">BBa_K678040</a>)</li>
-
We still have not succeed to make biobricks pyrG and pyrG-DR (use as a marker in fungi), despite two PCR attempts with a high annealing (59°) and low (56°) temperature.
+
<li><b>Device <a href="http://partsregistry.org/Part:BBa_K678066">BBa_K678066</a></b>:<br> plasmid_fun (<a href="http://partsregistry.org/Part:BBa_K678046">BBa_K678046</a>) + DMKP_P6 (<a href="http://partsregistry.org/Part:BBa_K678000">BBa_K678000</a>) + GFP_GOI (<a href="http://partsregistry.org/Part:BBa_K678028">BBa_K678028</a>) + RFP_TS (<a href="http://partsregistry.org/Part:BBa_K678032">BBa_K678032</a>) + T2 (<a href="http://partsregistry.org/Part:BBa_K678038">BBa_K678038</a>) + bleR (<a href="http://partsregistry.org/Part:BBa_K678044">BBa_K678044</a>)</li>
 +
<li><b>Device <a href="http://partsregistry.org/Part:BBa_K678067">BBa_K678067</a></b>:<br> plasmid_fun (<a href="http://partsregistry.org/Part:BBa_K678046">BBa_K678046</a>) + pAlc (<a href="http://partsregistry.org/Part:BBa_K678001">BBa_K678001</a>) + RFP_GOI (<a href="http://partsregistry.org/Part:BBa_K678031">BBa_K678031</a>) + RFP_TS (<a href="http://partsregistry.org/Part:BBa_K678032">BBa_K678032</a>) + T3 (<a href="http://partsregistry.org/Part:BBa_K678039">BBa_K678039</a>) + pyrG_DR (<a href="http://partsregistry.org/Part:BBa_K678041">BBa_K678041</a>)</li>
-
'''Work for iGEM Copenhagen team'''
+
<li>Device 31(Copenhagen): <br>plasmid_mam (<a href="http://partsregistry.org/Part:BBa_K678023">BBa_K678023</a>) + pIPTG (<a href="http://partsregistry.org/Part:BBa_R0010">BBa_R0010</a>) + 1A2 (<b>C7</b>) + Terminator (<a href="http://partsregistry.org/Part:BBa_B0015">BBa_B0015</a>) + amp_cas (<a href="http://partsregistry.org/Part:BBa_K678047">BBa_K678047</a>)</li>
-
We have established a collaboration with iGEM Copenhagen team, where we will create some biobricks for them and they can use our plug 'n' play system and they will save a lot of time.  
+
<li>Device 32 (Copenhagen): <br>plasmid_mam (<a href="http://partsregistry.org/Part:BBa_K678023">BBa_K678023</a>) + pIPTG (<a href="http://partsregistry.org/Part:BBa_R0010">BBa_R0010</a>) + 2C9 (<b>C8</b>) + Terminator (<a href="http://partsregistry.org/Part:BBa_B0015">BBa_B0015</a>) + amp_cas (<a href="http://partsregistry.org/Part:BBa_K678047">BBa_K678047</a>)</li>
-
So today we transformed competent E.coli cells with some of the standard biological parts from iGEM, so we later take make biobricks to match our system.
+
 
 +
<li>Device 7 (Copenhagen):<br> plasmid_mam (<a href="http://partsregistry.org/Part:BBa_K678023">BBa_K678023</a>) + pIPTG (<a href="http://partsregistry.org/Part:BBa_R0010">BBa_R0010</a>) + CYP79A2 (<a href="http://partsregistry.org/Part:BBa_K527001">BBa_K527001</a>) + Terminator (<a href="http://partsregistry.org/Part:BBa_B0015">BBa_B0015</a>) + amp_cas (<a href="http://partsregistry.org/Part:BBa_K678047">BBa_K678047</a>)</li>
 +
 
 +
<li>Device 8 (Copenhagen): <br>plasmid_mam (<a href="http://partsregistry.org/Part:BBa_K678023">BBa_K678023</a>) + pIPTG (<a href="http://partsregistry.org/Part:BBa_R0010">BBa_R0010</a>) + CYP79B2 (<a href="http://partsregistry.org/Part:BBa_K527002">BBa_K527002</a>) + Terminator (<a href="http://partsregistry.org/Part:BBa_B0015">BBa_B0015</a>) + amp_cas (<a href="http://partsregistry.org/Part:BBa_K678047">BBa_K678047</a>)</li>
 +
 
 +
</ul>
<br>
<br>
 +
<br>
 +
Mini-prep purifications and restriction analysis of plasmids from transformed <i>E.coli </i>.
-
===Friday 22.07.2011===
+
<a name="Thursday 18.08.2011"></a><h4>Thursday</h4>
-
===Monday 25.07.2011===
+
The coverslips covered with transfected U2OS cells were washed and fixated. We put the coverslips upside down on a glass slide and fixated them with nailpolish (very high fashion). We took them on a little journey to building 301 to get confocal pictures of them - and they looked great and green.
-
===Tuesday 26.07.2011===
+
<br>
-
=== Wednesday 27.07.2011===
+
<br>
 +
Transformation of <i>Aspergillus nidulans</i> for promotor characterization.
 +
<br>
 +
<br>
 +
<a name="Friday 19.08.2011"></a><h4>Friday</h4>
 +
<br>
 +
<b>USER cloning and transformation in <i>E.coli </i> today: </b>
 +
<br>
 +
<ul>
 +
<li><b>Device <a href="http://partsregistry.org/Part:BBa_K678066">BBa_K678066</a></b>:<br> plasmid_fun (<a href="http://partsregistry.org/Part:BBa_K678046">BBa_K678046</a>) + DMKP_P6 (<a href="http://partsregistry.org/Part:BBa_K678000">BBa_K678000</a>) + GFP_GOI (<a href="http://partsregistry.org/Part:BBa_K678028">BBa_K678028</a>) + RFP_TS (<a href="http://partsregistry.org/Part:BBa_K678032">BBa_K678032</a>) + T2 (<a href="http://partsregistry.org/Part:BBa_K678038">BBa_K678038</a>) + bleR (<a href="http://partsregistry.org/Part:BBa_K678044">BBa_K678044</a>)</li>
-
=='''August'''==
+
<li><b>Device <a href="http://partsregistry.org/Part:BBa_K678060">BBa_K678060</a></b>: <br>plasmid_fun (<a href="http://partsregistry.org/Part:BBa_K678046">BBa_K678046</a>) + pgdA (<a href="http://partsregistry.org/Part:BBa_K678024">BBa_K678024</a>) + GFP_module (<a href="http://partsregistry.org/Part:BBa_K678027">BBa_K678027</a>) + TtrpC (<a href="http://partsregistry.org/Part:BBa_K678036">BBa_K678036</a>) + pyrG (<a href="http://partsregistry.org/Part:BBa_K678040">BBa_K678040</a>)</li>
-
==='''Thursday 04.08.2011 ''' ===
+
<li><b>Device <a href="http://partsregistry.org/Part:BBa_K678068">BBa_K678068</a></b>:<br> plasmid_fun (<a href="http://partsregistry.org/Part:BBa_K678046">BBa_K678046</a>) + PAlc (<a href="http://partsregistry.org/Part:BBa_K678001">BBa_K678001</a>) + yA (<a href="http://partsregistry.org/Part:BBa_K678035">BBa_K678035</a>) + RFP_TS (<a href="http://partsregistry.org/Part:BBa_K678032">BBa_K678032</a>) + TtrpC (<a href="http://partsregistry.org/Part:BBa_K678036">BBa_K678036</a>) + amp_cas (<a href="http://partsregistry.org/Part:BBa_K678047">BBa_K678047</a>)</li>
-
Today we purified 22 biobricks, which have been obtained over the last week of laboratory work with PCR. The purification of DNA was done by using the illustra GFX PCR DNA and Gel Band Purification Kit (GE Healthcare) according to manufactures manual for purification of DNA from TAE and TBE agarose gels protocol, see protocol section for more information.
+
<br>
 +
<br>
-
Today, we also started cultivation of the two mammalian cell lines, HeLa and U2OS. They were defrosted and cultivated in in DMEM medium containing serum, penicillin and streptomycin. Each cell line was then transferred to a 25 cm2 culture flask, see protocol section.
+
<a name="Week 8"></a><h3><b>Week 8: 22.08.2011 - 28.08.2011</b></h3>
 +
<br>
 +
<a name="Monday 22.08.2011"></a><h4>Monday</h4>
 +
The U2OS cells were splitted and passed on to a new culture flask and to coverslips placed in the bottom of 2 6-well-plates.
 +
<br>
 +
<br>
 +
<a name="Tuesday 23.08.2011"></a><h4>Tuesday</h4>
 +
The U2OS cells were transfected with all the mammalian plasmides.Hopefully, we will get some really cool pictures soon with cells expressing RFP, CFP, YFP, GFP, and GFP targeted to the peroxisomes.
 +
<br>
 +
<br>
 +
Measurements of plasmids containing our devices before sending them to DNA Technology for sequencing. The measurements were done with Qubit Fluorometer from Invitrogen.
 +
<br>
 +
<br>
 +
More restriction analysis of plasmids were done.
 +
<br>
 +
<br> 
 +
<a name="Wednesday 24.08.2011"></a><h4>Wednesday</h4>
 +
The coverslips covered with transfected cells were washed and fixated. We put the coverslips upside down on a glass slide and fixated them with nailpolish. We took them to building 301 for confocal microscopy. The cells looked great -everything worked = we are done with the mammalian cells. Hip hip hurra!
 +
<br>
 +
<br>
 +
Transformation of <i>Aspergillus nidulans</i> for proof of concept.
 +
<br>
 +
<br>
 +
<a name="September"></a><h2><b>September</b></h2>
-
*pyrG TEMP
+
<a name="Week 9"></a><h3><b>Week 9: 29.08.2011 - 04.09.2011</b></h3>
 +
<a name="Tuesday 30.08.2011"></a><h4>Tuesday</h4>
 +
Plasmids and primers were sent to DNA Technology for sequencing.
 +
<br>
 +
<br>
 +
 +
<a name="Wednesday 31.08.2011"></a><h4>Wednesday</h4>
 +
PCR reaction of the shipping plasmid were done. USER tails were applied to the shipping plasmid, so it can be easy and quick assembled with the 2 BioBricks and the device.
 +
<br>
 +
<br>
-
==='''Friday 05.08.2011'''===
+
<img src="https://static.igem.org/mediawiki/2011/a/a0/01.09.2011.png" height="270px" alt="""/>
-
==='''Monday 08.08.2011'''===
 
-
U2OS cells
 
-
HeLa cells
 
-
==='''Wednesday 10.08.2011'''===
+
<br>
-
U2OS cells
+
<br>
-
HeLa cells
+
<a name="Thursday 1.09.2011"></a><h4> Thursday </h4>
 +
USER cloning of the two promoters and device 12 with the shipping plasmid were done.
 +
<br>
 +
<br>
 +
Due to the extensive use of biobrick plasmid_fun(<b>28</b>) and plasmid_mam(<b>30</b>) we made some more. 
 +
<a name="Week 10"></a><h3><b>Week 10: 05.09.2011 - 11.09.2011</b></h3>
 +
<a name="Monday 05.09.2011"></a><h4> Monday </h4>
-
==='''Friday 12.08.2011'''===
+
Restriction analysis of shipping plasmid was made to ensure that the USER cloning were done correctly. The shipping plasmid was sent to sequencing.
-
The U2OS cells are splitted and passed on to two new 75 cm2 culture flasks.  
+
<br>
-
Hopefully, they will be ready to pass on to cover slips on monday.
+
<br>
 +
Inoculation of fungi for β-galactosidase, Bradford assay and Fluorescence microscope.
 +
<br>
 +
<br>
 +
<a name="Tuesday 06.09.2011"></a><h4> Tuesday</h4>
 +
Today we checked out our fungi in fluorescence microscope, a beautiful sight it was! We have now proven that our system works in fungi as well as in mammalian cells.  
 +
<br>
 +
<br>
-
==='''Monday 15.08.2011'''===
+
<a name="Wednesday 07.09.2011"></a><h4>Wednesday </h4>
-
The U2OS are splitted and passed on to coverslips placed in a 6-well plate and a new 75 cm2 flask. They are placed in the incubator overnight - and tomorrow they will be ready for transfection.
+
-
==='''Tuesday 16.08.2011'''===
+
A whole day was spent in the lab doing the β-galactosidase and Bradford assay for characterisation of our two promoters.
-
Transfection of U2OS cells.
+
<br>
 +
<br>
 +
<a name="Friday 09.09.2011"></a><h4>Friday</h4>
-
=='''September'''==
+
This might be one of the last iGEM days in lab. We are measuring the DNA concentration of the biobricks, so they can be sent to MIT next week.
-
<br />
+
<br>
 +
<br>
 +
<a name="October"></a><h2><b>October</b></h2>
 +
<a name="November"></a><h2><b>November</b></h2>
 +
</ul>
 +
</td>
 +
</tr>
 +
</table>
 +
</div>
-
=='''October'''==
 
-
===Amsterdam here we come!!===
 
-
=='''November'''==
 
-
===MIT here we come===
+
 
 +
 
 +
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Latest revision as of 16:49, 21 September 2011




Lab notes

July



Week 1: 04.07.2011 - 10.07.2011

First week! Time was spent on designing the Plug 'n' Play assembly system, figuring out what 48 biobricks to construct, and searching for appropriate DNA templates.

Week 2: 11.07.2011 - 17.07.2011

Wednesday

The first batch of primers have been ordered, so we are waiting in excitement for them to arrive, so we can start our work in the lab.

Week 3: 18.07.2011 - 24.07.2011

Tuesday

First day in the lab and 48 biobricks to go. This is exciting!
We plan to construct 48 new BioBricks. We have now established 17 of them and ordered primers for them with USER tails. We started the day with 11 PCR reactions. 6 of them were correct, and they were purified with a purification Kit (GE Healthcare). Only 42 biobricks to go!

Biobricks in the bag:


Thursday

The failed PCR reactions from tuesday were repeated. This time we used a different annealing temperature, and....IT WORKED!

New biobricks in the bag:

The fragments will be frozen and later purified.

We still have not succeed to make BioBricks pyrG and pyrG-DR (used as a marker in fungi), despite different PCR attempts with a high annealing (59°) and low (56°) temperature.

Work for iGEM Copenhagen team
We have established collaboration with the iGEM team from Copenhagen University. We will costumize a number of their BioBricks to use with our Plug 'n' Play system. This is a great way of showing how easy it is to costumize the Plug 'n' Play system for any application, and the Copenhagen team will save a lot of time. Today we transformed competent E.coli cells with some of the standard biological parts from iGEM, BBa_R0010, Bba_B0015 and BBa_J52034, and later we will make them fit the Plug 'n' Play with DNA system.

Friday

We made a PCR of a troublesome BioBrick again - but this time with a small modification. We made a PCR mix with and without MgCl2 50mM in the reactions. And...IT WORKED with MgCl2 50mM!

Biobrick obtained:

The fragments will be frozen and later purified .

Week 4: 25.07.2011 - 31.07.2011


Monday

We got a new delivery of primers from IDT today. They are to be diluted and stocked, before use. We have had some difficulty with obtaining 5 of our new biobricks; SV40+ori, SV40 pA, LacZ, pyrG and pyrG-DR. We ran a touch PCR program, where the annealing temperature ran from 55° to 65° for each sample in each cycle.

The PCR reactions were successful, and following biobricks are ready for Plug'n'Play:

Tuesday

The primers recieved yesterday will be used for amplification of many many many new biobricks today.

Out of 15 PCR reactions 5 succeeded:


Wednesday

Today we have run 8 reactions, 3 standard and 5 touch PCR reactions on some of the biobricks that we still have difficulties obtaining.

One succeeded:

August


Week 5: 01.08.2011 - 07.08.2011

Monday

New week, new energy, and lots of team spirit! Today 15 PCR reactions were conducted, including parts for iGEM Copenhagen team. 14 succeeded. The annealing temperature was increased to 63°, and it worked like magic!

New biobricks in the bag:

Wednesday

There are still 2 BioBricks that we haven't been able to obtain with PCR: pyrG and pyrG-DR.
Today we tried again with gradient PCR to find the optimal melting temperature, but no magic happened.

Thursday

22 BioBricks, which have been obtained with PCR during the last week of laboratory work, was purified today. The purification of DNA was done by using the Illustra GFX PCR DNA and Gel Band Purification Kit (GE Healthcare). The procedure was carried out according to the manufactures protocol for purification of DNA from TAE and TBE agarose gels, see protocol section for more information.

Despite several attempts we have not been able to obtain BioBrick pyrG and pyrG-DR. Therefore we made a test with well-known primers without USER-tails (sometimes these USER-tails can be tricky). This was done to verify if something was wrong with the DNA template. The gel electrophoresis did not show any bands, and thereby it revealed that the DNA template was the source of error. Problem solved!

We also started cultivation of the mammalian cell line, U2OS, which is derived from a patient with osteosarcoma (bone cancer). They were defrosted and cultivated in DMEM medium containing serum, penicillin and streptomycin. The cell line was then transferred to a 25 cm2 culture flask, see protocol section.

Week 5: 08.08.2011 - 14.08.2011


Monday

Today we made the first USER cloning, we are very excited to see if the system works the way, we want it to!
All our biobricks are divided into devices, see protocol for USER MIX.
Device 1,2,3,5 and 6 was not registrated later.
The following devices were cloned today:

PCR reactions done today:


After recieving the new DNA template we finally succeeded in getting biobricks pyrG and pyrG-DR.
The U2OS cell line was splitted and passed on to new culture flasks. They looked really lovely in the microscope, which bodes well for future confocal microscopy. They seem to thrive in the mammalian cell lab, and they are passed on every second or third day, depending on the initial cell concentration.

Tuesday

E.coli was inoculated with the plasmides done by USER cloning yesterday.

PCR done today:


Purification with GFX kit of the following biobricks:

USER cloning done today:

Wednesday

The U2OS cells was investigated in the microscope. They had a confluency of about 90%, an therefore ready to pass on to new culture flasks. This was done according to the protocol (see protocol for passing and maintenance of mammalian cells")

Friday

Today 13 PCR reaction were carried out, some parts were for characterisation of two promoters: pAlc and DMKP-P6, and the rest were parts for the Copenhagen iGEM team.





The U2OS cells were splitted and passed on to two new 75 cm2 culture flasks. Hopefully, they will be ready to pass on to cover slips on monday.

Week 7: 15.08.2011 - 21.08.2011


Monday

The U2OS cells were splitted and passed on to a new 75 cm2 culture flask and to coverslips placed in a 6-well plate. They were placed in the incubator overnight - and will be ready for transfection with plasmides tomorrow.
In preparation for characterization of two promoters with β-galactosidase assay the plasmid p68 cut with restriction enzyme.

Purification of PCR products with GFX kit.

9 PCR reactions were done today




USER cloning and transformation in E.coli today:

Tuesday

We transfected U2OS cells with plasmides containing GFP and placed them in the incubator overnight.

USER cloning and transformation in E.coli today:

Wednesday


Inoculations of E.coli transformants with USER devices cloned yesterday.

Restriction analysis of device 4, 7, 8, and 27.

USER cloning and transformation in E.coli today:

Mini-prep purifications and restriction analysis of plasmids from transformed E.coli .

Thursday

The coverslips covered with transfected U2OS cells were washed and fixated. We put the coverslips upside down on a glass slide and fixated them with nailpolish (very high fashion). We took them on a little journey to building 301 to get confocal pictures of them - and they looked great and green.

Transformation of Aspergillus nidulans for promotor characterization.

Friday


USER cloning and transformation in E.coli today:
  • Device BBa_K678066:
    plasmid_fun (BBa_K678046) + DMKP_P6 (BBa_K678000) + GFP_GOI (BBa_K678028) + RFP_TS (BBa_K678032) + T2 (BBa_K678038) + bleR (BBa_K678044)
  • Device BBa_K678060:
    plasmid_fun (BBa_K678046) + pgdA (BBa_K678024) + GFP_module (BBa_K678027) + TtrpC (BBa_K678036) + pyrG (BBa_K678040)
  • Device BBa_K678068:
    plasmid_fun (BBa_K678046) + PAlc (BBa_K678001) + yA (BBa_K678035) + RFP_TS (BBa_K678032) + TtrpC (BBa_K678036) + amp_cas (BBa_K678047)


    • Week 8: 22.08.2011 - 28.08.2011


    Monday

    The U2OS cells were splitted and passed on to a new culture flask and to coverslips placed in the bottom of 2 6-well-plates.

    Tuesday

    The U2OS cells were transfected with all the mammalian plasmides.Hopefully, we will get some really cool pictures soon with cells expressing RFP, CFP, YFP, GFP, and GFP targeted to the peroxisomes.

    Measurements of plasmids containing our devices before sending them to DNA Technology for sequencing. The measurements were done with Qubit Fluorometer from Invitrogen.

    More restriction analysis of plasmids were done.

    Wednesday

    The coverslips covered with transfected cells were washed and fixated. We put the coverslips upside down on a glass slide and fixated them with nailpolish. We took them to building 301 for confocal microscopy. The cells looked great -everything worked = we are done with the mammalian cells. Hip hip hurra!

    Transformation of Aspergillus nidulans for proof of concept.

    September

    Week 9: 29.08.2011 - 04.09.2011

    Tuesday

    Plasmids and primers were sent to DNA Technology for sequencing.

    Wednesday

    PCR reaction of the shipping plasmid were done. USER tails were applied to the shipping plasmid, so it can be easy and quick assembled with the 2 BioBricks and the device.



    Thursday

    USER cloning of the two promoters and device 12 with the shipping plasmid were done.

    Due to the extensive use of biobrick plasmid_fun(28) and plasmid_mam(30) we made some more.

    Week 10: 05.09.2011 - 11.09.2011

    Monday

    Restriction analysis of shipping plasmid was made to ensure that the USER cloning were done correctly. The shipping plasmid was sent to sequencing.

    Inoculation of fungi for β-galactosidase, Bradford assay and Fluorescence microscope.

    Tuesday

    Today we checked out our fungi in fluorescence microscope, a beautiful sight it was! We have now proven that our system works in fungi as well as in mammalian cells.

    Wednesday

    A whole day was spent in the lab doing the β-galactosidase and Bradford assay for characterisation of our two promoters.

    Friday

    This might be one of the last iGEM days in lab. We are measuring the DNA concentration of the biobricks, so they can be sent to MIT next week.

    October

    November

Retrieved from "http://2011.igem.org/Team:DTU-Denmark-2/Notebook/Lab_notes"