Team:Freiburg/Notebook/lab light

From 2013.igem.org

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</p>
</p>
 +
<p class="first_order"><a href="https://2013.igem.org/Team:Freiburg/Notebook/crrna"> Targeting </a></p>
<p class="first_order"><a href="https://2013.igem.org/Team:Freiburg/Notebook/lab_effector"> Effector </a></p>
<p class="first_order"><a href="https://2013.igem.org/Team:Freiburg/Notebook/lab_effector"> Effector </a></p>
-
<p class="first_order"><a class="active" href="https://2013.igem.org/Team:Freiburg/Notebook/induction"> Induction </a> </p>
+
<p class="first_order"><a class="active" href="https://2013.igem.org/Team:Freiburg/Notebook/induction"> Effector Control </a> </p>
<p class="second_order_note"> <a class="active" id="link" href="https://2013.igem.org/Team:Freiburg/Notebook/lab_light"> Light </a> </p>
<p class="second_order_note"> <a class="active" id="link" href="https://2013.igem.org/Team:Freiburg/Notebook/lab_light"> Light </a> </p>
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<p class="third_order"> <a href="#august"> August </a> </p>
<p class="third_order"> <a href="#august"> August </a> </p>
<p class="third_order"> <a href="#september"> September </a> </p>
<p class="third_order"> <a href="#september"> September </a> </p>
 +
<p class="third_order"> <a href="#oktober"> October </a> </p>
-
<p class="second_order_note"> <a id="link" href="https://2013.igem.org/Team:Freiburg/Notebook/lab_hormon"> Hormon </a> </p>
+
<p class="second_order_note"> <a id="link" href="https://2013.igem.org/Team:Freiburg/Notebook/lab_hormon"> Hormone </a> </p>
-
<p class="first_order"><a href="https://2013.igem.org/Team:Freiburg/Notebook/crrna"> Targeting </a></p>
 
-
<p class="first_order"><a href="https://2013.igem.org/Team:Freiburg/Notebook/method"> uniBAss </a></p>
 
<p class="first_order"><a href="https://2013.igem.org/Team:Freiburg/Notebook/modeling"> Modeling </a></p>
<p class="first_order"><a href="https://2013.igem.org/Team:Freiburg/Notebook/modeling"> Modeling </a></p>
-
<p class="first_order"><a href="https://2013.igem.org/Team:Freiburg/Notebook/standardisation"> Standardisation </a></p>
+
<p class="first_order"><a href="https://2013.igem.org/Team:Freiburg/Notebook/method"> uniBAss </a></p>
-
<p class="first_order"> <a id="link" href="https://2013.igem.org/Team:Freiburg/protocols"> Protocols </a> </p>
+
 
 +
<p class="first_order"><a href="https://2013.igem.org/Team:Freiburg/Notebook/standardisation"> Standardization </a></p>
 +
<p class="first_order"> <a id="link" href="https://2013.igem.org/Team:Freiburg/protocols"> Material and Methods </a> </p>
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<div id="h1">
<div id="h1">
-
Induction - Light
+
Effector Control - Light
</div>
</div>
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       <h2> 9.8.2013 </h2>
       <h2> 9.8.2013 </h2>
-
      <h3> Midis of ... </h3>
 
       <h3> Test digest of pIG4100 </h3>
       <h3> Test digest of pIG4100 </h3>
       <h2> 10.8.2013 </h2>
       <h2> 10.8.2013 </h2>
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                           <p>Spectroscopic measurement every minute for 120 times (2h); wavelenght 405 nm.</p>
                           <p>Spectroscopic measurement every minute for 120 times (2h); wavelenght 405 nm.</p>
                            
                            
-
<td/> <img class="gelpic" src="https://static.igem.org/mediawiki/2013/0/02/28light2_0_freiburg_13.png"> </td>
+
<td/> <img src="https://static.igem.org/mediawiki/2013/0/02/28light2_0_freiburg_13.png"> </td>
-
<td/> <img class="gelpic" src="https://static.igem.org/mediawiki/2013/e/e6/29light2_0_freiburg_13.png"> </td>
+
<td/> <img src="https://static.igem.org/mediawiki/2013/e/e6/29light2_0_freiburg_13.png"> </td>
                      
                      
                     <p> numbers 1-6 are first six rows of the UV-b light approach -> look at transfections from 15.8.2013. numbers 7-12 are rows 1-6 of the red-light transfections from 15.8.2013 </p>
                     <p> numbers 1-6 are first six rows of the UV-b light approach -> look at transfections from 15.8.2013. numbers 7-12 are rows 1-6 of the red-light transfections from 15.8.2013 </p>
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<h3> Oligo annealing </h3>
<h3> Oligo annealing </h3>
<p> Targets T2, T3, T4, T5, T6, T7 and Max's random target were annealed.</p>
<p> Targets T2, T3, T4, T5, T6, T7 and Max's random target were annealed.</p>
-
<td/> <img class="gelpic" src="https://static.igem.org/mediawiki/2013/c/c7/31light2_0_freiburg_13.png"> </td>
+
<td/> <img src="https://static.igem.org/mediawiki/2013/c/c7/31light2_0_freiburg_13.png"> </td>
<h3> Ligation of new RNA plasmid </h3>
<h3> Ligation of new RNA plasmid </h3>
<p> Digested pIG3010 (50,1 ng/µl) and the annealed oliges were ligated with 3 molar amount of Insert. </p>
<p> Digested pIG3010 (50,1 ng/µl) and the annealed oliges were ligated with 3 molar amount of Insert. </p>
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<td/> <img class="gelpic" src="https://static.igem.org/mediawiki/2013/f/f8/32light2_0_freiburg_13.png"> </td>
<td/> <img class="gelpic" src="https://static.igem.org/mediawiki/2013/f/f8/32light2_0_freiburg_13.png"> </td>
-
<td/> <img class="gelpic" src="https://static.igem.org/mediawiki/2013/f/f4/33light2_0_freiburg_13.png"> </td>
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<td/> <img src="https://static.igem.org/mediawiki/2013/f/f4/33light2_0_freiburg_13.png"> </td>
-
<td/> <img class="gelpic" src="https://static.igem.org/mediawiki/2013/c/c6/34light2_0_freiburg_13.png"> </td>
+
<td/> <img src="https://static.igem.org/mediawiki/2013/c/c6/34light2_0_freiburg_13.png"> </td>
<h2> 30.8.2013 </h2>
<h2> 30.8.2013 </h2>
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<h3> SEAP assay </h3>
<h3> SEAP assay </h3>
<p> SEAP assay of UV light activation was performed. </p>
<p> SEAP assay of UV light activation was performed. </p>
-
<td/> <img class="gelpic" src="https://static.igem.org/mediawiki/2013/b/b4/35light2_0_freiburg_13.png"> </td>
+
<td/> <img src="https://static.igem.org/mediawiki/2013/b/b4/35light2_0_freiburg_13.png"> </td>
<div id="september">
<div id="september">
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</div>
</div>
-
<h2> 01.9.2013 </h2>
+
 
-
<h3> </h3>
+
-
<h2> 02.9.2013 </h2>
+
-
<h3> </h3>
+
<h2> 03.9.2013 </h2>
<h2> 03.9.2013 </h2>
<h3> Oligo annealing </h3>
<h3> Oligo annealing </h3>
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<h3> Transfection of HEKs for UV & BL activation and repression</h3>
<h3> Transfection of HEKs for UV & BL activation and repression</h3>
<p> HEK cells were transfected (standard protocol) at 19.30. Media was changed after 5 hours.</p>
<p> HEK cells were transfected (standard protocol) at 19.30. Media was changed after 5 hours.</p>
-
<td/> <img class="gelpic" src="https://static.igem.org/mediawiki/2013/2/23/36light2_0_freiburg_13.jpg"></td>
+
<td/> <img src="https://static.igem.org/mediawiki/2013/2/23/36light2_0_freiburg_13.jpg"></td>
-
<td/> <img class="gelpic" src="https://static.igem.org/mediawiki/2013/f/ff/37light2_0_freiburg_13.jpg"></td>
+
<td/> <img src="https://static.igem.org/mediawiki/2013/f/ff/37light2_0_freiburg_13.jpg"></td>
-
<td/> <img class="gelpic" src="https://static.igem.org/mediawiki/2013/5/55/38light2_0_freiburg_13.jpg"></td>
+
<td/> <img src="https://static.igem.org/mediawiki/2013/5/55/38light2_0_freiburg_13.jpg"></td>
-
<td/> <img class="gelpic" src="https://static.igem.org/mediawiki/2013/3/3e/39light2_0_freiburg_13.jpg"></td>
+
<td/> <img src="https://static.igem.org/mediawiki/2013/3/3e/39light2_0_freiburg_13.jpg"></td>
-
<td/> <img class="gelpic" src="https://static.igem.org/mediawiki/2013/8/86/40light2_0_freiburg_13.jpg"></td>
+
<td/> <img src="https://static.igem.org/mediawiki/2013/8/86/40light2_0_freiburg_13.jpg"></td>
-
<td/> <img class="gelpic" src="https://static.igem.org/mediawiki/2013/4/4d/41light2_0_freiburg_13.jpg"></td> <h3> T4 RNA plasmid</h3>
+
<td/> <img src="https://static.igem.org/mediawiki/2013/4/4d/41light2_0_freiburg_13.jpg"></td> <h3> T4 RNA plasmid</h3>
<p> 6 colonies were scratched out. </p>
<p> 6 colonies were scratched out. </p>
<h2> 09.09.2013 </h2>
<h2> 09.09.2013 </h2>
<h3> Minis of T4 </h3>
<h3> Minis of T4 </h3>
-
<p> 3 of 6 plasmids were sent for sequencing (1-3).
+
<p> 3 of 6 plasmids were sent for sequencing (1-3).
<h3> Light start </h3>
<h3> Light start </h3>
-
<p> Blue light was started at 21.30 with 450 nm and unsure intesity (4 colour box, 655 intensity number). Dark control is in a light box without light on.</p>
+
<p> Blue light was started at 21.30 with 450 nm and unsure intesity (4 colour box, 655 intensity  
-
<p> UV light was started at 20.30 Dark control is in the incubator (no UV light in the lab).</p>
+
 
 +
number). Dark control is in a light box without light on.</p>
 +
<p> UV light was started at 20.30. Dark control is in the incubator (no UV light in the lab).</p>
<h2>10.09.2013</h2>
<h2>10.09.2013</h2>
<h3>Sequencing results</h3>
<h3>Sequencing results</h3>
-
<p> Sequencing results were positive for Bla Target 4. Bla Targets 2 and 3 were cloned by Philipp who got positive results, too. Plasmids were retransformed and Midis were inoculated.<p>
+
<p> Sequencing results were positive for Bla Target 4. Bla Targets 2 and 3 were cloned by Philipp who  
 +
 
 +
got positive results, too. Plasmids were retransformed and Midis were inoculated.<p>
<h3>Seeding of HEK cells.</h3>
<h3>Seeding of HEK cells.</h3>
<p> 4 24 well plates were seeded with 65.000 HEK cells per well.</p>
<p> 4 24 well plates were seeded with 65.000 HEK cells per well.</p>
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<h2>11.09.2013</h2>
<h2>11.09.2013</h2>
<h3> Midis </h3>
<h3> Midis </h3>
-
<p> Midis of Bla target 2 (pIG4), Bla target 3 (pIG4, Bla target 4 (pIG4), EMX1 (pIG4 and VEGF4 (pIG4) were prepped.</p>
+
<p> Midis of Bla target 2 (pIG4), Bla target 3 (pIG4, Bla target 4 (pIG4), EMX1 (pIG4 and VEGF4  
 +
 
 +
(pIG4) were prepped.</p>
<h3> SEAP of UV and BL activation and repression</h3>
<h3> SEAP of UV and BL activation and repression</h3>
 +
 +
<div>
 +
<table class="imgtxt" width="500px">
 +
<tr>
 +
<td> <img class="imgtxt" width="500px"
 +
 +
src="https://static.igem.org/mediawiki/2013/4/4a/Light-seap-11-09-blue-freiburg-2013.PNG"> </td>
 +
</tr>
 +
<tr>
 +
<td> <b>Figure 1: SEAP measurement for blue light activation. </b><br>
 +
</td>
 +
</tr>
 +
</table>
 +
</div>
 +
 +
<div>
 +
<table class="imgtxt" width="500px">
 +
<tr>
 +
<td> <img class="imgtxt" width="500px"
 +
 +
src="https://static.igem.org/mediawiki/2013/8/83/Light-seap-11-09-uv-freiburg-2013.PNG"> </td>
 +
</tr>
 +
<tr>
 +
<td> <b>Figure 2: SEAP measurement for UV light activation.</b><br>
 +
</td>
 +
</tr>
 +
</table>
 +
</div>
<p> Activation was okay, repression was in overflow.</p>
<p> Activation was okay, repression was in overflow.</p>
<h3> Transfection for Activation-VP16 and Repression-KRAB target test row</h3>
<h3> Transfection for Activation-VP16 and Repression-KRAB target test row</h3>
-
<p> In order to test which target we should use, we made test rows with different targets and different target combinations. Medium was changed after 5 h.</p>
+
<p> In order to test which target we should use, we made test rows with different targets and  
-
<h4> Transfection plan</h4>
+
 
-
<td/> <img class="gelpic" src=""></td>
+
different target combinations. Medium was changed after 5 h.</p>
-
<h4> Transfection calculation</h4>
+
<div>
-
<td/> <img class="gelpic" src=""></td>
+
<table class="imgtxt" width="500px">
 +
<tr>
 +
<td> <img class="imgtxt" width="500px"
 +
 
 +
src="https://static.igem.org/mediawiki/2013/b/b1/Light-tranfection-plan-11-09-VP16-test1-freiburg-2013.PNG"> </td>
 +
</tr>
 +
<tr>
 +
<td> <b>Figure 3: Transfection plan: Target test row Activation 1</b><br>
 +
</td>
 +
</tr>
 +
</table>
 +
</div>
 +
<div>
 +
<table class="imgtxt" width="500px">
 +
<tr>
 +
<td> <img class="imgtxt" width="500px"
 +
 
 +
src="https://static.igem.org/mediawiki/2013/3/33/Light-tranfection-plan-11-09-VP16-test2-freiburg-2013.PNG"> </td>
 +
</tr>
 +
<tr>
 +
<td> <b>Figure 4: Transfection plan: Target test row Activation 1 </b><br>
 +
</td>
 +
</tr>
 +
</table>
 +
</div>
 +
<div>
 +
<table class="imgtxt" width="500px">
 +
<tr>
 +
<td> <img class="imgtxt" width="500px"
 +
 
 +
src="https://static.igem.org/mediawiki/2013/c/c7/Light-tranfection-plan-11-09-KRAB-test1-freiburg-2013.PNG"> </td>
 +
</tr>
 +
<tr>
 +
<td> <b>Figure 5: Transfection plan: Target test row repression 1. </b><br>
 +
</td>
 +
</tr>
 +
</table>
 +
</div>
 +
<div>
 +
<table class="imgtxt" width="500px">
 +
<tr>
 +
<td> <img class="imgtxt" width="500px"
 +
 
 +
src="https://static.igem.org/mediawiki/2013/5/5b/Light-tranfection-plan-11-09-KRAB-test2-freiburg-2013.PNG"> </td>
 +
</tr>
 +
<tr>
 +
<td> <b>Figure 6: Transfection plan: Target test row repression 2. </b><br>
 +
</td>
 +
</tr>
 +
</table>
 +
</div>
<h2>12.09.2013</h2>
<h2>12.09.2013</h2>
<h3> SEAP of Activation VP16 test row</h3>
<h3> SEAP of Activation VP16 test row</h3>
 +
<div>
 +
<table class="imgtxt" width="500px">
 +
<tr>
 +
<td> <img class="imgtxt" width="850px"
 +
 +
src="https://static.igem.org/mediawiki/2013/2/26/Light-seap-12-09-vp16-target-test-freiburg-2013.PNG"> </td>
 +
</tr>
 +
<tr>
 +
<td> <b>Figure 7: SEAP measurement for target test row for Cas9-VP16.</b><br>
 +
</td>
 +
</tr>
 +
</table>
 +
</div>
<h3> Seeding of HEK cells for light activation experiments</h3>
<h3> Seeding of HEK cells for light activation experiments</h3>
<h2>13.09.2013</h2>
<h2>13.09.2013</h2>
<h3> SEAP of Repression KRAB test row and UV and BL repression </h3>
<h3> SEAP of Repression KRAB test row and UV and BL repression </h3>
-
<p> Samples had to be diluted 1:10 because the constitutive CMV promoter is very strong.</p>
+
<p> Samples had to be diluted 1:10 because the constitutive CMV promoter is very strong. SEAP levels
-
<h3> Transfection for UV/BL/RL</h3>
+
 
 +
were still in overflow.</p>
 +
<h3> Transfection for UV/BL/RL activation</h3>
 +
<div>
 +
<table class="imgtxt" width="500px">
 +
<tr>
 +
<td> <img class="imgtxt" width="500px"
 +
 
 +
src="https://static.igem.org/mediawiki/2013/5/55/Light-tranfection-plan-13-09-blue-freiburg-2013.PNG"> </td>
 +
</tr>
 +
<tr>
 +
<td> <b>Figure 8: Transfection plan for blue light activation. plan</b><br>
 +
</td>
 +
</tr>
 +
</table>
 +
</div>
 +
<div>
 +
<table class="imgtxt" width="500px">
 +
<tr>
 +
<td> <img class="imgtxt" width="500px"
 +
 
 +
src="https://static.igem.org/mediawiki/2013/4/4d/Light-tranfection-plan-13-09-red-freiburg-2013.PNG"> </td>
 +
</tr>
 +
<tr>
 +
<td> <b>Figure 9: Transfection plan for red light activation.</b><br>
 +
</td>
 +
</tr>
 +
</table>
 +
</div>
 +
<div>
 +
<table class="imgtxt" width="500px">
 +
<tr>
 +
<td> <img class="imgtxt" width="500px"
 +
 
 +
src="https://static.igem.org/mediawiki/2013/8/8c/Light-tranfection-plan-13-09-UV-freiburg-2013.PNG"> </td>
 +
</tr>
 +
<tr>
 +
<td> <b>Figure 10: Transfection plan for UVB light activation.</b><br>
 +
</td>
 +
</tr>
 +
</table>
 +
</div>
<h2>14.09.2013</h2>
<h2>14.09.2013</h2>
<h3> Light start for UV/BL/RL</h3>
<h3> Light start for UV/BL/RL</h3>
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<h3> Light stop for UV and BL</h3>
<h3> Light stop for UV and BL</h3>
<h3> SEAP of UV and BL activation</h3>
<h3> SEAP of UV and BL activation</h3>
 +
<div>
 +
<table class="imgtxt" width="500px">
 +
<tr>
 +
<td> <img class="imgtxt" width="500px"
 +
 +
src="https://static.igem.org/mediawiki/2013/a/ab/Light-seap-13-09-blue-freiburg-2013.PNG"> </td>
 +
</tr>
 +
<tr>
 +
<td> <b>Figure 11: SEAP measurement for blue light activation.</b><br>
 +
 +
</td>
 +
</tr>
 +
</table>
 +
</div>
 +
<div>
 +
<table class="imgtxt" width="500px">
 +
<tr>
 +
<td> <img class="imgtxt" width="500px"
 +
 +
src="https://static.igem.org/mediawiki/2013/8/86/Light-seap-13-09-uv-freiburg-2013.PNG"> </td>
 +
</tr>
 +
<tr>
 +
<td> <b>Figure 12: SEAP measurement for UV light activation.</b><br>
 +
</td>
 +
</tr>
 +
</table>
 +
</div>
<h2>16.09.2013</h2>
<h2>16.09.2013</h2>
<h3> Light stop for RL</h3>
<h3> Light stop for RL</h3>
<h3> SEAP for RL</h3>
<h3> SEAP for RL</h3>
 +
<div>
 +
<table class="imgtxt" width="500px">
 +
<tr>
 +
<td> <img class="imgtxt" width="500px"
 +
 +
src="https://static.igem.org/mediawiki/2013/3/3e/Light-seap-16-09-red-freiburg-2013.PNG"> </td>
 +
</tr>
 +
<tr>
 +
<td> <b>Figure 13: SEAP measurement for red light activation.
 +
</b><br>
 +
 +
</td>
 +
</tr>
 +
</table>
 +
</div>
<h2>18.09.2013</h2>
<h2>18.09.2013</h2>
-
<h3> Medium change for CHOs</h3>
+
<h3> New charge of medium for CHOs</h3>
<p> Medium was complemented with FCS, Pen Strep and Gentamicin.</p>
<p> Medium was complemented with FCS, Pen Strep and Gentamicin.</p>
-
+
 
-
+
<h2>20.09.13</h2>
 +
<h3>New CHO-K1 & NIH cells</h3>
 +
<p> We received CHO-K1 cells from a freshly thawed cell batch. Thank you Shima from AG Ulbrich! These
 +
 
 +
cells were cultivated in DMEM.</p>
 +
<p> We received NIH cells.</p>
 +
<h2>24.09.13</h2>
 +
<h3>Seeding of CHO-K1 (from Toolbox) cells</h3>
 +
<p> 6 24 well plates were seeded with about 65.000 cells per well.</p>
 +
<h2>25.09.13</h2>
 +
<h3>Transfection for red/blue/UV light induction in CHO-K1 (from Toolbox)</h3>
 +
<p> CHO-K1 cells were transfected for red, blue and UV light induced gene activation. Cells that were
 +
 
 +
transfected for UV light were also transfected with Renilla. This was done for normalization, because UV light can kill the
 +
 
 +
cells.</p>
 +
<div>
 +
<table class="imgtxt" width="500px">
 +
<tr>
 +
<td> <img class="imgtxt" width="500px"
 +
 
 +
src="https://static.igem.org/mediawiki/2013/7/78/Light-tranfection-plan-25-09-blue-freiburg-2013.PNG"> </td>
 +
</tr>
 +
<tr>
 +
<td> <b>Figure 14: Transfection plan for blue light activation.</b><br>
 +
</td>
 +
</tr>
 +
</table>
 +
</div>
 +
<div>
 +
<table class="imgtxt" width="500px">
 +
<tr>
 +
<td> <img class="imgtxt" width="500px"
 +
 
 +
src="https://static.igem.org/mediawiki/2013/5/58/Light-tranfection-plan-25-09-red-freiburg-2013.PNG"> </td>
 +
</tr>
 +
<tr>
 +
<td> <b>Figure 15: Transfection plan for red light activation.</b><br>
 +
 
 +
</td>
 +
</tr>
 +
</table>
 +
</div>
 +
<div>
 +
<table class="imgtxt" width="500px">
 +
<tr>
 +
<td> <img class="imgtxt" width="500px"
 +
 
 +
src="https://static.igem.org/mediawiki/2013/2/2a/Light-tranfection-plan-25-09-UV-freiburg-2013.PNG"> </td>
 +
</tr>
 +
<tr>
 +
<td> <b>Figure 16: Transfection plan for UV light activation.</b><br>
 +
</td>
 +
</tr>
 +
</table>
 +
</div>
 +
<p> HTS medium was changed after 5 hours.</p>
 +
<h3>Seeding of NIH cells</h3>
 +
<p> 2 24 well plates were seeded with NIH cells (65.000 cells per well).</p>
 +
<h2>26.09.13</h2>
 +
<h3>Transfection for UV light induction in NIH cells</h3>
 +
<p> Transfection plan was the same as for CHOs on 25.09.</p>
 +
<h3>Light start for red/blue/UV light in CHO-K1 cells (from Toolbox)</h3>
 +
<p> 1 plate was placed in UV light (311 nm). 1 plate was placed in a red light box (660) after PCB
 +
 
 +
treatment. 1 plate was placed in a far-red light box (740 nm) after PCB treatment. One plate was placed in a blue light box
 +
 
 +
(460 nm).</p>
 +
<h2>27.09.13</h2>
 +
<h3> Light start for UV light in NIH cells </h3>
 +
<p> 1 plate of transfected NIH cells was placed in UV light.</p>
 +
<h3> Light stop for UV (CHO-K1 from toolbox) & blue light (CHO-K1) cells </h3>
 +
<p> 200 µl of supernatant of each well were removed and heated. After this the 96 well plate was
 +
 
 +
frozen (-20° C). SEAP measurement was done on the next day.</p>
 +
<h2>28.09.13</p>
 +
 
 +
<h3> SEAP measurement of UV (CHO-K1 from toolbox) & blue light (CHO-K1) cells</h3>
 +
 
 +
<div>
 +
<table class="imgtxt" width="700px">
 +
<tr>
 +
<td> <img class="imgtxt" width="700px"
 +
 
 +
src="https://static.igem.org/mediawiki/2013/f/f2/Light-seap-28-09-blue-freiburg-2013.PNG"> </td>
 +
</tr>
 +
<tr>
 +
<td> <b>Figure 17: SEAP measurement for blue light induced activation (CHO-K1)</b><br>
 +
</td>
 +
</tr>
 +
</table>
 +
</div>
 +
 
 +
 
 +
 
 +
<div>
 +
<table class="imgtxt" width="700px">
 +
<tr>
 +
<td> <img class="imgtxt" width="700px"
 +
 
 +
src="https://static.igem.org/mediawiki/2013/c/cb/Light-seap-28-09-uv-freiburg-2013.PNG"> </td>
 +
</tr>
 +
<tr>
 +
<td> <b>Figure 18: SEAP measurement for UVB light induced activation (CHO-K1).</b><br>
 +
</td>
 +
</tr>
 +
</table>
 +
</div>
 +
 
 +
<h3> Light stop for red light (CHO-K1 from toolbox) and UV light (NIH)</h3>
 +
<p> 200 µl of supernatant of each well were removed and heated. SEAP measurement was done according
 +
 
 +
to standard protocol.</p>
 +
<div>
 +
<table class="imgtxt" width="700px">
 +
<tr>
 +
<td> <img class="imgtxt" width="700px"
 +
 
 +
src="https://static.igem.org/mediawiki/2013/0/0b/Light-seap-28-09-red-freiburg-2013.PNG"> </td>
 +
</tr>
 +
<tr>
 +
<td> <b>Figure 19: SEAP measurement of red light induced activation (CHO-K1)</b><br>
 +
</td>
 +
</tr>
 +
</table>
 +
</div>
 +
<div>
 +
<table class="imgtxt" width="700px">
 +
<tr>
 +
<td> <img class="imgtxt" width="700px"
 +
 
 +
src="https://static.igem.org/mediawiki/2013/d/d0/Light-seap-28-09-uvNIH-freiburg-2013.PNG"> </td>
 +
</tr>
 +
<tr>
 +
<td> <b>Figure 20: SEAP measurement for UVB light induced activation (NIH).</b><br>
 +
</td>
 +
</tr>
 +
</table>
 +
</div>
 +
<h2>29.09.13</h2>
 +
<h3> Seeding of CHO-K1 from AG Ulbrich and NIH cells</h3>
 +
<p> 6 24 well plates with CHO-K1 were seeded and 6 24 well plates with NIH cells were seeded.</p>
 +
<h2>30.09.13</h2>
 +
<h3> Transfection of CHO-K1 & NIH cells - red, blue and UV light</h3>
 +
<p> Alltogether 12 24 well plates were transfected.</p>
 +
<h4> Transfection plans </h4>
 +
<div>
 +
<table class="imgtxt" width="500px">
 +
<tr>
 +
<td> <img class="imgtxt" width="500px"
 +
 
 +
src="https://static.igem.org/mediawiki/2013/5/55/Light-tranfection-plan-30-09-blue-freiburg-2013.PNG"> </td>
 +
</tr>
 +
<tr>
 +
<td> <b>Figure 21: Transfection plan: Blue light activation.</b><br>
 +
</td>
 +
</tr>
 +
</table>
 +
</div>
 +
<div>
 +
<table class="imgtxt" width="500px">
 +
<tr>
 +
<td> <img class="imgtxt" width="500px"
 +
 
 +
src="https://static.igem.org/mediawiki/2013/8/8f/Light-tranfection-plan-30-09-red-freiburg-2013.PNG"> </td>
 +
</tr>
 +
<tr>
 +
<td> <b>Figure 22: Transfection plan: Red light activation.</b><br>
 +
</td>
 +
</tr>
 +
</table>
 +
</div>
 +
<div>
 +
<table class="imgtxt" width="500px">
 +
<tr>
 +
<td> <img class="imgtxt" width="500px"
 +
 
 +
src="https://static.igem.org/mediawiki/2013/8/8e/Light-tranfection-plan-30-09-UV-freiburg-2013.PNG"> </td>
 +
</tr>
 +
<tr>
 +
<td> <b>Figure 23: Transfection plan: UVB light activation. </b><br>
 +
</td>
 +
</tr>
 +
</table>
 +
</div>
 +
<h4> transfection calculation </h4>
 +
<div>
 +
<table class="imgtxt" width="500px">
 +
<tr>
 +
<td> <img class="imgtxt" width="500px"
 +
 
 +
src="https://static.igem.org/mediawiki/2013/a/a5/Light-transfection-calc-30-09-blue1.PNG"> </td>
 +
</tr>
 +
<tr>
 +
<td> <b>Figure 24: Transfection calculation for blue light activation.</b><br>
 +
</td>
 +
</tr>
 +
</table>
 +
</div>
 +
 
 +
<div>
 +
<table class="imgtxt" width="500px">
 +
<tr>
 +
<td> <img class="imgtxt" width="500px"
 +
 
 +
src="https://static.igem.org/mediawiki/2013/7/7a/Light-transfection-calc-30-09-blue2.PNG"> </td>
 +
</tr>
 +
<tr>
 +
<td> <b>Figure 25: Transsfection calculation 2 for blue light activation. </b><br>
 +
</td>
 +
</tr>
 +
</table>
 +
</div>
 +
 
 +
<div>
 +
<table class="imgtxt" width="500px">
 +
<tr>
 +
<td> <img class="imgtxt" width="500px"
 +
 
 +
src="https://static.igem.org/mediawiki/2013/6/69/Light-transfection-calc-30-09-red1.PNG"> </td>
 +
</tr>
 +
<tr>
 +
<td> <b>Figure 26: Transfection calculation 1 for red light activation.</b><br>
 +
</td>
 +
</tr>
 +
</table>
 +
</div>
 +
 
 +
<div>
 +
<table class="imgtxt" width="500px">
 +
<tr>
 +
<td> <img class="imgtxt" width="500px"
 +
 
 +
src="https://static.igem.org/mediawiki/2013/d/d8/Light-transfection-calc-30-09-red2.PNG"> </td>
 +
</tr>
 +
<tr>
 +
<td> <b>Figure 27: Transfection calculation 2 for red light activation. </b><br>
 +
</td>
 +
</tr>
 +
</table>
 +
</div>
 +
 
 +
<div>
 +
<table class="imgtxt" width="500px">
 +
<tr>
 +
<td> <img class="imgtxt" width="500px"
 +
 
 +
src="https://static.igem.org/mediawiki/2013/8/85/Light-transfection-calc-30-09-uv1.PNG"> </td>
 +
</tr>
 +
<tr>
 +
<td> <b>Figure 28: Transfection calculation 1 for UVB light activation. </b><br>
 +
 
 +
</td>
 +
</tr>
 +
</table>
 +
</div>
 +
 
 +
<div>
 +
<table class="imgtxt" width="500px">
 +
<tr>
 +
<td> <img class="imgtxt" width="500px"
 +
 
 +
src="https://static.igem.org/mediawiki/2013/7/7e/Light-transfection-calc-30-09-uv2.PNG"> </td>
 +
</tr>
 +
<tr>
 +
<td> <b>Figure 29: Transfection calculation 2 for UVB light activation </b><br>
 +
</td>
 +
</tr>
 +
</table>
 +
</div>
 +
<div id="oktober">
 +
<p id="h2">
 +
October
 +
</p>
 +
</div>
 +
<h2>01.10.13</h2>
 +
<h3> Light start </h3>
 +
<p> Both transfected cell lines were illuminated: 1 CHO plate & 1 NIH plate with red light 660 nm, 1
 +
 
 +
CHO plate & 1 NIH plate with blue 465 nm and 1 CHO plate & 1 NIH plate with UV light 311 nm. For every cell line and light
 +
 
 +
system there was a dark control plate (red light's dark control is far red light of 740 nm because the system is reversible)
 +
 
 +
</p>
 +
<h2>02.10.13</h2>
 +
<h3> Light stop </h3>
 +
<p> Light was stoped for UV and blue light illumination (after 24 hours) (CHO and NIH cells). 200 µl
 +
 
 +
supernatant were removed from each well for SEAP measurements. SEAP measurement was done according to standard protocol.
 +
<div>
 +
<table class="imgtxt" width="700px">
 +
<tr>
 +
<td> <img class="imgtxt" width="700px" src="https://static.igem.org/mediawiki/2013/0/0a/2Freiburg-2013-SEAP-blue-activation-CHO.PNG"> </td>
 +
</tr>
 +
<tr>
 +
<td> <b>Figure 30: SEAP measurement for blue light activation in CHO-K1 cells.</b><br>
 +
HIER DANN ALLES ANDERE
 +
</td>
 +
</tr>
 +
</table>
 +
</div>
 +
<div>
 +
<table class="imgtxt" width="700px">
 +
<tr>
 +
<td> <img class="imgtxt" width="700px" src="https://static.igem.org/mediawiki/2013/2/23/2Freiburg-2013-SEAP-blue-activation-NIH.PNG"> </td>
 +
</tr>
 +
<tr>
 +
<td> <b>Figure 31: SEAP measurement for blue light activation in NIH cells.</b><br>
 +
 
 +
</td>
 +
</tr>
 +
</table>
 +
</div>
 +
<div>
 +
<table class="imgtxt" width="700px">
 +
<tr>
 +
<td> <img class="imgtxt" width="700px" src="https://static.igem.org/mediawiki/2013/9/92/3Freiburg-2013-SEAP-UV-activation-CHO.PNG"> </td>
 +
</tr>
 +
<tr>
 +
<td> <b>Figure 32: SEAP measurement for UVB light activation in CHO-K1 cells.</b><br>
 +
 +
</td>
 +
</tr>
 +
</table>
 +
</div>
 +
<div>
 +
<table class="imgtxt" width="700px">
 +
<tr>
 +
<td> <img class="imgtxt" width="700px" src="https://static.igem.org/mediawiki/2013/7/73/3Freiburg-2013-SEAP-UV-activation-NIH.PNG"> </td>
 +
</tr>
 +
<tr>
 +
<td> <b>Figure 33: SEAP measurement for UVB light activation in NIH cells.</b><br>
 +
</td>
 +
</tr>
 +
</table>
 +
</div>
 +
<h2>03.10.13</h2>
 +
<h3> Light stop </h3>
 +
<p> Light was stoped for red light illumantion. 200 µl supernatant were removed from each well for
 +
 
 +
SEAP measurements. SEAP measurement was done according to standard protocol.
 +
<div>
 +
<table class="imgtxt" width="700px">
 +
<tr>
 +
<td> <img class="imgtxt" width="700px" src="https://static.igem.org/mediawiki/2013/7/76/1Freiburg-2013-SEAP-red-activation-NIH.PNG"> </td>
 +
</tr>
 +
<tr>
 +
<td> <b>Figure 34: SEAP measurement for red light activation in NIH cells.</b><br>
 +
 +
</td>
 +
</tr>
 +
</table>
 +
</div>
 +
<div>
 +
<table class="imgtxt" width="700px">
 +
<tr>
 +
<td> <img class="imgtxt" width="700px" src="https://static.igem.org/mediawiki/2013/4/4b/1Freiburg-2013-SEAP-red-activation-CHO.PNG"> </td>
 +
</tr>
 +
<tr>
 +
<td> <b>Figure 35: SEAP measurement for red light activation in CHO-K1 cells.</b><br>
 +
 +
</td>
 +
</tr>
 +
</table>
 +
</div>
 +
 

Latest revision as of 05:16, 28 October 2013

Effector Control - Light

July

29.07.2013

Plasmids that we need as templates were transformed.

30.07.2013

Primers

Primers arrived today. Primers were diluted 1:10.

Minipreps

Transformed bacteria from yesterday with our template plasmids were minipreped.

  • pKM018: 83 ng/µl
  • pKM220: 216 ng/µl
  • pKM115: 177 ng/µl
  • pKM006: 118 ng/µl
  • pSAM200: 213 ng/µl
  • pSW42: 134 ng/µl
  • pSW45: 243 ng/µl
  • pIG2004: 212 ng/µl
  • T405: 213 ng/µl

Oligo Annealing of tetO crRNA

  • Oligos tetO13 1. CC and tetO13 2. CC were annealed
  • Therefore 20 µM diluted Primes were heated on 98° C for 4 minutes and then cooled down.

PCR list, program and composition

  • aIG4102: CMV Promotor from template T405 - Primers oIG4100F+R - 589 bp
  • aIG4103: Cas9 from template pIG2004 - Primers oIG4101F+R - 4170 bp
  • aIG4104: PIF and backbone from template pKM022- Primers oIG4102F+R - 3287 bp
  • aIG4105: KRAB from template pIG2004 - Primers oIG4103F+R - 390 bp
  • aIG4106: PhyB and backbone from template pKM018 - Primers oIG4104F+R - 4922 bp
  • aIG4107: UVR8 and backbone from template pKM168 - Primers oIG4105F+R - 3921 bp
  • aIG4108: COP1 and backbone frome template pKM115 - Primers oIG4106F+R - 3972 bp
  • aIG4109: CRY2 from template pSW42 - Primers oIG4107F+R - 1591 bp
  • aIG4110: Backbone from template pKM018 - Primers oIG4108F+R - 2924 bp
  • aIG4111: Backbone with VP16 from template pKM018 - Primers oIG4109F+R - 3282 bp
  • aIG4112: CIB from template pSW45 - Primers oIG4110F+R - 593 bp
  • aIG4113: Backbone from template pKM018 - Primers oIG4111F+R - 2881 bp
  • aIG4114: CIB from template pSW45 - Primers !! oIG4110F+oIG4112R - 638 bp
µl Substance
10 Q5-HF Reaction Buffer
200 ng Template
1 Primer forward
1 Primer reverse
4 dNTPs
1 DMSO
0,5 Q5-HF Polymerase
Add to 50 H2O
Temperature [° C] Time
98 5 Min
98 30s
60 (or optimized) 30
72 30-40/kb
72 10 Min
4 hold
  • 18 cycles

PCR results

  • aIG4102: 60° C - WORKED - GelExtraction: 87,9ng/ul
  • aIG4103: 60° C - 1 of 4 WORKED, Annealing Temperature lower? - GelExtraction: 50,5ng/ul Try 56°!
  • aIG4104: 60° C - DIDN'T WORK!!! Try with 58° - Was done, has to be tested on gel.
  • aIG4105: 60° C - WORKED - GelExtraction: 70,1ng/ul I and 57,7ng/ul II
  • aIG4106: 60° C - Was done, has to be tested on gel.
  • aIG4107: 60° C - ONLY SLIGHT BAND. TRY AGAIN!
  • aIG4108: 60° C - WRONG AMPLIFICATED BAND. Try again with different temperature!
  • aIG4109: 60° C - WORKED! - GelExtraction: 176,4ng/ul
  • aIG4110: 60° C - Was done, has to be tested on gel.
  • aIG4111: 60° C - WORKED! - GelExtraction: 84,4ng/ul
  • aIG4112: 60° C - Wasn't done yet.
  • aIG4113: 60° C - Was done, has to be tested on gel.
  • aIG4114: 60° C - Wan't done yet.

2-log Marker

aIG4102 (first 4) worked, 589 bp

aIG4105 (rest) worked, 390 bp

Roth Marker

aIG4103 only one PCR worked, 4170 bp

Roth Marker

aIG4104 didn't work

aIG4111 worked, 3282 bp

2-log Marker

aIG4109 (first two bands) worked, 1591 bp

aIG4107 (next two bands) didn't work, should have been 3921 bp

aIG4108 (one band) didnt work. Wrong band was amplified, should have been 3972 bp - another band of aIG4108 can be tested on a gel, didn't have space on this one.

31.07.13

PCR results

  • aIG4103: 54°-64° C - Everything WORKED!
  • aIG4104: 58° C - Gave three bands, upper band is supposed to be right. Was confirmed on a second gel with charging the gel only with a small amount of DNA.
  • aIG4106: 60° C - Didn't work. Try temperature gradient.
  • aIG4107: 54°-64° C - Everything Worked! But not very strong bands.
  • aIG4108: 54°-64° C - Didn't Work. Try seperate PCRs.
  • aIG4110: 60° C - Didn't work. Tried temperature gradient 54°-59° C - Worked with 55° and better with every additional ° C.
  • aIG4112: 60° C - Worked!
  • aIG4113: 60° C - Worked!
  • aIG4114: 60° C - Worked!

2-log marker

UPPER SIDE

aIG4103 (2 bands) - worked!

aIG4107 (2 bands) - worked!

aIG4106 (4 bands) - didn't work with 60° C, bands are too low!

LOWER SIDE

aIG4108 temperature gradient 54°-64° C - didn't work!

2-log marker

aIG4103 temperature gradient 54°-64° C - everything worked!

2-log marker

aIG4105 (2bands) - worked!

aIG4104 (2 bands) - upper band is considered to be the right band! - Worked!

aIG4110 - didn't work with 60° C.

2-log marker

aIG4107 temperature gradient 54°-64° C - worked, but only slight bands!

2-log marker

aIG4108 (2bands) - didn't work with 60° C!

aIG4113 (2 bands) - Worked!

aIG4110 - didn't work with 60° C.

2-log marker

aIG4110 (1 band) last column of temperature gradient (59° C) - worked!

aIG4112 (2 bands) - Worked!

aIG4114 - worked!.

2-log marker

aIG4110 temperature gradient (54°-59° C) - worked from 55° C upwards!

August

01.08.13

PCR results

  • aIG4104: Was gelexed and size was confirmed on a gel with a smaller amount of DNA. - Worked!
  • aIG4106: Temperature gradient 54° - 59° C and excess of reverse primer oIG4104R - Worked!
  • aIG4108: Seperate PCRs were tried out. Therefore the PCR was performed with template and at a time only one primer. After these PCRs (1x with forward Primer (58° C) and 1x with reverse Primer (62° C)) both columns were mixed and another PCR was performed. - Worked, but two bands appeared, upper band is considered to be the right fragment.

2-log marker

aIG4106 temperature gradient (54°-59° C) - worked!

2-log marker

aIG4108 seperate PCRs - worked, but other amplified products can be seen, too!

Gibsons

Fragments are mixed together to a total volume of 5µl and added to 15µl Gibson Master- Mix.

pIG4101: aIG4104- 0,83µl; aIG4103- 3,96µl; H2O- 0,21µl

pIG4103: aiG4107- 2,12µl; aiG4103- 2,88µl

pIG4105: aIG4110- 1,11µl; aIG4103- 3,51µl

pIG4106: aIG4111- 0,5µl; aIG4112- 0,3µl, H2O- 4,2ul

pIG4107: aIG4113- 0,4µl; aIG4105- 0,35µl; aIG4114- 0,35µl; H2O- 3,9µl

Gibson Mixes were transformed.

Ligation of pIG4100

Digest for pIG4100

µl type
10 DNA (pKM006 or oIG4102) ~ 1000 ng
4 Cut Smart Buffer
1 EcoRI HF
1 Nhe1 HF
Add to 40µl H2O
  • Temp.: 37°C
  • Incubation time: 1,5h

2-log marker

Digested aIG4102 with 601 bp

Digested pKM006 with 6393 bp and 126 bp.

Both were gelexed: aIG4102 19 ng/µl and pKM006 40 ng/µl

Ligation and Transformation of approach pIG4100

µl type
1 T4 Ligase
2 T4 Ligase buffer
3 fold molar amount of insert
2 Backbone pKM006
1,3 Insert aIG4102
13,7 H2O
  • Temp.: RT
  • Incubation time: 15 minutes

After the ligation, 2,5 µl if this approach were transformed into E.coli and scratched out.

02.08.13

Gibson & Ligation results

Colonies could be seen on every plate except for one of the two ligation plates. Clones were picked and scratched out -> 51 Minis

Gibson

pIG4102: aIG4105- 0,4µl; aIG4106- 2,00µl; H2O- 2,6 µl

pIG4104: aiG4108- 3,2µl; aiG4105- 0,5µl; H2O- 1,3µl

Gibson Mixes were transformed.

03.08.13

Gibson results

Colonies could be seen on one of two plates, each. Colonies were scratched out for mini preps. Therefore we had clones for every construct.

Minipreps

51 Minis - concentrations between 50 and 200 ng/µl.

04.08.13

Minipreps

15 Minis.

05.08.13

Test digest

Today we made a test digest session of our 66 minis with following enzymes.

Plasmid Enzymes Size
pIG4100 EcoRI-HF & EcoRV-HF 1571 bp (Insert CMV 600bp) & 5417 bp
pIG4101 EcoRV-HF & NotI-HF 1952 bp (half the Cas9) & 5456 bp
pIG4102 EcoRI-HF & NotI-HF 1966 bp (with PhyB) & 3274 bp
pIG4103 BamHI-HF & XhoI 5337 bp (Cas9, UVR8) & 2682 bp
pIG4104 NotI-HF & BamHI-HF 1451 bp (with COP1) & 2839 bp
pIG4105 EcoRI-HF & EcoRV-HF 3040 bp (Half the Cas9 and half CRY2) & 5529 bp
pIG4106 NotI-HF & XbaI 970 bp (CIB, VP16) & 2833 bp
pIG4107 PstI-HF & NotI-HF 976 bp (CIB, KRAB) & 2821 bp
µl type
~ 100-200 ng DNA
1 Cut Smart buffer
0,5 Enzyme 1
0,5 Enzyme 2
Add to 10µl H2O
  • Temp.: 37°C
  • Incubation time: 2h

2-log marker

Upper bands: pIG4100, expected sizes: Plasmid 3 and 5 were send to GATC

Bottom bands: pIG4101, expected sizes: Plasmid 4 and 7 were send to GATC

2-log marker

pIG4102, expected sizes, Plasmid 1 and 9 were send to GATC.

2-log marker

Upper bands left side: pIG4104, expected sizes: Plasmid 1 and 4 were send to GATC

Upper bands right side: pIG4105, expected sizes: Plasmid 1 and 6 were send to GATC

2-log marker

Upper bands: pIG4106,
not expected sizes: Plasmid 3 was send to GATC

Bottom bands: pIG4107, expected sizes: Plasmid 1 and 4 were send to GATC

For the sequencing we used GATC Primers: CMV-F for pIG4100 and EBV-RP (binds in pSV40) for all other plasmids.

Insertion of crRNA into RNA Plasmid

  1. First RNA Plasmid backbone was digested with BbsI for 3 hours. Enzyme was taken out of - 80° C.
  2. Then it was put on a gel and gelexed. Concentration: 50,5ng/ug. Backbone from Hormone- Group: 8,7ng/ul
  3. Ligation: Digested backbone from Hormone- Induction and self- digested backbone was used. Molar ratio of backbone : insert - 1 : 3.
  4. Mix: Backbone; Insert, 1ul T4- ligase, 2ul T4-ligase-buffer, H2O up to a total volume of 20ul
  5. Incubated for 15min at RT
  6. Transformed and scratched out.

06.08

Sequencing results

Sequencing results were
positive for pIG4101, pIG4102, pIG4103, pIG4105, pIG4106 and pIG4107
(sequenced with EBV-RP -> SV40 Terminator). pIG4100 didn't have the CMV promotor as an insert (-> new ligation) and pIG4104 didn't contain KRAB, although KRAB worked with pIG4102 (new Primers?)

Re-Trafos and inoculation for midi-preps

The positive Plasmids and already existing plasmids pKM018 and pKM115 were retransformed and put into 150 ml LB with 150 µl Ampicillin and incubated at 37° C over night.

New Primers for pIG4104

New Primers were designed for pIG4104.

Ligation of pIG4100

Ligation of pIG4100 was done again like on 31.07.13, only the ratio of backbone and insert was changed (3 µl backbone and 1,5 µl Insert).

2-log marker

Left band: Backbone, Right band: Inser

Result of Ligation of RNA Plasmid

There were clones on every plate. They were scratched out for minipreps.

New sequencing

Plasmids were send to GATC with different primers. Cas9 was sequenced completely in pIG4101.7. Other pIG4104 were screened for insertion of KRAB.

7.8.2013

Midipreps of pKM017; pIG4101.7 (?), pIG4102.1, pIG4103, pKM115, pIG4105.5, pIG4106.3, pIG4107.4

All midipreps were successfull

Concentrations:

pKM018: 474 ng/ul

pIG4101: 485 ng/ul

pIG4102: 528 ng /ul

pIG4103: 521 ng/ul

pKM115: 517 ng/ul

pIG4105.1: 492 ng /ul

pIG4106: 567 ng/ul

pIG4107: 476 ng /ul

Minipreps of RNA Plasmid pIG4201 and pIG4202

Minis were send to GATC. 3 of ligation with digested Backbone from Michi and tetO1 and 3 with tetO2. And 3 of ligation with newly digested Backbone from Gabi and tetO1 and 3 with tetO2.

Result of Ligation of pIG4100

There were clones on every plate. They were scratched out for minis.

8.8.2013

Sequencing of RNA Plasmid

1 of the 6 pIG4201 and 4 of the 6 pIG4202 were positiv with inserted crRNA.

Sequencing Order

Sequencing of pIG4107_2 and pIG4107_3 with Primer EBV-RP

Minis of pIG4100

12 Minis were prepped.

Re-Trafo for inoculation for Midi Preps

The following plasmids were retransformed: pIG1000, pIG4201, pIG4202

9.8.2013

Test digest of pIG4100

10.8.2013

Seeding of CHO cells

CHO- cells were seeded: 70.000 cells per well on 24 well- plates in HTS-medium

11.8.2013

Transfection of CHO cells

Protocol for 24- well plates:

1. Mix 40 µl Opti-MEM + 1.5 µl PEI-solution in a 1.5 ml Eppi

2.Prepare in another Eppi 0.5 µg of the DNA of interest

3.Add DNA to former Eppi, vortex thoroughly and incubate 15 min at RT

4.Resuspend solution gently and spread them drop-wise to the cells in the dish

5.Swing the dish in axis-form

6.Change of media 5h post transfection

12.8.2013

PCB- treatment of CHO-cells and illumination

Cells are treated with HTS-medium containing 15uM of phycocyanobilin

illumination started at 16.15. with 20uE light- quantity

plate 1 with activating red light (660nm)

plate 2 with inctivating far-red light (740nm), control

construction of pIG4104

pKM115 was digested with BamHI and BssHII for 4h. KRAB was amplified from pIG2004 with oIG4103FII and oIG4103RII

Subsequently the fragments were assembled via Gibson-cloning and transformed into E.coli

12.8.2013

Gibson results

Cell-colonies are picked and plated out anew

A mistake in the cloning strategy was discovered, new primers are designed

13.8.2013

Gibson results

CHO- cells are illuminated

14.8.2013

Seeding of CHO-cells

CHO- cells were seeded: 70.000 cells per well on 24 well- plates in HTS-medium

SEAP-measurements of illuminated cells

The results are all negative except for the positive control (-> see transfection Nr.6 of 11.8.2013)

The interaction with the cri-RNA may be responsible. The experiment is repeated with new target- sites

15.8.2013

Transfection of CHO-cells for red light and UV tests

The wiki- transfection protocoll is used:

1. Mix 40 µl Opti-MEM + 1.5 µl PEI-solution in a 1.5 ml Eppi

2.Prepare in another Eppi 0.5 µg of the DNA of interest

3.Add DNA to former Eppi, vortex thoroughly and incubate 15 min at RT

4.Resuspend solution gently and spread them drop-wise to the cells in the dish

5.Swing the dish in axis-form

6.Change of media 5h post transfection

The following pattern is transfected in 24- well plates:

16.8.2013

UV-b and red-/ far red light illumination of transfected cells from 15.8.2013

Cells for red- light illumination are illuminated with 20uE; 660nm or 720nm for 24h. Illumination is started at 14.20

Cells for UV-b light illumination are illiminated with 5uE; 311nm for 24h

17.8.2013

SEAP-measurements taken from illuminated- and untreated cells.

the following protocoll was obeyed for the whole process

  • 200 µl of cell supernatant were transfered form the 24-well plate into one 96-well plate (round bottom)
  • Incubation for 30 min at 65 °C.
  • Centrifugation for 1 min at 1250 g.
  • 100 µl of SEAP buffer were filled in the wells of a 96 well plate (flat bottom).
  • Addition of 80 µl supernatant from the round bottom plate to each well with SEAP buffer.
  • Addition of 20 µl pNPP (substrate).
  • 96 plate was immediately placed in the blade reader.
  • Spectroscopic measurement every minute for 120 times (2h); wavelenght 405 nm.

    numbers 1-6 are first six rows of the UV-b light approach -> look at transfections from 15.8.2013. numbers 7-12 are rows 1-6 of the red-light transfections from 15.8.2013

    the system has to be improved, the controls showed the desired effect, but very weak. In the following the expression of Cas9- constructs has to be checked and improved. The tetO cri-RNA showed an insufficient binding pattern. New target sites for the plasmid have to be construct

    20.8.2013

    Construction of new cri-RNAs

    6 new cri-RNAs are designed and ordered at sigma-aldrich. One new cri-RNA is provided by Max Stamnitz. All new cri-RNAs target pKM006!

    Cloning of pIG4100; pIG4104; pIG4106; pIG4107

    Primers for new cloning- strategies have arrived.

  • pIG4100: pKM006 is digested and the CMV-promotor from pAcGFP1 is amplified via PCR.
  • pIG4104: pKM115 is digested with BamHI and BssHII. KRAB is amplified via PCR from pIG2001
  • pIG4106: pKM018 is digested with EcoRI and NotI. CIB is amplified from pSW45
  • pIG4107: pIG4102 is digested with EcoRI and NotI. CIB is amplified from pSW45
  • 21.8.2013

    Cloning of pIG4100; pIG4104; pIG4106; pIG4107

    Products from digestions and PCRs are purified via gel run in 1% agarose and gelexed The digest of pKM115 for pIG4104 has not worked and is repeated executing the same protocol.

    Parts for pIG4100, pIG4106, pIG4107 are Gibson cloned and subsequently transformated into competent "TOP10" E.coli- cells

    22.8.2013

    Cloning of pIG4100; pIG4104; pIG4106; pIG4107

    Clones of Gibsons are picked and plated out. Later to be picked and test- digested. For each plasmid 2 samples were sent to GATC for sequencing.

    Gibson- cloning of pIG4104 is repeated with new digest and PCR-fragments. The constructs are subsequently transformated into competent "TOP10" E.coli- cells

    Transfection control: Is Cas9-VP16 expresse? How Strong? Is it toxic?

    HEK, HeLa and CHO- cells are seeded in 6-well plates

    23.8.2013

    Cloning of pIG4104

    Colonies are picked and plated out for mini and test- digest on 24.- or 25.8.13

    Transfection control: Is Cas9-VP16 expresse? How Strong? Is it toxic?

    different DNA- concentrations are used for transfection in different cell lines. pIG1000 containing Cas9-VP16 is used. As a already tested sample, pIG8004 containing Cas9-G9a is used as positive- control. The following DNA- concentrations are transfected: 2ug; 3ug; 4ug; 6ug. One sample is tested with and addition of 0,5% DMSO into the medium attempting to boost the expression

    The following pattern is transfected

    24.8.2013

    Oligo annealing

    Targets T2, T3, T4, T5, T6, T7 and Max's random target were annealed.

    Ligation of new RNA plasmid

    Digested pIG3010 (50,1 ng/µl) and the annealed oliges were ligated with 3 molar amount of Insert.

    25.8.2013

    Result of ligation

    Colonies were picked for mini preps.

    26.8.2013

    Minipreps of targets

    Minipreps were sent for sequencing

    27.8.2013

    Result of sequencing

    T2, T5 and T6 were positive.

    Seeding of cells for UV light activation experiment

    29.8.2013

    Transfection of CHOs for UV light activation

    30.8.2013

    Light start

    UV light was started at 15.30 o clock (two plexi glass panes)

    31.8.2013

    SEAP assay

    SEAP assay of UV light activation was performed.

    September

    03.9.2013

    Oligo annealing

    • 5µl Oligo forward 100 µM to get 20 µM concentration
    • 5 µl Oligo reverse 100 µM to get 20 µM concentration
    • 10 µl NEB buffer 2
    • 30 µl ddH2O

    Mix was heated to 98° C, Heatblock was turned off and Oligos were cooled down

    Digest of pIG3010

    • 10 µl pIG3010
    • 1 µl BbsI from -80° C
    • 5 µl NEB buffer 2.1
    • up to 50 µl ddH2O

    Digestion was put on a gel and purified (10 ng/µl).

    Ligation of Target 3,4 and VEGF and transformation

    Digested pIG3010 (10 ng/µl) and the annealed oliges were ligated with 3 molar amount of Insert.

    04.9.2013

    Result of Ligation

    Colonies were picked

    Gibson of pIG4104

    Wrong primers were used for the last pIG4104 plasmid (was seen with the last sequencing). Therefore we made a new Gibson approach.

    05.9.2013

    Minipreps of T3, T4 and VEGF (plates) and pIG4104 (fluid culture)

    Minipreps were send for sequencing (Primers for RNA Plasmid (in pIG3010) BGH-reverse and EBV-RP for pIG4104.)

    06.9.2013

    Sequencing results

    pIG4104-3 was positive, T3, T4 and VEGF RNA plasmids were negative.

    New Ligation of T4 target into standardized RNA plasmid

    Procedure was done by standard protocol.

    Seeding of CHO cells

    4 24-well plates were ceeded with 65000 CHO cells per well.

    07.9.2013

    Status of CHO cells

    CHO cells were all contaminated with bacteria again. Media was plated in a 10 cm dish. Result: No contamination here.

    seeding of HEK cells

    HEK cells were seeded for UV & BL activation and repression experiments at around 19/19.30. 4 24-well plates and 65000 cells per well.

    08.09.2013

    Transfection of HEKs for UV & BL activation and repression

    HEK cells were transfected (standard protocol) at 19.30. Media was changed after 5 hours.

    T4 RNA plasmid

    6 colonies were scratched out.

    09.09.2013

    Minis of T4

    3 of 6 plasmids were sent for sequencing (1-3).

    Light start

    Blue light was started at 21.30 with 450 nm and unsure intesity (4 colour box, 655 intensity number). Dark control is in a light box without light on.

    UV light was started at 20.30. Dark control is in the incubator (no UV light in the lab).

    10.09.2013

    Sequencing results

    Sequencing results were positive for Bla Target 4. Bla Targets 2 and 3 were cloned by Philipp who got positive results, too. Plasmids were retransformed and Midis were inoculated.

    Seeding of HEK cells.

    4 24 well plates were seeded with 65.000 HEK cells per well.

    Light stop

    Lightning of UV and BL were stopped after about 24 h and supernatants were taken and frozen.

    11.09.2013

    Midis

    Midis of Bla target 2 (pIG4), Bla target 3 (pIG4, Bla target 4 (pIG4), EMX1 (pIG4 and VEGF4 (pIG4) were prepped.

    SEAP of UV and BL activation and repression

    Figure 1: SEAP measurement for blue light activation.
    Figure 2: SEAP measurement for UV light activation.

    Activation was okay, repression was in overflow.

    Transfection for Activation-VP16 and Repression-KRAB target test row

    In order to test which target we should use, we made test rows with different targets and different target combinations. Medium was changed after 5 h.

    Figure 3: Transfection plan: Target test row Activation 1
    Figure 4: Transfection plan: Target test row Activation 1
    Figure 5: Transfection plan: Target test row repression 1.
    Figure 6: Transfection plan: Target test row repression 2.

    12.09.2013

    SEAP of Activation VP16 test row

    Figure 7: SEAP measurement for target test row for Cas9-VP16.

    Seeding of HEK cells for light activation experiments

    13.09.2013

    SEAP of Repression KRAB test row and UV and BL repression

    Samples had to be diluted 1:10 because the constitutive CMV promoter is very strong. SEAP levels were still in overflow.

    Transfection for UV/BL/RL activation

    Figure 8: Transfection plan for blue light activation. plan
    Figure 9: Transfection plan for red light activation.
    Figure 10: Transfection plan for UVB light activation.

    14.09.2013

    Light start for UV/BL/RL

    15.09.2013

    Light stop for UV and BL

    SEAP of UV and BL activation

    Figure 11: SEAP measurement for blue light activation.
    Figure 12: SEAP measurement for UV light activation.

    16.09.2013

    Light stop for RL

    SEAP for RL

    Figure 13: SEAP measurement for red light activation.

    18.09.2013

    New charge of medium for CHOs

    Medium was complemented with FCS, Pen Strep and Gentamicin.

    20.09.13

    New CHO-K1 & NIH cells

    We received CHO-K1 cells from a freshly thawed cell batch. Thank you Shima from AG Ulbrich! These cells were cultivated in DMEM.

    We received NIH cells.

    24.09.13

    Seeding of CHO-K1 (from Toolbox) cells

    6 24 well plates were seeded with about 65.000 cells per well.

    25.09.13

    Transfection for red/blue/UV light induction in CHO-K1 (from Toolbox)

    CHO-K1 cells were transfected for red, blue and UV light induced gene activation. Cells that were transfected for UV light were also transfected with Renilla. This was done for normalization, because UV light can kill the cells.

    Figure 14: Transfection plan for blue light activation.
    Figure 15: Transfection plan for red light activation.
    Figure 16: Transfection plan for UV light activation.

    HTS medium was changed after 5 hours.

    Seeding of NIH cells

    2 24 well plates were seeded with NIH cells (65.000 cells per well).

    26.09.13

    Transfection for UV light induction in NIH cells

    Transfection plan was the same as for CHOs on 25.09.

    Light start for red/blue/UV light in CHO-K1 cells (from Toolbox)

    1 plate was placed in UV light (311 nm). 1 plate was placed in a red light box (660) after PCB treatment. 1 plate was placed in a far-red light box (740 nm) after PCB treatment. One plate was placed in a blue light box (460 nm).

    27.09.13

    Light start for UV light in NIH cells

    1 plate of transfected NIH cells was placed in UV light.

    Light stop for UV (CHO-K1 from toolbox) & blue light (CHO-K1) cells

    200 µl of supernatant of each well were removed and heated. After this the 96 well plate was frozen (-20° C). SEAP measurement was done on the next day.

    28.09.13

    SEAP measurement of UV (CHO-K1 from toolbox) & blue light (CHO-K1) cells

    Figure 17: SEAP measurement for blue light induced activation (CHO-K1)
    Figure 18: SEAP measurement for UVB light induced activation (CHO-K1).

    Light stop for red light (CHO-K1 from toolbox) and UV light (NIH)

    200 µl of supernatant of each well were removed and heated. SEAP measurement was done according to standard protocol.

    Figure 19: SEAP measurement of red light induced activation (CHO-K1)
    Figure 20: SEAP measurement for UVB light induced activation (NIH).

    29.09.13

    Seeding of CHO-K1 from AG Ulbrich and NIH cells

    6 24 well plates with CHO-K1 were seeded and 6 24 well plates with NIH cells were seeded.

    30.09.13

    Transfection of CHO-K1 & NIH cells - red, blue and UV light

    Alltogether 12 24 well plates were transfected.

    Transfection plans

    Figure 21: Transfection plan: Blue light activation.
    Figure 22: Transfection plan: Red light activation.
    Figure 23: Transfection plan: UVB light activation.

    transfection calculation

    Figure 24: Transfection calculation for blue light activation.
    Figure 25: Transsfection calculation 2 for blue light activation.
    Figure 26: Transfection calculation 1 for red light activation.
    Figure 27: Transfection calculation 2 for red light activation.
    Figure 28: Transfection calculation 1 for UVB light activation.
    Figure 29: Transfection calculation 2 for UVB light activation

    October

    01.10.13

    Light start

    Both transfected cell lines were illuminated: 1 CHO plate & 1 NIH plate with red light 660 nm, 1 CHO plate & 1 NIH plate with blue 465 nm and 1 CHO plate & 1 NIH plate with UV light 311 nm. For every cell line and light system there was a dark control plate (red light's dark control is far red light of 740 nm because the system is reversible)

    02.10.13

    Light stop

    Light was stoped for UV and blue light illumination (after 24 hours) (CHO and NIH cells). 200 µl supernatant were removed from each well for SEAP measurements. SEAP measurement was done according to standard protocol.

    Figure 30: SEAP measurement for blue light activation in CHO-K1 cells.
    HIER DANN ALLES ANDERE
    Figure 31: SEAP measurement for blue light activation in NIH cells.
    Figure 32: SEAP measurement for UVB light activation in CHO-K1 cells.
    Figure 33: SEAP measurement for UVB light activation in NIH cells.

    03.10.13

    Light stop

    Light was stoped for red light illumantion. 200 µl supernatant were removed from each well for SEAP measurements. SEAP measurement was done according to standard protocol.

    Figure 34: SEAP measurement for red light activation in NIH cells.
    Figure 35: SEAP measurement for red light activation in CHO-K1 cells.