http://2014.igem.org/wiki/index.php?title=Team:BGU_Israel/Notebook1&feed=atom&action=historyTeam:BGU Israel/Notebook1 - Revision history2024-03-28T09:03:17ZRevision history for this page on the wikiMediaWiki 1.16.5http://2014.igem.org/wiki/index.php?title=Team:BGU_Israel/Notebook1&diff=328619&oldid=prevPriels at 15:36, 17 October 20142014-10-17T15:36:24Z<p></p>
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</table>Prielshttp://2014.igem.org/wiki/index.php?title=Team:BGU_Israel/Notebook1&diff=328196&oldid=prevPriels at 15:28, 17 October 20142014-10-17T15:28:43Z<p></p>
<a href="http://2014.igem.org/wiki/index.php?title=Team:BGU_Israel/Notebook1&diff=328196&oldid=306535">Show changes</a>Prielshttp://2014.igem.org/wiki/index.php?title=Team:BGU_Israel/Notebook1&diff=306535&oldid=prevPriels at 06:48, 17 October 20142014-10-17T06:48:18Z<p></p>
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</table>Prielshttp://2014.igem.org/wiki/index.php?title=Team:BGU_Israel/Notebook1&diff=276725&oldid=prevStav shamir at 15:17, 16 October 20142014-10-16T15:17:55Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>mRuby2: excitation - 559 nm, emission - 600 nm </p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>mRuby2: excitation - 559 nm, emission - 600 nm </p></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><u>Results:</u></p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><u>Results:</u></p></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><p><img src="https://static.igem.org/mediawiki/2014/6/6a/BGU14notefig1.png" style="height:<del class="diffchange diffchange-inline">130px</del>"/></p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p><img src="https://static.igem.org/mediawiki/2014/6/6a/BGU14notefig1.png" style="height:<ins class="diffchange diffchange-inline">150px</ins>"/></p></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>Co-expression of mRuby2 and eGFP. All three pictures show the same cells. Picture A shows only eGFP expressing cells, picture B shows only mRuby2 expressing cells, and picture C show all cells. </p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>Co-expression of mRuby2 and eGFP. All three pictures show the same cells. Picture A shows only eGFP expressing cells, picture B shows only mRuby2 expressing cells, and picture C show all cells. </p></div></td></tr>
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</table>Stav shamirhttp://2014.igem.org/wiki/index.php?title=Team:BGU_Israel/Notebook1&diff=276712&oldid=prevStav shamir at 15:17, 16 October 20142014-10-16T15:17:34Z<p></p>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><p><img src="https://static.igem.org/mediawiki/2014/6/6a/BGU14notefig1.png" style="height:<del class="diffchange diffchange-inline">1300px</del>"/></p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p><img src="https://static.igem.org/mediawiki/2014/6/6a/BGU14notefig1.png" style="height:<ins class="diffchange diffchange-inline">130px</ins>"/></p></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>Co-expression of mRuby2 and eGFP. All three pictures show the same cells. Picture A shows only eGFP expressing cells, picture B shows only mRuby2 expressing cells, and picture C show all cells. </p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>Co-expression of mRuby2 and eGFP. All three pictures show the same cells. Picture A shows only eGFP expressing cells, picture B shows only mRuby2 expressing cells, and picture C show all cells. </p></div></td></tr>
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</table>Stav shamirhttp://2014.igem.org/wiki/index.php?title=Team:BGU_Israel/Notebook1&diff=276700&oldid=prevStav shamir at 15:17, 16 October 20142014-10-16T15:17:05Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><u>Results:</u></p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><u>Results:</u></p></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><p><img src="https://static.igem.org/mediawiki/2014/6/6a/BGU14notefig1.png" style="height:<del class="diffchange diffchange-inline">200px</del>"/></p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p><img src="https://static.igem.org/mediawiki/2014/6/6a/BGU14notefig1.png" style="height:<ins class="diffchange diffchange-inline">1300px</ins>"/></p></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>Co-expression of mRuby2 and eGFP. All three pictures show the same cells. Picture A shows only eGFP expressing cells, picture B shows only mRuby2 expressing cells, and picture C show all cells. </p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>Co-expression of mRuby2 and eGFP. All three pictures show the same cells. Picture A shows only eGFP expressing cells, picture B shows only mRuby2 expressing cells, and picture C show all cells. </p></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
</table>Stav shamirhttp://2014.igem.org/wiki/index.php?title=Team:BGU_Israel/Notebook1&diff=276678&oldid=prevStav shamir at 15:16, 16 October 20142014-10-16T15:16:33Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><u>Results:</u></p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><u>Results:</u></p></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><p><img src="https://static.igem.org/mediawiki/2014/6/6a/BGU14notefig1.png" style="height:<del class="diffchange diffchange-inline">100px</del>"/></p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p><img src="https://static.igem.org/mediawiki/2014/6/6a/BGU14notefig1.png" style="height:<ins class="diffchange diffchange-inline">200px</ins>"/></p></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>Co-expression of mRuby2 and eGFP. All three pictures show the same cells. Picture A shows only eGFP expressing cells, picture B shows only mRuby2 expressing cells, and picture C show all cells. </p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>Co-expression of mRuby2 and eGFP. All three pictures show the same cells. Picture A shows only eGFP expressing cells, picture B shows only mRuby2 expressing cells, and picture C show all cells. </p></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
</table>Stav shamirhttp://2014.igem.org/wiki/index.php?title=Team:BGU_Israel/Notebook1&diff=255104&oldid=prevPriels at 21:01, 15 October 20142014-10-15T21:01:55Z<p></p>
<a href="http://2014.igem.org/wiki/index.php?title=Team:BGU_Israel/Notebook1&diff=255104&oldid=217958">Show changes</a>Prielshttp://2014.igem.org/wiki/index.php?title=Team:BGU_Israel/Notebook1&diff=217958&oldid=prevPriels: Created page with "{{Team:BGU_Israel/Menu}} <html> <section> <div style="height:12px"> </div> <div style="margin-bottom:10px"> <img src="https://static.igem.org/mediawiki/2014/0/07/BGU..."2014-10-13T19:56:30Z<p>Created page with "{{Team:BGU_Israel/Menu}} <html> <section> <div style="height:12px"> </div> <div style="margin-bottom:10px"> <img src="https://static.igem.org/mediawiki/2014/0/07/BGU..."</p>
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<th align="center"bgcolor="#B4DEE0"><a onClick="show(w6)" href="#">Week 6</a></th><br />
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<th align="center" bgcolor="#B4DEE0"><a onClick="show(w7)" href="#">Week 7</a></th><br />
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<th align="center" bgcolor="#B4DEE0"><a onClick="show(w9)" href="#">Week 9</a></th><br />
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<div id="june" class="textCont" style="height:630px;display:none"><br />
<h3 style="border-bottom:dashed;border-color:#000000">June</h3><br />
<br><br />
<div align="center" style="float:left" class="prevNext" onClick="show(may)"><img src="https://static.igem.org/mediawiki/2014/8/8f/BGU14finger-point3.gif" width="26px"/>&nbsp;Prev</div><br />
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<div class="col2" style=" width:400px;"><br />
<h3>General</h3><br />
<p>No Activity *****</p> <br />
</div><br />
</div><br />
</div><br />
<br />
<!--july--><br />
<div id="july" class="textCont" style="height:630px;display:none"><br />
<h3 style="border-bottom:dashed;border-color:#000000">July</h3><br />
<br><br />
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<h3>General</h3><br />
<p>No Activity *****</p> <br />
</div><br />
<br />
</div><br />
</div><br />
<br />
<div id="w1" class="textCont" style="height:630px;display:none"><br />
<h3 style="border-bottom:dashed;border-color:#000000">Week 1: July 27th - August 2nd</h3><br />
<br><br />
<div align="center" style="float:left" class="prevNext" onClick="show(july)"><img src="https://static.igem.org/mediawiki/2014/8/8f/BGU14finger-point3.gif" width="26px"/>&nbsp;Prev</div><br />
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<div class="col2" style=" width:400px;"><br />
<h3>Lab</h3><br />
<p><b>Intelligent Medication</b><br />
<p><u>Propagation of pcDNA3-mRuby2</u></p><br />
<p>Starters were taken from a pcDNA3-mRuby2 bacterial stab (acquired form Addgene) and incubated in LB containing 100 µg/ml ampicillin for 24 hours at 37˚c.<br />
Plasmid DNA was extracted from using a miniprep kit.</p><br />
</p> <br />
</div><br />
<div class="col2" style=" width:400px;margin-left:160px" ><br />
<h3>Human Practice</h3><br />
</div><br />
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</div><br />
</div><br />
<br />
<!--August--><br />
<div id="august" class="textCont" style="height:630px;display:none"><br />
<h3 style="border-bottom:dashed;border-color:#000000">August</h3><br />
<br><br />
<div align="center" style="float:left" class="prevNext" onClick="show(w1)"><img src="https://static.igem.org/mediawiki/2014/8/8f/BGU14finger-point3.gif" width="26px"/>&nbsp;Prev</div><br />
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<div class="textContNotebook" style="height:460px; background-image:"images/notebook.jpg""><br />
<br />
<div class="col2" style=" width:400px;"><br />
<h3>General</h3><br />
<p>*****</p> <br />
</div><br />
</div><br />
</div> <br />
<br />
<div id="w2" class="textCont" style="height:1050px;display:none"><br />
<h3 style="border-bottom:dashed;border-color:#000000">Week 2: August 3rd- August 9th</h3><br />
<br><br />
<div align="center" style="float:left" class="prevNext" onClick="show(august)"><img src="https://static.igem.org/mediawiki/2014/8/8f/BGU14finger-point3.gif" width="26px"/>&nbsp;Prev</div><br />
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<div class="textContNotebook" style="height:900px; background-image:"images/notebook.jpg""><br />
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<div class="col2" style=" width:400px;"><br />
<h3>Lab</h3><br />
<p><strong>Intelligent Medication</strong></p> <br />
<p><u>Validation of co-expression of mRuby2 and eGFP in CT26 eGFP cell line</u></p><br />
<p>Goal: A preliminary experiment to validate we can visualize the co-expression of both mRuby2 and eGFP in CT26 eGFP cell line using confocal microscopy. <br />
Method:<br />
20,000 cells were plated in each well of a chamber slide and incubated for 24 hours at 37˚c.<br />
4 wells were transfected with pcDNA3-mRuby2 using Lipofectamine 2000 (protocol), and incubated for 48 hours at 37˚c. The other 4 wells were mock transfected with Lipofectamine 2000 and incubated in the same conditions for negative control.<br />
After 48 hours, the transfected cells were viewed at the confocal microscope. <br />
eGFP: excitation - 488 nm, emission - 509 nm <br />
mRuby2: excitation - 559 nm, emission - 600 nm </p><br />
<p><u>Results:</u></p><br />
<p><img src="https://static.igem.org/mediawiki/2014/6/6a/BGU14notefig1.png" style="height:100px"/></p><br />
<p>Co-expression of mRuby2 and eGFP. All three pictures show the same cells. Picture A shows only eGFP expressing cells, picture B shows only mRuby2 expressing cells, and picture C show all cells. </p><br />
<br />
<p><u>Conclusions:</u><br><br />
mRuby2 and eGFP were co-expressed in CT26 eGFP cells and viewed successfully using confocal microscopy.</p> <br />
</div><br />
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<h3>Human Practice</h3><br />
</div><br />
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</div><br />
</div> <br />
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<div id="w3" class="textCont" style="height:630px;display:none"><br />
<h3 style="border-bottom:dashed;border-color:#000000">Week 3 </h3><br />
<br><br />
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<h3>Lab</h3><br />
<p>No Activity</p><br />
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</div><br />
<div class="col2" style=" width:400px;margin-left:160px" ><br />
<h3>Human Practice</h3><br />
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</div><br />
</div><br />
<br />
<div id="w4" class="textCont" style="height:630px;display:none"><br />
<h3 style="border-bottom:dashed;border-color:#000000">Week 4</h3><br />
<br><br />
<div align="center" style="float:left" class="prevNext" onClick="show(w3)"><img src="https://static.igem.org/mediawiki/2014/8/8f/BGU14finger-point3.gif" width="26px"/>&nbsp;Prev</div><br />
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<h3>Lab</h3><br />
<p>No Activity</p> <br />
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<h3>Human Practice</h3><br />
</div><br />
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<div id="w5" class="textCont" style="height:4100px;display:none"><br />
<h3 style="border-bottom:dashed;border-color:#000000">Week 5: August 31th-September 6th</h3><br />
<br><br />
<div align="center" style="float:left" class="prevNext" onClick="show(w4)"><img src="https://static.igem.org/mediawiki/2014/8/8f/BGU14finger-point3.gif" width="26px"/>&nbsp;Prev</div><br />
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<div class="col2" style="width:400px"><br />
<h3>Lab</h3><br />
<p><strong>Intelligent Medication</strong></p> <br />
<p><u>Functional silencing experiment</u></p><br />
<p>Goal: Silencing eGFP in CT26 eGFP cells co - transfected with pcDNA3 mRuby2 and our scRNA construct. The scRNA construct is designed to be activated by mRuby2 mRNA, becoming a dicer substrate and finally silence eGFP. <br />
Method:<br />
We gave a different treatment for each of our four groups:<br />
</p><br />
<p align="center"><img src="https://static.igem.org/mediawiki/2014/9/9e/BGU14notefig2.png" style="height:150px"/></p><br />
<p>We conducted the experiment both in 24 well plate (for flow cytometry) and in a chamber slide (for confocal microscopy).<br><br />
The cells were plated on the appropriate plates for the different tests (100,000 cells per well in the 24 well plate and 15,000 cells per well in the chamber slide) and incubated for 24 hours at 37˚c.<br> <br />
After 24 hours, the incubated cells were transfected (according to the different treatments detailed above) with one or more of the following: </p><br />
<ul style="list-style:disc"><br />
<li>pcDNA3 mRuby2 - 1.0 µg</li><br />
<li>scRNA – both detection part A + hair pin part B - 100 pmol for each of the parts (A+B)</li><br />
</ul><br />
<p>Transfection was done by lipofectamine (protocol), and then the cells were incubated for 48 hours at 37˚c.<br><br />
Preparations for image stream were done by the general protocol (protocol).<br />
</p><br />
<p><u>Results:</u></p><br />
<p>Image stream (flow cytometry):<br />
<p><strong>Treatment 1 – Negative control</strong><br />
<p align="center"><img src="https://static.igem.org/mediawiki/2014/3/34/BGU14notefig3.png" style="height:340px"/></p><br />
</p><br />
<p><strong>Treatment 2 – transfected with pcDNA3 mRuby2</strong></p><br />
<p align="center"><img src="https://static.igem.org/mediawiki/2014/f/fc/BGU14notefig4.png" style="height:340px"/></p><br />
<p><strong>Treatment 3 – transfected with scRNA</strong><br />
<p align="center"><img src="https://static.igem.org/mediawiki/2014/3/36/BGU14notefig5.png" style="height:340px"/></p><br />
<p><strong>Treatment 4 – transfected with pcDNA3 mRuby2 & scRNA</strong><br />
<p align="center"><img src="https://static.igem.org/mediawiki/2014/3/3a/BGU14notefig6.png" style="height:340px"/></p><br />
<p align="center"><img src="https://static.igem.org/mediawiki/2014/3/34/BGU14notefig7.PNG" style="height:175px"/></p><br />
<p>Confocal microscope:<br><br />
Co-expression of mRuby2 and eGFP was validated in all relevant wells.</p><br />
<p align="center"><img src="https://static.igem.org/mediawiki/2014/d/d4/BGU14notefig8.png" style="height:200px"/></p><br />
<p dir="LTR"><u>Conclusions:</u></p><br />
<ul style="list-style:disc"><br />
<li><span dir="LTR"> </span>GFP negative population: The first parameter to compare that comes to mind when looking for silencing is the percentage of GFP negative cells of the entire population – if a significantly bigger portion of the population is GFP negative, then the silencing worked. However, when looking at the population distribution of the different control groups, we can see no significant difference in the GFP negative population. This might not the result of unsuccessful silencing – we are using a stable GFP expressing cell line, but no matter how stable it is, always there will be a portion of the cells that do not actually express GFP. Additionally, a successful silencing is not expected to completely shut down the expression of GFP in single cells. This creates the necessity to evaluate the expression of GFP using another parameter. </li><br />
<li><span dir="LTR"> </span>Mean intensity of GFP positive cells: This parameter will show difference between a negative control and a successful silencing in light of the above. However, cells treated with scRNA showed only a very small difference in this parameter, so we could not observe a successful silencing. </li><br />
</ul><br />
<p dir="LTR">For the next try, the following changes were implemented:</p><br />
<ol type="1"><br />
<li><span dir="LTR"> </span>double the amount of scRNA in the transfection</li><br />
<li><span dir="LTR"> </span>Change the protocol of preparing Lipofectamine – scRNA mix to allow for better interaction between the 2 parts of the construct, and prepare the Lipofectamine- DNA mix separately.</li><br />
</ol><br />
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<h3>Human Practice</h3><br />
</div><br />
<br />
<br />
</div><br />
</div><br />
<br />
<!--September--><br />
<div id="september" class="textCont" style="height:630px;display:none"><br />
<h3 style="border-bottom:dashed;border-color:#000000">September</h3><br />
<br><br />
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<h3>General</h3><br />
<p>*****</p> <br />
</div><br />
</div><br />
</div><br />
<br />
<br />
<div id="w6" class="textCont" style="height:1400px;display:none"><br />
<h3 style="border-bottom:dashed;border-color:#000000">Week 6: September 7th-September 13th</h3><br />
<br><br />
<div align="center" style="float:left" class="prevNext" onClick="show(september)"><img src="https://static.igem.org/mediawiki/2014/8/8f/BGU14finger-point3.gif" width="26px"/>&nbsp;Prev</div><br />
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<div class="textContNotebook" style="height:1270px; background-image:"images/notebook.jpg""><br />
<br />
<div class="col2" style=" width:400px;"><br />
<h3>Lab</h3><br />
<p><strong>Intelligent Medication</strong><br><br />
Goal: A second try of silencing eGFP in CT26 eGFP cells co - transfected with pcDNA3 mRuby2 and our scRNA construct. The scRNA construct is designed to be activated by mRuby2 mRNA, becoming a dicer substrate and finally silence eGFP. <br><br />
<u>Details:</u><br><br />
The changes concluded from former results:</p><br />
<ol type="1"><br />
<li><span dir="LTR"> </span>Half of calls amount was seeded (50,000 cells per well)</li><br />
<li><span dir="LTR"> </span>The cells were transfected with double amount of scRNA:</li><br />
<ul style="list-style:disc"><br />
<li><span dir="LTR"> </span>pcDNA3-mRuby2- <strong><u>1.0 µg</u></strong> </li><br />
<li><span dir="LTR"> </span> scRNA – both detection part A + hair pin part B- <strong><u>200 pmol</u></strong> for each of the parts (A+B) </li><br />
</ul><br />
<li><span dir="LTR"> </span>This time, the lipofectamin mixes were prepared separately for the pcDNA3-mRuby2 and for the scRNA (both A and B parts together), so that both of the mixes will make its content ready and prepared for transfection.</li><br />
</ol><br />
<p>The rest of the protocol remained unchanged (<a href="https://www.dropbox.com/s/k03gi5spggb0937/03-%20transfection%20by%20Lipofectamine.docx?dl=0">protocol</a>). <br><br />
<u>Notes:</u><br><br />
During this transfection a mistake occurred. In one of the plates, an incorrect of volume of Lipofectamine-scRNA was added due to a measurement error. Consequently, this plate had unknown concentrations of nucleic acids and lipofectamine, with a positive deviation in volume.<br><br />
<u>Results:</u><br><br />
Most of the cells died, probably due to the increased volume of Lipofectamine that was added by mistake.</p><br />
<p><strong><u>Biobricks</u></strong><br><br />
<em>Goal: The ordered parts for Biobricks preparation had arrived</em> <em>in the weekend so preparations of plates and medium for the Biobricks construction were done.</em><br><br />
<u>Details:</u></p><br />
<ol type="1" ><br />
<li><span dir="LTR"> </span>LB agar plates with ampicillin (100 µg/ml) or Chloramphenicol (25 µg/ml) were made following the <a href="https://www.dropbox.com/s/k04kop5o8zjzh0v/05-Making%20an%20LB%20plate.docx?dl=0">LB agar plates protocol</a>.</li><br />
<li><span dir="LTR"> </span>SOC medium for bacterial transformation was prepared. SOC medium content is: </li><br />
<ul style="list-style:disc"><br />
<li><span dir="LTR"> </span>0.5% (w/v) yeast extract</li><br />
<li><span dir="LTR"> </span>2% (w/v) tryptone</li><br />
<li><span dir="LTR"> </span>10 mM NaCl</li><br />
<li><span dir="LTR"> </span>2.5 mM KCl</li><br />
<li><span dir="LTR"> </span>20 mM MgSO4</li><br />
<li><span dir="LTR"> </span>Glucose 20%</li><br />
</ul><br />
</ol><br />
<p>Preparation followed by the <a href="https://www.dropbox.com/s/5r9zltzkn5p2vsd/06-SOC%20medium%20prep.docx?dl=0">SOC medium prep protocol</a>.</p><br />
</div><br />
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<h3>Human Practice</h3><br />
</div><br />
<br />
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</div><br />
</div> <br />
<br />
<div id="w7" class="textCont" style="height:4020px;display:none"><br />
<h3 style="border-bottom:dashed;border-color:#000000">Week 7:September 14th-September 20th</h3><br />
<br><br />
<div align="center" style="float:left" class="prevNext" onClick="show(w6)"><img src="https://static.igem.org/mediawiki/2014/8/8f/BGU14finger-point3.gif" width="26px"/>&nbsp;Prev</div><br />
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<br />
<div class="col2" style=" width:400px;"><br />
<h3>Lab</h3><br />
<p><strong><u>Biobricks- </u></strong><br><br />
<strong><em>First trial -</em></strong><em> the DNA parts that were used in Biobricks preparation are: Adiponectin (pUC</em> <em>57 Adiponectin), DsbA-L (pUC 57 DsbA-L), HSP70 promotor (pUC 57 HSP70), HSP70 promotor (with modifications) (pUC 57 HSP70 modified), UCP1 (pcDNA3.1 UCP1) and pSB1C3 backbone. The process included- transformation</em><span dir="RTL"> </span><span dir="RTL"> </span><em><span dir="RTL"><span dir="RTL"> </span><span dir="RTL"> </span></span><span dir="LTR"> </span><span dir="LTR"> </span><span dir="LTR"> </span><span dir="LTR"> </span>-&gt; extraction and creating a glycerol stock-&gt; restriction-&gt; gel electrophoresis-&gt; gel products extraction-&gt; ligation-&gt; transformation.</em><br><br />
<u>Details: </u><br><br />
DNA parts and backbone were digested with restriction enzymes: Pst1 and EcoR1<br><br />
The transformation was done according to the <a href="https://www.dropbox.com/s/p6fau982fhzeihe/08-Transformation%20to%20competent%20bactiria.docx?dl=0">general transformation protocol</a>.<br><br />
At first, heat-shock transformation to chemically competent BH5α bacteria with the following DNA parts (separately): </p><br />
<ul style="list-style:disc"><br />
<li><span dir="LTR"> </span>pUC 57 Adiponectin</li><br />
<li><span dir="LTR"> </span>pUC 57 HSP70 </li><br />
<li><span dir="LTR"> </span>pUC 57 HSP70 modified</li><br />
<li><span dir="LTR"> </span>pUC 57 DsbA-L</li><br />
<li><span dir="LTR"> </span>pcDNA3.1 UCP1</li><br />
</ul><br />
<p>The transformed bacteria were incubated for 24 hr at 37˚c.<br><br />
After 24 hr incubation, colonies from each plate were transferred to liquid medium and incubated for 24 hr at 37˚c. <br><br />
Plasmid DNA was extracted using a miniprep kit and concentrations were measured using nanodrop. <br><br />
The plasmids were digested with restriction enzymes Pst1 and EcoR1, following <a href="https://www.dropbox.com/s/bvv0w3rod9dabvu/09-restriction.docx?dl=0">general restriction protocol</a>. <br><br />
The digested DNA parts were tested by gel electroporation, following <a href="https://www.dropbox.com/s/r98k3xv1svh48ys/10-Gel%20Elctrophoresis.docx?dl=0">general gel electrophoresis protocol</a>.<br><br />
<u>Results:</u><br><br />
The wanted bands didn&rsquo;t appear on the gel.<br><br />
<u>Conclusions:</u><br><br />
Since the desired results didn&rsquo;t appear on the gel, it was assumed that the restriction didn&rsquo;t work. The reason is probably technical inexperience of using restriction enzymes and inappropriate pipetation and spin down absence. </p><br />
<p><strong>Intelligent Medication</strong><br>Goal: Third try of trying to silence eGFP in CT26 eGFP cells – this time we also made a positive control with anti eGFP siRNA from our lab. Additionally we simplified the silencing process – using our mechanism, if transfected with 2 parts of the RNA construct silencing will take place only in the presence of a specific mRNA. This time we used only one part (part B – hairpin) that is supposed to have a silencing effect without the presence of the specific mRNA. We also tested our samples for silencing using RT PCR and flow cytometry (Image Stream).<br><br />
<u>Details:</u><br><br />
The changes concluded from former results:</p><br />
<ol type="1"><br />
<li><span dir="LTR"> </span>Added positive control and used only part B hairpin:</li><br />
<ul style="list-style:disc"><br />
<li><span dir="LTR"> </span>Anti eGFP siRNA - 200 pmol</li><br />
<li><span dir="LTR"> </span>scRNA part B hairpin - 200 pmol </li><br />
</ul><br />
<li><span dir="LTR"> </span>Seeded cells amount was doubled, back to 100,000 cells per well.</li><br />
</ol><br />
<p>The rest of the protocol remained unchanged (<a href="https://www.dropbox.com/s/k03gi5spggb0937/03-%20transfection%20by%20Lipofectamine.docx?dl=0">general lipofectamin protocol</a>).<br><br />
<u>Results:</u><br><br />
<strong>Image stream:</strong><br><br />
<strong>scRNA part B (hairpin) only</strong></p><br />
<p align="center"><img src="https://static.igem.org/mediawiki/2014/d/d9/BGU14notefig9.png" style="height:330px"/></p><br />
<p><strong>Positive control - anti GFP siRNA</strong></p><br />
<p align="center"><img src="https://static.igem.org/mediawiki/2014/3/32/BGU14notefig10.png" style="height:330px"/></p><br />
<p><strong>Negative control-</strong></p><br />
<p align="center"><img src="https://static.igem.org/mediawiki/2014/2/28/BGU14notefig11.png" style="height:330px"/></p><br />
<p><strong>Representative image stream pictures:</strong></p><br />
<p align="center"><img src="https://static.igem.org/mediawiki/2014/6/69/BGU14notefig12.png" style="height:400px"/></p><br />
<p dir="LTR"><strong>RealTime PCR:</strong><br>The housekeeping genes showed unstable expression and thus could not be used as reliable control, rendering the results of the entire experiments unreliable. <br><br />
<u>Conclusions:</u></p><br />
<ul style="list-style:disc"><br />
<li><span dir="LTR"> </span>The mean intensity in the GFP positive population of the positive control (anti GFP siRNa) is lower by 28 % of that in the negative control. However, this sequence was tested before and resulted in much higher silencing rates. With this, and in addition to previous results, we assume that flow cytometry, the way we use it, tests silencing in the protein level. Therefore it is likely that the proteins (eGFP is known to be stable) in the sample did not have enough time to degrade. Therefore, it was decided that image stream is not efficient test for the scRNA experiments and the appropriate test would deal with mRNA levels.</li><br />
<li><span dir="LTR"> </span>The treatment containing scRNa part B only (hairpin silencing sequence) didn't show any lower GFP intensity in the GFP positive population, concerning the negative control.</li><br />
<li><span dir="LTR"> </span>It was assumed that as image stream <u>Real Time PCR results</u>: Since the housekeeping genes showed unstable expression no significant result could be drawn regarding neither gene expression nor gene silencing from the RealTime PCR this time.</li><br />
</ul><br />
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<h3>Human Practice</h3><br />
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<div id="w8" class="textCont" style="height:1550px;display:none"><br />
<h3 style="border-bottom:dashed;border-color:#000000">Week 8: September 21th-September 27th</h3><br />
<br><br />
<div align="center" style="float:left" class="prevNext" onClick="show(w7)"><img src="https://static.igem.org/mediawiki/2014/8/8f/BGU14finger-point3.gif" width="26px"/>&nbsp;Prev</div><br />
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<h3>Lab</h3><br />
<p><strong><u>Biobricks- </u></strong><br><br />
<em>Trying again the experiment from September 7th.</em><br><br />
<u>Details: </u><br><br />
DNA parts and backbone were digested with restriction enzymes: Pst1 and EcoR1<br><br />
The transformation was done according to the <a href="https://www.dropbox.com/s/p6fau982fhzeihe/08-Transformation%20to%20competent%20bactiria.docx?dl=0">general transformation protocol</a>.<br><br />
At first, heat-shock transformation to chemically competent BH5α bacteria with the following DNA parts (separately): </p><br />
<ul style="list-style:disc"><br />
<li><span dir="LTR"> </span>pUC 57 Adiponectin</li><br />
<li><span dir="LTR"> </span>pUC 57 HSP70 </li><br />
<li><span dir="LTR"> </span>pUC 57 HSP70 modified</li><br />
<li><span dir="LTR"> </span>pUC 57 DsbA-L</li><br />
<li><span dir="LTR"> </span>pcDNA3.1 UCP1</li><br />
</ul><br />
<p>The transformed bacteria were incubated for 24 hr at 37˚c.<br><br />
After 24 hr incubation, colonies from each plate were transferred to liquid medium and incubated for 24 hr at 37˚c. <br><br />
Plasmid DNA was extracted using a miniprep kit and concentrations were measured using nanodrop. <br><br />
The plasmids were digested with restriction enzymes Pst1 and EcoR1, following <a href="https://www.dropbox.com/s/bvv0w3rod9dabvu/09-restriction.docx?dl=0">general restriction protocol</a>. <br><br />
The digested DNA parts were tested by gel electroporation, following <a href="https://www.dropbox.com/s/r98k3xv1svh48ys/10-Gel%20Elctrophoresis.docx?dl=0">general gel electrophoresis protocol</a>.<br><br />
Ligation products were transformed by heat-shock to chemically competent DH5α bacteria, plated on cmp LB agar plates and incubated for 24 hr at 37˚c.<br><br />
<u>Results: </u><br><br />
All parts were successfully restricted (unfortunately the picture taken was not in good quality).<u></u><br><br />
No colonies grew on the plates.<br><br />
<u>Conclusions:</u><br><br />
Since no colonies grew on the plates, but restriction went well, the problem might lie either in the ligation process or the competent cells. For the next try, a new competent cells stock was used.<strong></strong></p><br />
<strong><em><br clear="all"><br />
</em></strong><br />
<p><strong><u>Biobricking third try:</u></strong><br><br />
<u>Details: </u><strong></strong><br><br />
DNA parts and pSB1C3 backbone (from pSB1C3 RFP and pSB1C3 amilcp) were digested with restriction enzymes: Pst1 and EcoR1<br><br />
The ligation and transformation was done according to the previous protocols.<br><br />
<u>Results: </u><br><br />
All parts were successfully restricted but again, no colonies grew on the plates.<br><br />
<u>Conclusions:</u><br><br />
Since no colonies grew on the plates even though different competent cells stock was used, and restriction went well, we assume that the competent cells are not the problem and for the next try, we would use a new DNA ligase.<strong></strong></p><br />
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<h3>Human Practice</h3><br />
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<h3 style="border-bottom:dashed;border-color:#000000">Week 9: September 28th- October 4th</h3><br />
<br><br />
<div align="center" style="float:left" class="prevNext" onClick="show(w8)"><img src="https://static.igem.org/mediawiki/2014/8/8f/BGU14finger-point3.gif" width="26px"/>&nbsp;Prev</div><br />
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<h3>Lab</h3><br />
<p><strong><u>Biobricks</u></strong><br><br />
<strong><em>Forth trial, first for this week –</em></strong><em> With a new ligase enzyme, the rest of the process is identical to the last times.</em><br><br />
<u>Details: </u><br><br />
DNA parts and backbone were digested with restriction enzymes: Pst1 and EcoR1<br><br />
The transformation was done according to the <a href="https://www.dropbox.com/s/p6fau982fhzeihe/08-Transformation%20to%20competent%20bactiria.docx?dl=0">general transformation protocol</a>.<br><br />
<u>Results: </u><br><br />
Restriction gel:</p><br />
<p ><img src="https://static.igem.org/mediawiki/2014/2/26/BGU14notefig13.png" style="height:207px"/><br />
<img src="https://static.igem.org/mediawiki/2014/6/6f/BGU14notefig14.png" style="height:207px"/></p><br />
<p>No colonies grew on the plates.<br><br />
<u>Conclusions:</u><br><br />
Since still unsuccessful. We will try a different kit for purifying restriction products. <strong></strong><br><br />
<strong><em>Fifth trial, second for this week </em></strong><br><br />
<u>Details: </u><br><br />
DNA parts and backbone were digested with restriction enzymes: Pst1 and EcoR1<br><br />
The transformation was done according to the <a href="https://www.dropbox.com/s/p6fau982fhzeihe/08-Transformation%20to%20competent%20bactiria.docx?dl=0">general transformation protocol</a>.<br><br />
<u>Results: </u><br><br />
All parts were successfully restricted, but again no colonies grew on the plates.<br><br />
<u>Conclusions:</u><br><br />
Since a lot of changes was already done, and the time became critical for iGEM deadline, it was decided to try looking for a different lab and get some help.<br><br />
<strong><em>Sixth trial, third for this week –</em></strong><em> with the former changes, we moved to Prof.&nbsp;</em>Lital<em><strong> </strong></em>Alfonta<em>'s lab, using the lab members' knowledge and experience. The transformation was changed from Heat-Shock transformation to electroporation transformation. </em><br><br />
<u>Details: </u><br><br />
DNA parts and backbone were digested with restriction enzymes: Pst1 and EcoR1<br><br />
The transformation was done according to the <a href="https://www.dropbox.com/s/p6fau982fhzeihe/08-Transformation%20to%20competent%20bactiria.docx?dl=0">general transformation protocol</a>.<br><br />
The gel products extraction, ligation and transformation was done at <em>Prof.&nbsp;</em>Lital<em><strong> </strong></em>Alfonta<em>'s lab</em> with instruction of her lab members.<span dir="RTL"> </span><br><br />
<u>Results: </u><br><br />
All parts were successfully restricted, electroporation time contants were all above 5 ms, but again no colonies grew on the plates.<br><br />
<u>Conclusions:</u></p><br />
We will try again with the same settings and a different insert to vector ratio.<br />
<p align="center" >&nbsp;</p> <br />
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<h3>Human Practice</h3><br />
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<!--October--><br />
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<h3 style="border-bottom:dashed;border-color:#000000">October</h3><br />
<br><br />
<div align="center" style="float:left" class="prevNext" onClick="show(w9)"><img src="https://static.igem.org/mediawiki/2014/8/8f/BGU14finger-point3.gif" width="26px"/>&nbsp;Prev</div><br />
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<h3>General</h3><br />
<p>*****</p> <br />
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<div id="w10" class="textCont" style="height:1020px;display:none"><br />
<h3 style="border-bottom:dashed;border-color:#000000">Week 10: October 5th- October 11th</h3><br />
<br><br />
<div align="center" style="float:left" class="prevNext" onClick="show(october)"><img src="https://static.igem.org/mediawiki/2014/8/8f/BGU14finger-point3.gif" width="26px"/>&nbsp;Prev</div><br />
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<h3>Lab</h3><br />
<p><strong><u>Biobricks- </u></strong><br><br />
<strong><em>Seventh and last trial, first for this week –</em></strong><em> with the former changes, working in different lab the insert vector ratio has been changed.</em><br><br />
<u>Details: </u><br><br />
The transformation was done according to the <a href="https://www.dropbox.com/s/p6fau982fhzeihe/08-Transformation%20to%20competent%20bactiria.docx?dl=0">general transformation protocol</a>.<br><br />
Protocol changes:</p><br />
<ul style="list-style:disc"><br />
<li><span dir="LTR"> </span>Transformation by electroporation.</li><br />
<li><span dir="LTR"> </span>The gel products extraction, ligation and transformation was done at <em>Prof.&nbsp;</em>Lital<em><strong> </strong></em>Alfonta<em>'s lab</em> with instruction of her lab members.</li><br />
<li><span dir="LTR"> </span>This time, the insert to vector ratio was raised to 4:1 instead of 3:1 <span dir="RTL"> </span></li><br />
</ul><br />
<p><u>Results: </u><br><br />
The restriction worked, but again- No colonies grew on the plates.<br><br />
<u>Conclusions:</u><br><br />
Since it was the last chance for preparing and sending the Biobricks to iGEM, unfortunately we have no more time to try and make this work. We suspect that step that prevented successful transformation was the ligation.<br><br />
<strong>Intelligent Medication</strong><br><br />
Goal: Fourth try of trying to silence eGFP in CT26 eGFP – we repeated the exact protocol form the third try, but this time we will only try to analyze results by real time PCR. <br><br />
<u>Details:</u><br><br />
Same as in third try.<br><br />
<u>Results:</u><br><br />
RealTime PCR:<br><br />
???<br><br />
<u>Conclusions:</u><br><br />
???</p><br />
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<h3>Human Practice</h3><br />
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<h3 style="border-bottom:dashed;border-color:#000000">Week 11: October 12th-October18th</h3><br />
<br><br />
<div align="center" style="float:left" class="prevNext" onClick="show(w10)"><img src="https://static.igem.org/mediawiki/2014/8/8f/BGU14finger-point3.gif" width="26px"/>&nbsp;Prev</div><br />
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<h3>Lab</h3><br />
<p><strong><u>PTPN1- </u></strong></p><br />
<p><strong><u>UCP1-</u></strong><br><br />
<em>In order to show uncoupling activity of UCP1 in the mitochondria, HepG2 cells were transfected with UCP1 by lipofectamin and tested for mitochondrial membrane potential with TMRM reagent.</em><br><br />
<u>Details:</u><br><br />
HepG2 cell-line cells were seeded (75,000 cells per well) using DMEM medium (details? ) and incubated for 24 hr at 37˚c.<br><br />
After 24 hr, the incubated cells were transfected with ucp1 (formal name?) and incubated for 24 hr at 37˚c. </p><br />
<p>After 24 hr incubation - **************NEED TO FINISH THIS WEEK*********** </p><br />
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<h3>Human Practice</h3><br />
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<h3 style="border-bottom:dashed;border-color:#000000">Week 12</h3><br />
<br><br />
<div align="center" style="float:left" class="prevNext" onClick="show(w11)"><img src="https://static.igem.org/mediawiki/2014/8/8f/BGU14finger-point3.gif" width="26px"/>&nbsp;Prev</div><br />
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<h3>Lab</h3><br />
<p>No Activity</p> <br />
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<h3>Human Practice</h3><br />
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<h3 style="border-bottom:dashed;border-color:#000000">Week 13</h3><br />
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<div align="center" style="float:left" class="prevNext" onClick="show(w12)"><img src="https://static.igem.org/mediawiki/2014/8/8f/BGU14finger-point3.gif" width="26px"/>&nbsp;Prev</div><br />
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<h3>Lab</h3><br />
<p>No Activity</p> <br />
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<h3>Human Practice</h3><br />
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