Difference between revisions of "Team:Wageningen UR/Notebook/ProteinEngineering"

 
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<h4><a href="https://2016.igem.org/Team:Wageningen_UR/Description/Specificity#Isolates">Main Results</a></h4>
 
<h4><a href="https://2016.igem.org/Team:Wageningen_UR/Description/Specificity#Isolates">Main Results</a></h4>
 
</div>
 
</div>
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<li class="menu-item">
 
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<a href="#april">April</a>
 
 
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<li class="menu-item">
 
<a href="#may">May</a>   
 
<a href="#may">May</a>   
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<a href="#august">August</a>
 
<a href="#august">August</a>
 
<li class="menu-item">
 
<li class="menu-item">
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<a href="#september">September</a>   
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<a href="#september">September</a>
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<li class="menu-item">
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<a href="#october">October</a>   
 
</li>     
 
</li>     
 
</html>
 
</html>
 
{{Wageningen_UR/menu}}
 
{{Wageningen_UR/menu}}
 
<html>
 
<html>
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<section id="april">
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<p>These experiments were performed by Linea Muhsal.</p>
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<h1><b>April</b></h1>
+
</section>
 +
<section id="may">
 +
<h1><b>May</b></h1>
 
<h2><b>Week 1</b></h2>
 
<h2><b>Week 1</b></h2>
−
<p></p>
+
<p>Setting up the lab, preparing media, preparing electrocompetent cells, primer design etc.</p>
 
<h2><b>Week 2</b></h2>
 
<h2><b>Week 2</b></h2>
−
<p></p>
+
<p>PCR of Cry3Aa from <i>Bacillus thuringiensis var. tenebrionis</i> and Cry1Ab from <i>Bacillus thuringiensis Berliner 1915</i>. Success with PCR of Cry3Aa, but not Cry1Ab. Creation of vector pBbA7c-Cry3Aa.</p>
−
<p></p>
+
−
<p></p>
+
−
<p></p>
+
 
<h2><b>Week 3</b></h2>
 
<h2><b>Week 3</b></h2>
−
<p></p>
+
<p>I tried to transform the created vector pBbA7c-Cry3Aa into Lemo21. No success.</p>
 
<h2><b>Week 4</b></h2>
 
<h2><b>Week 4</b></h2>
−
<p> </p><p>
+
<p>A lot of time spent on trying to clone the vector pBbA7c-Cry3Aa into Lemo21. Inconclusive results.</p>
−
 
+
−
</section>
+
−
<section id="may">
+
−
<h1><b>May</b></h1>
+
−
<h2><b>Week 5</b></h2>
+
−
<p></p>
+
−
<h2><b>Week 6</b></h2>
+
−
<p></p>
+
−
<h2><b>Week 7</b></h2>
+
−
<p></p>
+
−
<h2><b>Week 8</b></h2>
+
−
<p></p>
+
 
</section>
 
</section>
 
<section id="june">
 
<section id="june">
 
<h1><b>June</b></h1>
 
<h1><b>June</b></h1>
−
<h2><b>Week 9-12</b></h2>
+
<h2><b>Week 5-8</b></h2>
−
<p></p>
+
<p>No chance of working in the lab due to moving of the lab.</p>
 
</section>
 
</section>
 
<section id="july">
 
<section id="july">
 
<h1><b>July</b></h1>
 
<h1><b>July</b></h1>
−
<h2><b>Week 13</b></h2>
+
<h2><b>Week 9</b></h2>
−
<p></p>
+
<p>A fresh start with a lot of work for setting up the lab again. I learned the basics of phage work in this and the next week.</p>
−
<h2><b>Week 14</b></h2>
+
<h2><b>Week 10</b></h2>
−
<p></p>
+
<p>Continuation of learning how to work with bacteriophages. Mite feeding experiments with fluorophores: Mites do take up fluorophores with their food! But only a small amount of them (~16 %).</p>
 
</p>
 
</p>
−
<h2><b>Week 15</b></h2>
+
<h2><b>Week 11</b></h2>
−
<p></p>
+
<p>Feeding experiment with mealworms. Mealworms are easy to feed, easy to dissect and easy to keep. They take up fluorescein when fed with carrots dipped in it.</p>
−
<h2><b>Week 16</b></h2>
+
<h2><b>Week 12</b></h2>
−
<p </p>
+
<p>vacation</p>
−
 
+
−
<p>
+
  
 
</section>
 
</section>
 
<section id="august">
 
<section id="august">
 
<h1><b>August</b></h1>
 
<h1><b>August</b></h1>
−
<h2><b>Week 17</b></h2>
+
<h2><b>Week 13</b></h2>
−
<p></p>
+
<p> SDS-PAGE of mealworm proteins, mite proteins, and mealworm gut proteins.Proteins can be seen in all of the samples. There is a distinct difference in protein pattern in the gut and the overall mealworm sample. This indicates that using only the midgut for experiments enhances the chances of finding a specific toxin. Attempts to do so in mites failed due to the small size of Varroa destructor.</p>
−
  </p>
+
 
−
<h2><b>Week 18-19</b></h2>
+
<figure>
−
<p></p>
+
<img src="https://static.igem.org/mediawiki/2016/4/4a/T--Wageningen_UR--mealwormSDS.jpg">
−
<h2><b>Week 20</b></h2>
+
<figcaption>Figure 1. SDS-PAGE of mealworm proteins. M: marker, 1: protein extract mealworm 10 x diluted, 2: protein extract mealworm 100 x diluted, 3: protein extract mealworm gut 10 x diluted, 4: protein extract mealworm gut 100 x diluted.</figcaption>
−
<p></p>
+
</figure><br/>
 +
 
 +
<figure>
 +
<img src="https://static.igem.org/mediawiki/2016/8/8e/T--Wageningen_UR--miteSDS.jpg">
 +
<figcaption>Figure 2. SDS-PAGE of mite proteins. M: marker, 1: protein extract mite undiluted, 2: protein extract mite 10 x diluted, 3: protein extract mealworm gut 100 x diluted.</figcaption>
 +
</figure><br/>
 +
 
 +
<h2><b>Week 14-15</b></h2>
 +
<p>The titre of the provided phage library was to be determined. Attempts to do so via "tear-drop assay", where one X-Gal/IPTG plate can be used to characterize 4 samples, failed. This is probably due to the high mobility of the phage M13KE. The titre was obtained by using the protocol listed here (LINK). </p>
 +
<p>In vivo phage display on mites and mealworms has been performed. Titre dropped drastically after each round.</p>
 +
<h2><b>Week 16</b></h2>
 +
<p>In vivo phage display on mealworms with propagation of phages inbetween the rounds. During this, unexpected, lytic phages appeared on the titre plates. Experiments were put on hold.</p>
 
</section>
 
</section>
 
<section id="september">
 
<section id="september">
 
<h1><b>September<b></h1>
 
<h1><b>September<b></h1>
−
<h2><b>Week 21</b></h2>
+
<h2><b>Week 17</b></h2>
−
<p> </p>
+
<p>Investigation into the source of contamination. Phages appear to originate from the mealworms. In vivo phage display experiments were cancelled. </p>
 
<p>
 
<p>
 
</p>
 
</p>
−
<h2><b>Week 22</b></h2>
+
<h2><b>Week 18</b></h2>
−
<p>
+
<p>Construction of Cry3Aa-expression plasmids with random binding site mutations. Transformation into Lemo21.
−
 
+
</p>
 +
<h2><b>Week 19</b></h2>
 +
<p>In vitro phage binding display.
 +
</p>
 +
<h2><b>Week 20</b></h2>
 +
<p>Expression and first screening of Cry3Aa mutants.
 
</section>
 
</section>
 +
<section id="october">
 +
<h1><b>October<b></h1>
 +
<h2><b>Week 21</b></h2>
 +
<p>Final toxicity analysis of Cry3Aa mutants. Data analysis.</p>
 
</html>
 
</html>
 
{{Wageningen_UR/footer}}
 
{{Wageningen_UR/footer}}

Latest revision as of 23:58, 19 October 2016

MENU ▤
Wageningen UR iGEM 2016

 

These experiments were performed by Linea Muhsal.

May

Week 1

Setting up the lab, preparing media, preparing electrocompetent cells, primer design etc.

Week 2

PCR of Cry3Aa from Bacillus thuringiensis var. tenebrionis and Cry1Ab from Bacillus thuringiensis Berliner 1915. Success with PCR of Cry3Aa, but not Cry1Ab. Creation of vector pBbA7c-Cry3Aa.

Week 3

I tried to transform the created vector pBbA7c-Cry3Aa into Lemo21. No success.

Week 4

A lot of time spent on trying to clone the vector pBbA7c-Cry3Aa into Lemo21. Inconclusive results.

June

Week 5-8

No chance of working in the lab due to moving of the lab.

July

Week 9

A fresh start with a lot of work for setting up the lab again. I learned the basics of phage work in this and the next week.

Week 10

Continuation of learning how to work with bacteriophages. Mite feeding experiments with fluorophores: Mites do take up fluorophores with their food! But only a small amount of them (~16 %).

Week 11

Feeding experiment with mealworms. Mealworms are easy to feed, easy to dissect and easy to keep. They take up fluorescein when fed with carrots dipped in it.

Week 12

vacation

August

Week 13

SDS-PAGE of mealworm proteins, mite proteins, and mealworm gut proteins.Proteins can be seen in all of the samples. There is a distinct difference in protein pattern in the gut and the overall mealworm sample. This indicates that using only the midgut for experiments enhances the chances of finding a specific toxin. Attempts to do so in mites failed due to the small size of Varroa destructor.

Figure 1. SDS-PAGE of mealworm proteins. M: marker, 1: protein extract mealworm 10 x diluted, 2: protein extract mealworm 100 x diluted, 3: protein extract mealworm gut 10 x diluted, 4: protein extract mealworm gut 100 x diluted.

Figure 2. SDS-PAGE of mite proteins. M: marker, 1: protein extract mite undiluted, 2: protein extract mite 10 x diluted, 3: protein extract mealworm gut 100 x diluted.

Week 14-15

The titre of the provided phage library was to be determined. Attempts to do so via "tear-drop assay", where one X-Gal/IPTG plate can be used to characterize 4 samples, failed. This is probably due to the high mobility of the phage M13KE. The titre was obtained by using the protocol listed here (LINK).

In vivo phage display on mites and mealworms has been performed. Titre dropped drastically after each round.

Week 16

In vivo phage display on mealworms with propagation of phages inbetween the rounds. During this, unexpected, lytic phages appeared on the titre plates. Experiments were put on hold.

September

Week 17

Investigation into the source of contamination. Phages appear to originate from the mealworms. In vivo phage display experiments were cancelled.

Week 18

Construction of Cry3Aa-expression plasmids with random binding site mutations. Transformation into Lemo21.

Week 19

In vitro phage binding display.

Week 20

Expression and first screening of Cry3Aa mutants.

October

Week 21

Final toxicity analysis of Cry3Aa mutants. Data analysis.