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

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<section id="may">
 
<section id="may">
 
<h1><b>May</b></h1>
 
<h1><b>May</b></h1>
−
<h2><b>Week 1</b></h2>
+
<h2><b>02 - 12 May</b></h2>
−
<p>Setting up the lab, preparing media, preparing electrocompetent cells, primer design etc.</p>
+
<p>First attempt in constructing the inverter part out of biobricks Bba_P0140 and Bba_I13521. This has been done via the standard iGEM 3A assembly protocol [Link]. The transformation resulted in one red colony.  
−
<h2><b>Week 2</b></h2>
+
<br><br>
−
<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>
+
This colony has been checked by placing the constitutive promoter J23119 in front of it via another 3A assembly. If the construct is correct, the result should be white colonies.  
−
<h2><b>Week 3</b></h2>
+
<br><br>
−
<p>I tried to transform the created vector pBbA7c-Cry3Aa into Lemo21. No success.</p>
+
However, this was not the case. Sequencing and PCR results showed that the construct only contained Bba_P0140. </p>
−
<h2><b>Week 4</b></h2>
+
<h2><b>13 - 24 May</b></h2>
−
<p>A lot of time spent on trying to clone the vector pBbA7c/Cry3Aa into Lemo21. Inconclusive results.</p>
+
<p>Creation of the riboswitch part out of <I>Bascillis subtilis </I> and <I>Escherichia coli</I>. The primers used for this can be found in table X. Extraction of genomic DNA from B. subtilis and E. coli was done with the use of the GeneJet genomic DNA purification kit and an overnight culture of B. subtilis strain 168 that has grown in B. subtilis medium and an overnight culture of E. coli strain DH5𝛂 in normal LB medium. The extraction was done according to the supplied Gram-positive and Gram-negative bacteria standard protocol.
−
</section>
+
<br><br>
−
<section id="june">
+
For the PCR the standard protocol of New England Biolab has been used. For the retrieval of the vitamin b12 riboswitch out of <I>Escherichia coli</I> DH5alpha an annealing temperature of 66° was used and for the guanine riboswitch out of <I>Bascillis subtilis </I> an annealing temperature of 63°. PCR and sequencing results showed that both constructs were correctly retrieved.
−
<h1><b>June</b></h1>
+
<br><br>
−
<h2><b>Week 5-8</b></h2>
+
Via 3A-assembly the riboswitch part and the inverter part have been assembled. This resulted in three types of colonies: white, pink and red. The correct construct should be around 2500 base pairs.  
−
<p>No chance of working in the lab due to moving of the lab.</p>
+
<br><br>
−
</section>
+
PCR amplification and sequencing results showed that the pink colonies where the correct construct. </p>
−
<section id="july">
+
<h2><b>05 - 20 July </b></h2>
−
<h1><b>July</b></h1>
+
<p>To provide a basic proof of principle of the designed construct, a colony containing the B/GRinv plasmid was grown overnight in 10 mL LB medium. The concentration range of guanine to test the guanine riboswitch construct were based on the concentrations used in previous experiments with guanine riboswitches from B. Subtilis. The concentration range of vitamin b12 was based on the concentrations normally used for testing bacteria that are used to determine the amount of vitamin b12 in human blood. Five tubes containing a concentration of f 0, 200 ng L-1 , 2 μg L-1, 20 μg L-1, 200 μg L-1 and 2 mg L-1 vitamin b12 and 0, 0.01, 0.05, 0.1, 0.5 and 1 mg mL-1 guanine were used.
−
<h2><b>Week 9</b></h2>
+
−
<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 10</b></h2>
+
−
<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>
+
−
<h2><b>Week 11</b></h2>
+
−
<p>Feeding experiment with mealworms. Mealworms are easy to feed, easy to dissect and easy to keep. They take up fluorescine when fed with carrots dipped in it.</p>
+
−
<h2><b>Week 12</b></h2>
+
−
<p>vacation</p>
+
  
 +
These cultures were spin down at 4700 RPM for 3 minutes in order to compare the colouration of the pellets.  </p>
 +
<h2><b>05 September - 05 October</b></h2>
 +
<p>For the second proof of principle experiment, agar plates were overgrown with E. coli containing the B/GRinv. In the middle of the plate a small scoop (0,5 cm in diameter) was taken out and filled with 100 µL of a 50 mg mL-1 guanine solution or 100 µL of a 200 mL L-1 vitamin b12 solution to create a decreasing gradient from the middle.
 +
To provide clear images showing the expressed mRFP, pictures were taking using the G:Box gel imager of Syngene. The RFP filter protocol  provided with the software programme was used. </p>
 
</section>
 
</section>
−
<section id="august">
 
−
<h1><b>August</b></h1>
 
−
<h2><b>Week 13</b></h2>
 
−
<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>
 
  
−
<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 id="september">
 
−
<h1><b>September<b></h1>
 
−
<h2><b>Week 17</b></h2>
 
−
<p>Investigation into the source of contamination. Phages appear to originate from the mealworms. In vivo phage display experiments were cancelled. </p>
 
−
<p>
 
−
</p>
 
−
<h2><b>Week 18</b></h2>
 
−
<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 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>
 
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Revision as of 23:34, 12 October 2016

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Wageningen UR iGEM 2016

 

May

02 - 12 May

First attempt in constructing the inverter part out of biobricks Bba_P0140 and Bba_I13521. This has been done via the standard iGEM 3A assembly protocol [Link]. The transformation resulted in one red colony.

This colony has been checked by placing the constitutive promoter J23119 in front of it via another 3A assembly. If the construct is correct, the result should be white colonies.

However, this was not the case. Sequencing and PCR results showed that the construct only contained Bba_P0140.

13 - 24 May

Creation of the riboswitch part out of Bascillis subtilis and Escherichia coli. The primers used for this can be found in table X. Extraction of genomic DNA from B. subtilis and E. coli was done with the use of the GeneJet genomic DNA purification kit and an overnight culture of B. subtilis strain 168 that has grown in B. subtilis medium and an overnight culture of E. coli strain DH5𝛂 in normal LB medium. The extraction was done according to the supplied Gram-positive and Gram-negative bacteria standard protocol.

For the PCR the standard protocol of New England Biolab has been used. For the retrieval of the vitamin b12 riboswitch out of Escherichia coli DH5alpha an annealing temperature of 66° was used and for the guanine riboswitch out of Bascillis subtilis an annealing temperature of 63°. PCR and sequencing results showed that both constructs were correctly retrieved.

Via 3A-assembly the riboswitch part and the inverter part have been assembled. This resulted in three types of colonies: white, pink and red. The correct construct should be around 2500 base pairs.

PCR amplification and sequencing results showed that the pink colonies where the correct construct.

05 - 20 July

To provide a basic proof of principle of the designed construct, a colony containing the B/GRinv plasmid was grown overnight in 10 mL LB medium. The concentration range of guanine to test the guanine riboswitch construct were based on the concentrations used in previous experiments with guanine riboswitches from B. Subtilis. The concentration range of vitamin b12 was based on the concentrations normally used for testing bacteria that are used to determine the amount of vitamin b12 in human blood. Five tubes containing a concentration of f 0, 200 ng L-1 , 2 μg L-1, 20 μg L-1, 200 μg L-1 and 2 mg L-1 vitamin b12 and 0, 0.01, 0.05, 0.1, 0.5 and 1 mg mL-1 guanine were used. These cultures were spin down at 4700 RPM for 3 minutes in order to compare the colouration of the pellets.

05 September - 05 October

For the second proof of principle experiment, agar plates were overgrown with E. coli containing the B/GRinv. In the middle of the plate a small scoop (0,5 cm in diameter) was taken out and filled with 100 µL of a 50 mg mL-1 guanine solution or 100 µL of a 200 mL L-1 vitamin b12 solution to create a decreasing gradient from the middle. To provide clear images showing the expressed mRFP, pictures were taking using the G:Box gel imager of Syngene. The RFP filter protocol provided with the software programme was used.