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

(Created page with "{{Wageningen_UR}} {{Wageningen_UR/header}} <html> <div class="menu-head"> <h4><a href="https://2016.igem.org/Team:Wageningen_UR/Description/Specificity#Isolates">Main Results</...")
 
Line 22: Line 22:
 
<section id="june">
 
<section id="june">
 
<h1><b>June</b></h1>
 
<h1><b>June</b></h1>
−
<h2><b>Week 5-8</b></h2>
+
<h2><b>Week 1-3</b></h2>
−
<p>No chance of working in the lab due to moving of the lab.</p>
+
<p>Literature research on pDusk and pDawn system as well as mazEF toxin antitoxin system.</p>
 +
<h2><b>Week 4</b></h2>
 +
<p>Getting familiar with Matlab and setting backbone model structure</p>
 
</section>
 
</section>
 
<section id="july">
 
<section id="july">
 
<h1><b>July</b></h1>
 
<h1><b>July</b></h1>
−
<h2><b>Week 9</b></h2>
+
<h2><b>Week 5-6</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>
+
<p>First dynamic model structure of pDusk and pDawn was built. Light unit conversion was is given here: <br/><br/>
−
<h2><b>Week 10</b></h2>
+
h = 6.63*10<sup>-34</sup> ([Js] Planck's constant)<br/>
−
<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>
+
c = 2.988*10<sup>8</sup> ([m/s] speed of light)<br/>
 +
lambda<sub>nm</sub> = 470 (% [nm] wavelength)<br/>
 +
N<sub>A</sub> = 6.022*10<sup>23</sup> ([mol<sup>-1</sup>] Avogadro's constant)<br/>
 +
<br/>
 +
N<sub>interm</sub> = N<sub>initial</sub>*10<sup>-2</sup> ([µW*cm^<sup>-2</sup>] → [W*m<sup>-2</sup>])
 +
E<sub>p</sub> = h*c/lambda<sub>nm</sub> (distinct energy quanta of a photon)
 +
N<sub>p</sub> = N<sub>interm</sub>*lambda<sub>nm</sub>*10<sup>6</sup>/(1.988*10<sup>-16</sup>) (number of photons; lambda in [nm])
 +
E<sub>q</sub> = N<sub>p</sub>/N<sub>A</sub> (photonflux N = E<sub>q</sub> in [µmol*m<sup>-2</sup>*s<sup>-1</sup>])
 +
N = E<sub>q</sub>*60*60 ([µmol*m<sup>-2</sup>*s<sup>-1</sup>] → [µmol*m^-2*h^-1])<br/><br/>
 +
</p>
 +
<h2><b>Week 7-8</b></h2>
 +
<p>Vacation</p>
 
</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>
 
  
 
</section>
 
</section>
 
<section id="august">
 
<section id="august">
 
<h1><b>August</b></h1>
 
<h1><b>August</b></h1>
−
<h2><b>Week 13</b></h2>
+
<h2><b>Week 9-11</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>
+
<p>Improved the equations for pDusk and pDawn. And added the equations for mazE and mazF.</p>
 +
<h2><b>Week 12</b></h2>
 +
<p>Got first useful model results for pDusk and pDawn individually. At this point, there was no weighted means approach yet.</p>
 +
<p>In vivo phage display on mites and mealworms has been performed. Titre dropped drastically after each round.</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>
 
<section id="september">
 
<section id="september">
 
<h1><b>September<b></h1>
 
<h1><b>September<b></h1>
−
<h2><b>Week 17</b></h2>
+
<h2><b>Week 13-14</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>Realised, that we should combine both the pDusk and pDawn scoring function with a weighted means approach. Started to rewrite the whole script.</p>
−
<p>
+
<h2><b>Week 15</b></h2>
−
</p>
+
<p>Vacation</p>
−
<h2><b>Week 18</b></h2>
+
<h2><b>Week 16</b></h2>
−
<p>Construction of Cry3Aa-expression plasmids with random binding site mutations. Transformation into Lemo21.
+
<p>Noticed problem with the complex formation. Read up on the complex formation of mazEF. There are actually several stages of the complex. After some iterations, we concluded to model the complex as done in the current <a href="https://static.igem.org/mediawiki/2016/c/c2/T--Wageningen_UR--Detailed_Methods_Kill_Switch.pdf">equations.</a></p>
−
</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">
 
<section id="october">
 
<h1><b>October<b></h1>
 
<h1><b>October<b></h1>
−
<h2><b>Week 21</b></h2>
+
<h2><b>Week 17-18</b></h2>
−
<p>Final toxicity analysis of Cry3Aa mutants. Data analysis.</p>
+
<p>Worked on updating last adjustments. Rewrote all Matlab files to a readable format - noticed quite some small bugs on the way. Ran the analysis and the evaluation. Got some preliminary results from the lab! Implemented that in my analysis last minute.</p>
 +
<h2><b>Week 19</b></h2>
 +
<p>The Wiki Week.</p>
 
</html>
 
</html>
 
{{Wageningen_UR/footer}}
 
{{Wageningen_UR/footer}}

Revision as of 02:44, 19 October 2016

MENU ▤
Wageningen UR iGEM 2016

 

June

Week 1-3

Literature research on pDusk and pDawn system as well as mazEF toxin antitoxin system.

Week 4

Getting familiar with Matlab and setting backbone model structure

July

Week 5-6

First dynamic model structure of pDusk and pDawn was built. Light unit conversion was is given here:

h = 6.63*10-34 ([Js] Planck's constant)
c = 2.988*108 ([m/s] speed of light)
lambdanm = 470 (% [nm] wavelength)
NA = 6.022*1023 ([mol-1] Avogadro's constant)

Ninterm = Ninitial*10-2 ([µW*cm^-2] → [W*m-2]) Ep = h*c/lambdanm (distinct energy quanta of a photon) Np = Ninterm*lambdanm*106/(1.988*10-16) (number of photons; lambda in [nm]) Eq = Np/NA (photonflux N = Eq in [µmol*m-2*s-1]) N = Eq*60*60 ([µmol*m-2*s-1] → [µmol*m^-2*h^-1])

Week 7-8

Vacation

August

Week 9-11

Improved the equations for pDusk and pDawn. And added the equations for mazE and mazF.

Week 12

Got first useful model results for pDusk and pDawn individually. At this point, there was no weighted means approach yet.

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

September

Week 13-14

Realised, that we should combine both the pDusk and pDawn scoring function with a weighted means approach. Started to rewrite the whole script.

Week 15

Vacation

Week 16

Noticed problem with the complex formation. Read up on the complex formation of mazEF. There are actually several stages of the complex. After some iterations, we concluded to model the complex as done in the current equations.

October

Week 17-18

Worked on updating last adjustments. Rewrote all Matlab files to a readable format - noticed quite some small bugs on the way. Ran the analysis and the evaluation. Got some preliminary results from the lab! Implemented that in my analysis last minute.

Week 19

The Wiki Week.