Team:Evry/Notebook/Test

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<h1 id="week3">Week 3: 1<sup>st</sup> July - 7<sup>th</sup> July</h1>
<h1 id="week3">Week 3: 1<sup>st</sup> July - 7<sup>th</sup> July</h1>
 +
<h2> Tuesday, July 2nd </h2>
 +
<p>We prepared the BL21 and BG1655 strains to be competent.</p>
 +
 +
 +
<h2> Wednesday, July 3rd </h2>
 +
<p>The two strains have been contaminated  and are not usable.</p>
 +
 +
 +
<h2> Thursday, July 4th </h2>
 +
<p>Genomic DNA extraction of the BL21 and BG1655 strains have been done using two different methods.
 +
We received the primers we have ordered. They have been prepared to do a PCR. In order to get our genes of interest we did a PCR using as the DNA template the genomic DNA extracted from BL21 and BG1655.</p>
 +
 +
 +
<h2>Friday, July 5th</h2>
 +
<p>To ensure the amplification of our genes by PCR, we did a agarose gel electrophoresis. To do so, we first prepared a TAE 50X stock solution. By diluting it, we use a 1X TAE solution to do a 1% agarose gel. We obtain the following gel. That showed that only two of our genes have been amplificated by PCR. A new PCR will be made.</p>
<!--*************************************** WEEK 4 ****************************************************************-->
<!--*************************************** WEEK 4 ****************************************************************-->
<h1 id="week4">Week 4: 8<sup>th</sup> July - 14<sup>th</sup> July</h1>
<h1 id="week4">Week 4: 8<sup>th</sup> July - 14<sup>th</sup> July</h1>
 +
<h2> Monday, July 8th </h2>
 +
<p>We prepared the BL21 and BG1655 strains to be competent.</p>
 +
 +
<h2> Tuesday, July 9th </h2>
 +
 +
<table id='team' cellspacing='10' align='center'>
 +
  <tr>   
 +
    <td>
 +
      <img src="https://static.igem.org/mediawiki/2013/e/e1/WP_20130711_004.jpg" alt="Petri box" width=200/>                   
 +
      <img src="https://static.igem.org/mediawiki/2013/b/b7/WP_20130711_006.jpg" alt="Petri box" width=192/>
 +
      <p>
 +
        Both strain are not contaminated.
 +
      </p>
 +
    </td>
 +
 +
    <td> 
 +
      <img src="https://static.igem.org/mediawiki/2013/f/f1/WP_20130711_002.jpg" alt="Petri box" width=170/>                   
 +
      <img src="https://static.igem.org/mediawiki/2013/c/cf/WP_20130711_003.jpg" alt="Petri box" width=151/>
 +
      <p>
 +
        BL21 are highly competent, BG1655 are little competent.
 +
      </p> 
 +
    </td>
 +
  </tr>
 +
</table>
 +
 +
<h2> Wednesday, July 10th </h2>
 +
 +
<h3>PCR procedure optimization</h3>
 +
 +
<p>
 +
<b><u>Aim:</u> Define the number of matrix of optimal DNA to amplify a gene by PCR.</b><br/><br/>
 +
 +
<b><u>Data:<br/></u>
 +
</p>
 +
<ul>
 +
<li> [primer] = --- ng/µl<br/>
 +
<li> size(primer) = --- nt<br/>
 +
</ul></b><br/>
 +
 +
<p>
 +
For PCR we chose to use … ng of primer (V=…µL). As we  know the size of our primers, we could define the number of primers that we have to use to begin the reaction.
 +
We define the number of recquired matrix DNA in terms of concentration of sample with the following formula:
 +
<br/><br/>
 +
 +
<b> AJOUTER LA FORMULE </b><br/><br/>
 +
 +
Analysis of Agarose gel's results:<br/><br/>
 +
 +
<b> AJOUTER LA PHOTO LEGENDEE </b> <br/>
 +
</p>
 +
 +
<h3> Extraction of natural promoter sequence </h3>
 +
 +
<p>
 +
<b><u>Aim:</u></b>  We want to have sequences genes' promoters' sequences under the control of FUR (Ferric Uptake Regulation)transcription factor. <br/><br/>
 +
We remake PCR samples that failed the previous week.<br/>
 +
</p>
 +
  <ul>
 +
    <li> Fec A
 +
    <li> Ent C
 +
    <li> Fec C
 +
    <li> Ace B
 +
  </ul>
 +
<br/>
 +
<p>
 +
PCR products are placed on gel and purificate. After, PCR products are tested with nanodrop.<br/><br/>
 +
 +
</p>
 +
 +
<table id='Dosage PCR' cellspacing='10' align='center'>
 +
  <tr>   
 +
    <td>
 +
      <p>
 +
        <b>Fec A</b> (BG1655)<br/>
 +
        [c] = 23.7 ng/µl<br/>
 +
        260/280 = 1.90<br/>
 +
      </p>
 +
    </td>
 +
 +
    <td>
 +
      <p>
 +
        <b>Fec A</b> (BL21)<br/>
 +
        [c] = 53.1 ng/µl<br/>
 +
        260/280 = 1.73<br/>
 +
      </p>
 +
    </td>
 +
 +
    <td>
 +
      <p>
 +
        <b>Ent C</b> (BG1655)<br/>
 +
        [c] = 52.4 ng/µl<br/>
 +
        260/280 = 1.71<br/>
 +
      </p>
 +
    </td>
 +
 +
    <td>
 +
      <p>
 +
        <b>Ent C</b> (BL21)<br/>
 +
        [c] = 50.0 ng/µl<br/>
 +
        260/280 = 1.94<br/>
 +
      </p>
 +
    </td>
 +
 +
    <td>
 +
      <p>
 +
        <b>Ace B</b> (BG1655)<br/>
 +
        [c] = 46.5 ng/µl<br/>
 +
        260/280 = 1.89<br/>
 +
      </p>
 +
    </td>
 +
 +
    <td>
 +
      <p>
 +
        <b>Fec C</b> (BG1655)<br/>
 +
        [c] = 34.2 ng/µl<br/>
 +
        260/280 = 1.91<br/>
 +
      </p>
 +
    </td>
 +
  </tr>
 +
 +
</table>
 +
 +
 +
<img src="https://static.igem.org/mediawiki/2013/9/9b/PCR_10-07.png" alt="pcr 10-07" width=250 />
 +
 +
<h3>Tris-HCl solution preparation(1M) : Stock solution 100X</h3>
 +
 +
<p>
 +
<b><u>Aim:</u></b> Tris-HCl solution will be use in a final concentration of 10 mM to resuspend our primers.<br/>
 +
</p>
 +
<u>Preparation of the stock solution (1 M, pH 7.5) for a volume of 50 mL:</u><br/>
 +
  <ol>
 +
<br><br/>
 +
    <li> Disolve 4,6g of Trisbase in 30 mL of distilled water.
 +
    <li> Adjust pH with concentrated HCl(~4 mL) until pH=7,5.
 +
    <li>Add distilled water until 50 mL then put in autoclave.
 +
  </ol> <br/>
 +
<p>
 +
<b><u>Note: </u> If the solution has a yellow coloration, do the preparation again with better Trisbase.</b>
 +
<br/><br/>
 +
<u>Preparation of the solution (10mM, pH 7.5) for a volume of 50 mL.</u><br/>
 +
Take 50µL of the stock solution at 1M and add 49.5 mL of water for the dilution.
 +
</p>
 +
 +
<h3>Kanamycin preparation</h3>
 +
 +
<p>
 +
Preparation of 3 tubes of 1.5 mL of Kanamycin.
 +
</p>
 +
 +
<h2>Thursday, July 11</h2>
 +
 +
<h3>Preparation of primers solution:</h3>
 +
 +
  <ol>
 +
    <li>Centrifugate tubes at 8000 rpm, begin 30 seconds.
 +
    <li>Add 250 µL of Tris HCl (10 mM); [Primer] = 100µM
 +
  </ol>
 +
<p>
 +
We prepare a diluted solution at 5 µM from to the stock solution, for PCR reactions. <br/>
 +
Dilution at 1/20: We take 5 µL of the stock solution (100 µM) and we diluate in 95 µL of tris-HCl (10 mM).
 +
 +
<h3>Transformation of BL21:</h3>
 +
 +
<p>
 +
Chemical transformation of BL21 with Cyrille's samples:
 +
 +
  <ul>
 +
    <li> Terminator (T)
 +
    <li> Promotor (P)
 +
    <li> Plasmid 1K3
 +
    <li> Plasmid 1C3
 +
    <li> sfGFP
 +
  </ul>
 +
 +
<p>
 +
These transformations allow us to do glycerols of these constrctions.
 +
</p>
 +
 +
<h3>PCR on E.coli genom (BG1655 strain)</h3>
 +
 +
  <ul>
 +
    <li> Fep A (Primers P021 and P022)
 +
    <li> Fes (Primers P023 and P024)
 +
    <li> sdh C (Primers P025 and  P026)
 +
    <li> ybi L (Primers P027 and P028)
 +
    <li> ync E (Primers P029 and P030)
 +
  </ul>
 +
 +
 +
<img src="https://static.igem.org/mediawiki/2013/7/75/PCR_11-07.png" alt="pcr 11-07" width=250 />
 +
 +
<h2>Friday, July 12</h2>
 +
 +
<h3>Transformations test</h3>
 +
 +
  <ul>
 +
    <li> Terminator (T) = OK
 +
    <li> Promotor (P) = OK
 +
    <li> Plasmid 1K3 = OK
 +
    <li> Plasmid 1C3 = OK
 +
    <li> sfGFP = OK
 +
  </ul>
 +
 +
<p>
 +
<b><u>Note :</u> The negative control was suspect.</b> <br/><br/>
 +
 +
Petri dishes are le on the bench at room temperature during the week-end, colonies will be reisolate next week. <br/>
 +
</p>
 +
 +
<h3>Migration of samples of PCR extraction.</h3>
 +
 +
<p>
 +
<img src="https://static.igem.org/mediawiki/2013/b/b1/PCR_12-07.png" alt="pcr 12-07" width=300 /> <br/><br/>
 +
 +
 +
The promoter sequence of genes Fep A, Fes, Sdh C, ybi L and ync E were extracted successfully.<br/>
 +
Samples were purified and, after, tested with nanodrop.<br/>
 +
</p>
 +
 +
<table id='Dosage PCR' cellspacing='10' align='center'>
 +
  <tr>   
 +
    <td>
 +
      <p>
 +
        <b>Fep A</b> (BG1655)<br/>
 +
        [c] = 38.3 ng/µl<br/>
 +
        260/280 = 1.93<br/>
 +
      </p>
 +
    </td>
 +
 +
    <td>
 +
      <p>
 +
        <b>Fes</b> (BG1655)<br/>
 +
        [c] = 45.5 ng/µl<br/>
 +
        260/280 = 1.89<br/>
 +
      </p>
 +
    </td>
 +
 +
    <td>
 +
      <p>
 +
        <b>Sdh C</b> (BG1655)<br/>
 +
        [c] = 28.5 ng/µl<br/>
 +
        260/280 = 1.86<br/>
 +
      </p>
 +
    </td>
 +
    <td>
 +
      <p>
 +
        <b>ybi L</b> (BG1655)<br/>
 +
        [c] = 31.0 ng/µl<br/>
 +
        260/280 = 2.09<br/>
 +
      </p>
 +
    </td>
 +
 +
    <td>
 +
      <p>
 +
        <b>ync E</b> (BG1655)<br/>
 +
        [c] = 25.1 ng/µl<br/>
 +
        260/280 = 1.93<br/>
 +
      </p>
 +
    </td>
 +
  </tr>
 +
</table>
 +
 +
<h1>Voir s'il ne faut pas faire le tableau recap des extractions</h1>
<!--*************************************** WEEK 5 ****************************************************************-->
<!--*************************************** WEEK 5 ****************************************************************-->
<h1 id="week5">Week 5: 15<sup>th</sup> July - 21<sup>st</sup> July</h1>
<h1 id="week5">Week 5: 15<sup>th</sup> July - 21<sup>st</sup> July</h1>
 +
 +
<h1>Week 5: 15<sup>th</sup> July - 21<sup>st</sup> July</h1>
 +
 +
<h2> Monday, July 15th </h2>
 +
 +
<h2> Tuesday, July 16th </h2>
 +
      <img src="https://static.igem.org/mediawiki/2013/c/c9/PCR_optimisation_16-07.png" alt="PCR Optimisation" width=300/> 
 +
 +
<h2> Wednesday, July 17th </h2>
 +
<p>We prepared the TOP10 to be competent.</p><br/>
 +
 +
<p>We launch our first Golden Gates.</p>
 +
 +
<h2> Thursday, July 18th </h2>
 +
 +
<h2> Friday, July 19th </h2>
<!--*************************************** WEEK 6 ****************************************************************-->
<!--*************************************** WEEK 6 ****************************************************************-->
<h1 id="week6">Week 6: 22<sup>nd</sup> July - 28<sup>th</sup> July</h1>
<h1 id="week6">Week 6: 22<sup>nd</sup> July - 28<sup>th</sup> July</h1>
 +
 +
<h2> Monday, July 22nd </h2>
 +
 +
<p>
 +
We did a PCR on the seven products of golden gate 1 using the VR and VF2 primers. We mixed :
 +
</p>
 +
<ul>
 +
<li>10 uL of One Taq Buffer 10X
 +
<li>1 uL of 10 mM dNTPs
 +
<li>1 uL of each VF2 and VR primers
 +
<li>35,5 uL of H2O
 +
<li>1 uL of the One Taq enzyme
 +
<li>1 uL of the golden gate products
 +
</ul>
 +
</p>
 +
 +
<p>
 +
We used the following PCR program :
 +
</p>
 +
 +
<p>
 +
<ol>
 +
<li> 95°C for 3 min
 +
<li> 95°C for 15 sec
 +
<li> 55°C for 30 sec
 +
<li> 68°C for 2 min
 +
<li> 68°C for 10 min
 +
</ol>
 +
</p>
 +
 +
<p>
 +
<b><u>Note:</u> We repeated step 2, 3 and 4 29 times.</b>
 +
</p>
 +
 +
 +
<p>
 +
The gel migration didn't reveal any band. We supposed that it hadn't worked due to the annealing temperature we used.
 +
Indeed, with the help of the New England BioLabs Tm Calculator website, we have found that the optimal annealing temperature for those primers was 53°C.     
 +
</p>
 +
 +
 +
<h2> Tuesday, July 23rd </h2>
 +
 +
<p>
 +
In order to test the best annealing temperature for our following PCRs, we did a PCR on our 5 first golden gate products with two different annealing temperature : 53 or 51°C. We also prepared two positiv controls for each conditions with either bacteria transformed by empty PSB1A3 plasmid or 1 uL of the miniprep plasmid. This was realised in order to verify if the problem of our first PCR was due to a bad lysis of our bacteria. What is more, we prepared one negativ control with an annealing temperature of 55°C.</p></br>
 +
 +
(Mettre photo gel)</br>
 +
 +
 +
<p>According to our results, we can assume that our first PCR was badly realised due to a handling error and not because of the annealing temperature we used.</p></br>
 +
 +
 +
 +
<p>In the meantime, we did the Golden Gate 1 again in order to obtain better ligation results. We transformed TOP 10 E. coli with the golden gate products and plated them on LB-Agar with carbenicillin antibiotic. The petri dishes have been let overnight at 37°C.
 +
</p>
 +
 +
 +
<h2> Wednesday, July 24th </h2>
 +
 +
<p> We did a colony PCR from colony obtained on the petri dishes plated with golden gate 2 products transformed bacteria. We chose one white colony of each of the first ten different FUR BS constructions. The positiv control was prepared by using the PSB1A3 plasmid. The same PCR program was used but we were out of One Taq so we utilize Dream Taq. </p>
 +
 +
<p>We re isolate colony of bacteria transformed with our golden gate 1 products.</p>
 +
 +
 +
 +
<h2> Thursday, July 25th </h2>
 +
 +
<p> We did a 1% gel in order to migrate our PCR products. It revealed that our PCR hadn't work. We then realised that we hadn't changed the elongation temperature of our PCR program. Indeed, the dream taq functions at 72°C.           
 +
 +
<p> We did a PCR colony with the white colony obtained after we plated our golden gate 1 products transformed bacteria.
 +
<p>
 +
We used the following PCR program :
 +
</p>
 +
 +
<p>
 +
<ol>
 +
<li> 95°C for 3 min
 +
<li> 95°C for 15 sec
 +
<li> 55°C for 30 sec
 +
<li> 72°C for 2 min
 +
<li> 72°C for 10 min
 +
</ol>
 +
</p>
 +
 +
<p>(mettre photo gel)</p>
 +
 +
<p> Considering our gel, we can reasonably think that our golden gate had worked properly. We then did miniprep.
 +
 +
<table id='team' cellspacing='20' align='center'>
 +
  <tr>   
 +
    <td>
 +
      <img src="https://static.igem.org/mediawiki/2013/1/13/PCR_25-07_FecA_AceB_FepA.png" alt="Clone 1-4 FecA AceB FepA" width=400/>                   
 +
      <img src="https://static.igem.org/mediawiki/2013/8/87/PCR_26-07_FecA_AceB_FepA.png" alt="Clone 5-8 FecA AceB FepA" width=400/>
 +
 +
    </td>
 +
 +
  </tr>
 +
</table>
 +
 +
 +
<table id='team' cellspacing='20' align='center'>
 +
  <tr>   
 +
    <td>
 +
      <img src="" alt="Clone 1-4 FecA AceB FepA" width=200/>                   
 +
      <img src="" alt="Clone 5-8 FecA AceB FepA" width=50/>
 +
 +
    </td>
 +
 +
  </tr>
 +
</table>
 +
 +
 +
<h2> Friday, July 26th </h2>
 +
 +
We used nanodrop to measure the concentration of plasmid of our minipreps. The concentrations obtained were extremely low. We decided to do it again, but we got the same low results.
<!--*************************************** WEEK 7 ****************************************************************-->
<!--*************************************** WEEK 7 ****************************************************************-->
<h1 id="week7">Week 7: 29<sup>th</sup> July - 4<sup>th</sup> August</h1>
<h1 id="week7">Week 7: 29<sup>th</sup> July - 4<sup>th</sup> August</h1>
 +
 +
<h2>Construction of plasmid N°1</h2>
 +
<p>We make an electrophoresis with 5 µL of plamsid to check the plasmid purification made on the last friday.</p>
 +
 +
<h1> Mettre l'image légendée ici </h1>
 +
 +
<p>There is not the 3 bandes that we sould see, so to check another time, we make a digestion with Pst I and EcoR I:
 +
  <ul>
 +
    <li> NEB Buffer 10X  5 µL
 +
    <li> BSA 10X  5 µL
 +
    <li> DNA  2 µg
 +
    <li> PstI  2 µL
 +
    <li> EcoRI  2 µL
 +
    <li> Water  50 µL
 +
  </ul>
 +
 +
After another electrophoresis, we obtain no band.<p>
 +
 +
<h3>Sequencing preparation</h3>
 +
 +
<table cellpadding="10" cellspacing="0" align='center' border="1">
 +
<thead>
 +
  <tr>
 +
  <th align="center">
 +
    Name
 +
  </th>
 +
  <th align="center">
 +
    Clone
 +
  </th>
 +
  <th align="center">
 +
    Concentration
 +
  </th>
 +
  <th align="center">
 +
    260/280
 +
  </th>
 +
  <th align="center">
 +
    260/230
 +
  </th>
 +
  <th align="center">
 +
    Sequencing
 +
  </th>
 +
  </tr>
 +
</thead>
 +
 +
<tbody>
 +
  <tr>
 +
  <td rowspan="4" align="center">
 +
    <p>AceB Fur Binding Site
 +
    <br/>(with sfGFP)</p>
 +
  </td>
 +
  <td align="center">
 +
    1
 +
  </td>
 +
  <td align="center">
 +
    166.1 ng/µL
 +
  </td>
 +
  <td align="center">
 +
    1.86
 +
  </td>
 +
  <td align="center">
 +
    1.76
 +
  </td>
 +
  <td align="center">
 +
    Good
 +
  </td>
 +
  </tr>
 +
  <tr>
 +
  <td align="center">
 +
    2
 +
  </td>
 +
  <td align="center">
 +
    192.0 ng/µL
 +
  </td>
 +
  <td align="center">
 +
    1.62
 +
  </td>
 +
  <td align="center">
 +
    1.02
 +
  </td>
 +
  <td align="center">
 +
    Good
 +
  </td>
 +
  </tr>
 +
  <tr>
 +
  <td align="center">
 +
    3
 +
  </td>
 +
  <td align="center">
 +
    123.5 ng/µL
 +
  </td>
 +
  <td align="center">
 +
    1.87
 +
  </td>
 +
  <td align="center">
 +
    1.75
 +
  </td>
 +
  <td align="center">
 +
    Good
 +
  </td>
 +
  </tr>
 +
  <tr>
 +
  <td align="center">
 +
    4
 +
  </td>
 +
  <td align="center">
 +
    128.0 ng/µL
 +
  </td>
 +
  <td align="center">
 +
    1.82
 +
  </td>
 +
  <td align="center">
 +
    1.66
 +
  </td>
 +
  <td align="center">
 +
    Good
 +
  </td>
 +
  </tr>
 +
 +
  <tr>
 +
  <td rowspan="4" align="center">
 +
    <p>FepA Fur Binding Site
 +
    <br/>(with sfGFP)</p>
 +
  </td>
 +
  <td align="center">
 +
    1
 +
  </td>
 +
  <td align="center">
 +
    21.1 ng/µL
 +
  </td>
 +
  <td align="center">
 +
    2.06
 +
  </td>
 +
  <td align="center">
 +
    -
 +
  </td>
 +
  <td align="center">
 +
    Good
 +
  </td>
 +
  </tr>
 +
  <tr>
 +
  <td align="center">
 +
    2
 +
  </td>
 +
  <td align="center">
 +
    22.4 ng/µL
 +
  </td>
 +
  <td align="center">
 +
    1.96
 +
  </td>
 +
  <td align="center">
 +
    -
 +
  </td>
 +
  <td align="center">
 +
    Good
 +
  </td>
 +
  </tr>
 +
  <tr>
 +
  <td align="center">
 +
    3
 +
  </td>
 +
  <td align="center">
 +
    55.4 ng/µL
 +
  </td>
 +
  <td align="center">
 +
    1.87
 +
  </td>
 +
  <td align="center">
 +
    1.58
 +
  </td>
 +
  <td align="center">
 +
    Good
 +
  </td>
 +
  </tr>
 +
  <tr>
 +
  <td align="center">
 +
    4
 +
  </td>
 +
  <td align="center">
 +
    68.5 ng/µL
 +
  </td>
 +
  <td align="center">
 +
    1.89
 +
  </td>
 +
  <td align="center">
 +
    1.67
 +
  </td>
 +
  <td align="center">
 +
    Good
 +
  </td>
 +
  </tr>
 +
 +
  <tr>
 +
  <td rowspan="4" align="center">
 +
    <p>Fes Fur Binding Site
 +
    <br/>(with sfGFP)</p>
 +
  </td>
 +
  <td align="center">
 +
    1
 +
  </td>
 +
  <td align="center">
 +
    39.9 ng/µL
 +
  </td>
 +
  <td align="center">
 +
    1.87
 +
  </td>
 +
  <td align="center">
 +
    1.80
 +
  </td>
 +
  <td align="center">
 +
    Good
 +
  </td>
 +
  </tr>
 +
  <tr>
 +
  <td align="center">
 +
    2
 +
  </td>
 +
  <td align="center">
 +
    40.6 ng/µL
 +
  </td>
 +
  <td align="center">
 +
    1.74
 +
  </td>
 +
  <td align="center">
 +
    -
 +
  </td>
 +
  <td align="center">
 +
    No sequencing
 +
  </td>
 +
  </tr>
 +
  <tr>
 +
  <td align="center">
 +
    3
 +
  </td>
 +
  <td align="center">
 +
    68.4 ng/µL
 +
  </td>
 +
  <td align="center">
 +
    1.87
 +
  </td>
 +
  <td align="center">
 +
    1.63
 +
  </td>
 +
  <td align="center">
 +
    Good
 +
  </td>
 +
  </tr>
 +
  <tr>
 +
  <td align="center">
 +
    4
 +
  </td>
 +
  <td align="center">
 +
    20.8 ng/µL
 +
  </td>
 +
  <td align="center">
 +
    2.05
 +
  </td>
 +
  <td align="center">
 +
    -
 +
  </td>
 +
  <td align="center">
 +
    No sequencing
 +
  </td>
 +
  </tr>
 +
 +
  <tr>
 +
  <td rowspan="4" align="center">
 +
    <p>ybiL Fur Binding Site
 +
    <br/>(with sfGFP)</p>
 +
  </td>
 +
  <td align="center">
 +
    1
 +
  </td>
 +
  <td align="center">
 +
    58.2 ng/µL
 +
  </td>
 +
  <td align="center">
 +
    1.87
 +
  </td>
 +
  <td align="center">
 +
    1.73
 +
  </td>
 +
  <td align="center">
 +
    Good
 +
  </td>
 +
  </tr>
 +
  <tr>
 +
  <td align="center">
 +
    2
 +
  </td>
 +
  <td align="center">
 +
    16.3 ng/µL
 +
  </td>
 +
  <td align="center">
 +
    1.95
 +
  </td>
 +
  <td align="center">
 +
    -
 +
  </td>
 +
  <td align="center">
 +
    No sequencing
 +
  </td>
 +
  </tr>
 +
  <tr>
 +
  <td align="center">
 +
    3
 +
  </td>
 +
  <td align="center">
 +
    18.8 ng/µL
 +
  </td>
 +
  <td align="center">
 +
    1.95
 +
  </td>
 +
  <td align="center">
 +
    -
 +
  </td>
 +
  <td align="center">
 +
    No sequencing
 +
  </td>
 +
  </tr>
 +
  <tr>
 +
  <td align="center">
 +
    4
 +
  </td>
 +
  <td align="center">
 +
    45.5 ng/µL
 +
  </td>
 +
  <td align="center">
 +
    1.90
 +
  </td>
 +
  <td align="center">
 +
    1.81
 +
  </td>
 +
  <td align="center">
 +
    Good
 +
  </td>
 +
  </tr>
 +
 +
  <tr>
 +
  <td rowspan="4" align="center">
 +
    <p>FecA Fur Binding Site
 +
    <br/>(with sfGFP)</p>
 +
  </td>
 +
  <td align="center">
 +
    1
 +
  </td>
 +
  <td align="center">
 +
    20.0 ng/µL
 +
  </td>
 +
  <td align="center">
 +
    1.96
 +
  </td>
 +
  <td align="center">
 +
    -
 +
  </td>
 +
  <td align="center">
 +
    Good
 +
  </td>
 +
  </tr>
 +
  <tr>
 +
  <td align="center">
 +
    2
 +
  </td>
 +
  <td align="center">
 +
    27.5 ng/µL
 +
  </td>
 +
  <td align="center">
 +
    1.77
 +
  </td>
 +
  <td align="center">
 +
    -
 +
  </td>
 +
  <td align="center">
 +
    No sequencing
 +
  </td>
 +
  </tr>
 +
  <tr>
 +
  <td align="center">
 +
    3
 +
  </td>
 +
  <td align="center">
 +
    31.5 ng/µL
 +
  </td>
 +
  <td align="center">
 +
    1.94
 +
  </td>
 +
  <td align="center">
 +
    1.81
 +
  </td>
 +
  <td align="center">
 +
    Good
 +
  </td>
 +
  </tr>
 +
  <tr>
 +
  <td align="center">
 +
    4
 +
  </td>
 +
  <td align="center">
 +
    57.0 ng/µL
 +
  </td>
 +
  <td align="center">
 +
    1.89
 +
  </td>
 +
  <td align="center">
 +
    1.64
 +
  </td>
 +
  <td align="center">
 +
    Good
 +
  </td>
 +
  </tr>
 +
 +
  <tr>
 +
  <td rowspan="3" align="center">
 +
    <p>yncE Fur Binding Site
 +
    <br/>(with sfGFP)</p>
 +
  </td>
 +
  <td align="center">
 +
    1
 +
  </td>
 +
  <td align="center">
 +
    92.4 ng/µL
 +
  </td>
 +
  <td align="center">
 +
    1.90
 +
  </td>
 +
  <td align="center">
 +
    1.75
 +
  </td>
 +
  <td align="center">
 +
    Good
 +
  </td>
 +
  </tr>
 +
  <tr>
 +
  <td align="center">
 +
    2
 +
  </td>
 +
  <td align="center">
 +
    64.7 ng/µL
 +
  </td>
 +
  <td align="center">
 +
    1.89
 +
  </td>
 +
  <td align="center">
 +
    1.66
 +
  </td>
 +
  <td align="center">
 +
    Good
 +
  </td>
 +
  </tr>
 +
  <tr>
 +
  <td align="center">
 +
    3
 +
  </td>
 +
  <td align="center">
 +
    38.6 ng/µL
 +
  </td>
 +
  <td align="center">
 +
    1.91
 +
  </td>
 +
  <td align="center">
 +
    1.80
 +
  </td>
 +
  <td align="center">
 +
    Good
 +
  </td>
 +
  </tr>
 +
</tbody>
 +
</table>
 +
 +
 +
<h2>Construction of plasmid N°2</h2>
 +
 +
<p>
 +
Our plasmid N°2 is building with a synthetic promote sequence which is composed of:
 +
  <ul>
 +
    <li> Andersen's promotor </li>
 +
    <li> Fur Binding Site (15 different)</li>
 +
    <li> RBS </li>
 +
    <li> sfGFP </li>
 +
    <li> Terminator </li>
 +
  </ul>
 +
</p>
 +
 +
<h3>Golden Gate</h3>
 +
 +
<p>
 +
In order to associate these sequences we performed a golden gate, using for each sample :
 +
  <ul>
 +
    <li> 80 ng of Andersen's promotor
 +
    <li> 80 ng of Fur Binding Site
 +
    <li> 80 ng of RBS
 +
    <li> 80 ng of sfGFP
 +
    <li> 80 ng of Terminator
 +
    <li> 1.5 µL of T4 buffer (10X)
 +
    <li> 15 Unit of T4 ligase
 +
    <li> 2.5 Unit of Bsa I
 +
  </ul>
 +
</p>
 +
<p>
 +
The Golden Gate products are chemically transformed into E. coli Top 10 strains. After the transformation process, bacteria are plated into LB medium with carbenicillin and icubated overnigth at 37°C.
 +
</p>
 +
 +
 +
<p>
 +
Golden Gates plates are composed at 98% of red colonies.
 +
The problem was probably on the equimolarity ratio between our different part which were not totally respected.
 +
We isolated white colonies (4 maximum by each sample) on LB medium with carbenicillin and we incubated them overnight at 37°c.
 +
</p>
 +
</br></br>
 +
 +
<p>
 +
We made pre culture (V = 10 mL), using bacterial colonies isolated from our plates of Golden Gate transformation. Then minipreps and glycerol conservation were made using these pre cultures.
 +
</p>
 +
 +
<table width="100%" cellpadding="10" align='center' border="1">
 +
  <tr>   
 +
    <th align="center">
 +
        <b>Fur Binding Site</b>
 +
    </th>
 +
    <th align="center">
 +
        <b>Sequence</b>
 +
    </th>
 +
    <th align="center">
 +
        <b>Clone</b>
 +
    </th>   
 +
    <th align="center">
 +
        <b>Concentration</b>
 +
    </th>
 +
  </tr>
 +
 +
  <tr>   
 +
    <td rowspan="3" align="center">
 +
      <p>
 +
        Fur BS 1
 +
      </p>
 +
    </td>
 +
    <td rowspan="3" align="center">
 +
      <p>
 +
        ATTATTGATAACTATTTG
 +
      </p>
 +
    </td> 
 +
    <td align="center">
 +
      <p>
 +
        Clone n°1
 +
      </p>
 +
    </td>
 +
    <td align="center">
 +
      <p>
 +
        80.8 ng/µL
 +
      </p>
 +
    </td>
 +
  </tr>
 +
  <tr>
 +
    <td align="center">
 +
      <p>
 +
        Clone n°2
 +
      </p>
 +
    </td>
 +
    <td align="center">
 +
      <p>
 +
        83.8 ng/µL
 +
      </p>
 +
    </td>
 +
  </tr>
 +
  <tr>
 +
    <td align="center">
 +
      <p>
 +
        Clone n°4
 +
      </p>
 +
    </td>
 +
    <td align="center">
 +
      <p>
 +
        90.3 ng/µL
 +
      </p>
 +
    </td>
 +
  </tr>
 +
 +
  <tr>   
 +
    <td rowspan="3" align="center">
 +
      <p>
 +
        Fur BS 2
 +
      </p>
 +
    </td>
 +
    <td rowspan="3" align="center">
 +
      <p>
 +
        GATAACTATTTGCATTTGCA
 +
      </p>
 +
    </td> 
 +
    <td align="center">
 +
      <p>
 +
        Clone n°1
 +
      </p>
 +
    </td>
 +
    <td align="center">
 +
      <p>
 +
        68.6 ng/µL
 +
      </p>
 +
    </td>
 +
  </tr>
 +
  <tr>
 +
    <td align="center">
 +
      <p>
 +
        Clone n°2
 +
      </p>
 +
    </td>
 +
    <td align="center">
 +
      <p>
 +
        77.4 ng/µL
 +
      </p>
 +
    </td>
 +
  </tr>
 +
  <tr>
 +
    <td align="center">
 +
      <p>
 +
        Clone n°3
 +
      </p>
 +
    </td>
 +
    <td align="center">
 +
      <p>
 +
        68.0 ng/µL
 +
      </p>
 +
    </td>
 +
  </tr>
 +
 +
  <tr>   
 +
    <td rowspan="3" align="center">
 +
      <p>
 +
        Fur BS 3
 +
      </p>
 +
    </td>
 +
    <td rowspan="3" align="center">
 +
      <p>
 +
        CAAATGCAAATAGTTATCCA
 +
      </p>
 +
    </td> 
 +
    <td align="center">
 +
      <p>
 +
        Clone n°1
 +
      </p>
 +
    </td>
 +
    <td align="center">
 +
      <p>
 +
        79.4 ng/µL
 +
      </p>
 +
    </td>
 +
  </tr>
 +
  <tr>
 +
    <td align="center">
 +
      <p>
 +
        Clone n°2
 +
      </p>
 +
    </td>
 +
    <td align="center">
 +
      <p>
 +
        110.5 ng/µL
 +
      </p>
 +
    </td>
 +
  </tr>
 +
  <tr>
 +
    <td align="center">
 +
      <p>
 +
        Clone n°3
 +
      </p>
 +
    </td>
 +
    <td align="center">
 +
      <p>
 +
        457.4 ng/µL
 +
      </p>
 +
    </td>
 +
  </tr>
 +
 +
  <tr>   
 +
    <td rowspan="2" align="center">
 +
      <p>
 +
        Fur BS 4
 +
      </p>
 +
    </td>
 +
    <td rowspan="2" align="center">
 +
      <p>
 +
        CAAATAGTTATCAATAATCA
 +
      </p>
 +
    </td> 
 +
    <td align="center">
 +
      <p>
 +
        Clone n°1
 +
      </p>
 +
    </td>
 +
    <td align="center">
 +
      <p>
 +
        73.2 ng/µL
 +
      </p>
 +
    </td>
 +
  </tr>
 +
  <tr>
 +
    <td align="center">
 +
      <p>
 +
        Clone n°2
 +
      </p>
 +
    </td>
 +
    <td align="center">
 +
      <p>
 +
        66.2 ng/µL
 +
      </p>
 +
    </td>
 +
  </tr>
 +
 +
  <tr>   
 +
    <td align="center">
 +
      <p>
 +
        Fur BS 5
 +
      </p>
 +
    </td>
 +
    <td align="center">
 +
      <p>
 +
        GTAAATTAATATTATTTACA
 +
      </p>
 +
    </td> 
 +
    <td align="center">
 +
      <p>
 +
        Clone n°1
 +
      </p>
 +
    </td>
 +
    <td align="center">
 +
      <p>
 +
        57.2 ng/µL
 +
      </p>
 +
    </td>
 +
  </tr>
 +
 +
  <tr>   
 +
    <td align="center">
 +
      <p>
 +
        Fur BS 6
 +
      </p>
 +
    </td>
 +
    <td align="center">
 +
      <p>
 +
        AATAATGCTTCTCATTTTCA
 +
      </p>
 +
    </td> 
 +
    <td align="center">
 +
      <p>
 +
        Clone n°1
 +
      </p>
 +
    </td>
 +
    <td align="center">
 +
      <p>
 +
        63.8 ng/µL
 +
      </p>
 +
    </td>
 +
  </tr>
 +
 +
  <tr>   
 +
    <td align="center">
 +
      <p>
 +
        Fur BS 7
 +
      </p>
 +
    </td>
 +
    <td align="center">
 +
      <p>
 +
        ATAAATGATAATCATTATCA
 +
      </p>
 +
    </td> 
 +
    <td align="center">
 +
      <p>
 +
        Clone n°1
 +
      </p>
 +
    </td>
 +
    <td align="center">
 +
      <p>
 +
        70.6 ng/µL
 +
      </p>
 +
    </td>
 +
  </tr>
 +
 +
  <tr>   
 +
    <td rowspan="4" align="center">
 +
      <p>
 +
        Fur BS 8
 +
      </p>
 +
    </td>
 +
    <td rowspan="4" align="center">
 +
      <p>
 +
        GAAAATAATTCTTATTTCCA
 +
      </p>
 +
    </td> 
 +
    <td align="center">
 +
      <p>
 +
        Clone n°1
 +
      </p>
 +
    </td>
 +
    <td align="center">
 +
      <p>
 +
        75.6 ng/µL
 +
      </p>
 +
    </td>
 +
  </tr>
 +
  <tr>
 +
    <td align="center">
 +
      <p>
 +
        Clone n°2
 +
      </p>
 +
    </td>
 +
    <td align="center">
 +
      <p>
 +
        87.2 ng/µL
 +
      </p>
 +
    </td>
 +
  </tr>
 +
  <tr>
 +
    <td align="center">
 +
      <p>
 +
        Clone n°3
 +
      </p>
 +
    </td>
 +
    <td align="center">
 +
      <p>
 +
        48.8 ng/µL
 +
      </p>
 +
    </td>
 +
  </tr>
 +
  <tr>
 +
    <td align="center">
 +
      <p>
 +
        Clone n°4
 +
      </p>
 +
    </td>
 +
    <td align="center">
 +
      <p>
 +
        57.8 ng/µL
 +
      </p>
 +
    </td>
 +
  </tr>
 +
 +
  <tr>   
 +
    <td align="center">
 +
      <p>
 +
        Fur BS 9
 +
      </p>
 +
    </td>
 +
    <td align="center">
 +
      <p>
 +
        AGCACTTATTATTATTTTCA
 +
      </p>
 +
    </td> 
 +
    <td align="center">
 +
      <p>
 +
        Clone n°1
 +
      </p>
 +
    </td>
 +
    <td align="center">
 +
      <p>
 +
        58.6 ng/µL
 +
      </p>
 +
    </td>
 +
  </tr>
 +
 +
  <tr>   
 +
    <td align="center">
 +
      <p>
 +
        Fur BS 10
 +
      </p>
 +
    </td>
 +
    <td align="center">
 +
      <p>
 +
        GATAATTGTTATCGTTTGCA
 +
      </p>
 +
    </td> 
 +
    <td align="center">
 +
      <p>
 +
        No Clone
 +
      </p>
 +
    </td>
 +
    <td align="center">
 +
      <p>
 +
        -
 +
      </p>
 +
    </td>
 +
  </tr>
 +
 +
  <tr>   
 +
    <td rowspan="3" align="center">
 +
      <p>
 +
        Fur BS 11
 +
      </p>
 +
    </td>
 +
    <td rowspan="3" align="center">
 +
      <p>
 +
        GATAATGCTTATCAAAATCA
 +
      </p>
 +
    </td> 
 +
    <td align="center">
 +
      <p>
 +
        Clone n°2
 +
      </p>
 +
    </td>
 +
    <td align="center">
 +
      <p>
 +
        75.6 ng/µL
 +
      </p>
 +
    </td>
 +
  </tr>
 +
  <tr>
 +
    <td align="center">
 +
      <p>
 +
        Clone n°3
 +
      </p>
 +
    </td>
 +
    <td align="center">
 +
      <p>
 +
        398.0 ng/µL
 +
      </p>
 +
    </td>
 +
  </tr>
 +
  <tr>
 +
    <td align="center">
 +
      <p>
 +
        Clone n°4
 +
      </p>
 +
    </td>
 +
    <td align="center">
 +
      <p>
 +
        57.4 ng/µL
 +
      </p>
 +
    </td>
 +
  </tr>
 +
 +
  <tr>   
 +
    <td align="center">
 +
      <p>
 +
        Fur BS 12
 +
      </p>
 +
    </td>
 +
    <td align="center">
 +
      <p>
 +
        CAAAATTATTATCACTTTCA
 +
      </p>
 +
    </td> 
 +
    <td align="center">
 +
      <p>
 +
        Clone n°1
 +
      </p>
 +
    </td>
 +
    <td align="center">
 +
      <p>
 +
        50.0 ng/µL
 +
      </p>
 +
    </td>
 +
  </tr>
 +
 +
  <tr>   
 +
    <td align="center">
 +
      <p>
 +
        Fur BS 13
 +
      </p>
 +
    </td>
 +
    <td align="center">
 +
      <p>
 +
        AATAATGATTACCATTCCCA
 +
      </p>
 +
    </td> 
 +
    <td align="center">
 +
      <p>
 +
        Clone n°2
 +
      </p>
 +
    </td>
 +
    <td align="center">
 +
      <p>
 +
        70.7 ng/µL
 +
      </p>
 +
    </td>
 +
  </tr>
 +
 +
  <tr>   
 +
    <td rowspan="4" align="center">
 +
      <p>
 +
        Fur BS 14
 +
      </p>
 +
    </td>
 +
    <td rowspan="4" align="center">
 +
      <p>
 +
        GAAATTGTTTTTGATTTTCA
 +
      </p>
 +
    </td> 
 +
    <td align="center">
 +
      <p>
 +
        Clone n°1
 +
      </p>
 +
    </td>
 +
    <td align="center">
 +
      <p>
 +
        42.7 ng/µL
 +
      </p>
 +
    </td>
 +
  </tr>
 +
  <tr>
 +
    <td align="center">
 +
      <p>
 +
        Clone n°2
 +
      </p>
 +
    </td>
 +
    <td align="center">
 +
      <p>
 +
        32.8 ng/µL
 +
      </p>
 +
    </td>
 +
  </tr>
 +
  <tr>
 +
    <td align="center">
 +
      <p>
 +
        Clone n°3
 +
      </p>
 +
    </td>
 +
    <td align="center">
 +
      <p>
 +
        37.8 ng/µL
 +
      </p>
 +
    </td>
 +
  </tr>
 +
  <tr>
 +
    <td align="center">
 +
      <p>
 +
        Clone n°4
 +
      </p>
 +
    </td>
 +
    <td align="center">
 +
      <p>
 +
        45.7 ng/µL
 +
      </p>
 +
    </td>
 +
  </tr>
 +
 +
  <tr>   
 +
    <td rowspan="3" align="center">
 +
      <p>
 +
        Fur BS 15
 +
      </p>
 +
    </td>
 +
    <td rowspan="3" align="center">
 +
      <p>
 +
        GTTAATTGTAATGATTTTCA
 +
      </p>
 +
    </td> 
 +
    <td align="center">
 +
      <p>
 +
        Clone n°1
 +
      </p>
 +
    </td>
 +
    <td align="center">
 +
      <p>
 +
        37.0 ng/µL
 +
      </p>
 +
    </td>
 +
  </tr>
 +
  <tr>
 +
    <td align="center">
 +
      <p>
 +
        Clone n°2
 +
      </p>
 +
    </td>
 +
    <td align="center">
 +
      <p>
 +
        53.2 ng/µL
 +
      </p>
 +
    </td>
 +
  </tr>
 +
  <tr>
 +
    <td align="center">
 +
      <p>
 +
        Clone n°3
 +
      </p>
 +
    </td>
 +
    <td align="center">
 +
      <p>
 +
        196.0 ng/µL
 +
      </p>
 +
    </td>
 +
  </tr>
 +
 +
</table>
 +
 +
 +
<h2>Construction of plasmid N°3</h2>
 +
 +
<p>
 +
29/07/13
 +
 +
</br>
 +
We received the primers ordered on friday the 26th of July and started to dilute them into TrisCL at 10 mM. Secondly, we diluted the previous stock solution at a rate of 1/20th to obtain a final concentration of 5 µM for our intermediate solution.
 +
</br>
 +
 +
</br>
 +
Then, for our plasmid three construction, we need to first extract the 6 enterobactin gene (EntA, EntB, EntC, EntD, EntE and EntF). The ordered primers from friday will theoretically extract them in the appropriate Golden Gate format with their own RBS upstream (designed from Salis RBS). This will allow us to construct our two N°3 plasmids, one containing EntA, EntD and EntF, and the other one EntB, EntC and EntE. The genes are spread like this to obtain two equivalent plasmids xxx
 +
</br>
 +
 +
</br>
 +
We proceeded to a genomic extraction of the 6 genes of interest and migrated to PCR products on a 1% gel. We successfully extracted 5 out of the 6.
 +
</br>
 +
 +
</br>
 +
30/07/13
 +
 +
</br>
 +
We annealled the oligonucleotides of the PL-LacO part in the golden gate format. Also, we extracted the sfGFP with the RBS upstream which will allow us to construct the control positive plasmid 3 for future TECAN experiments.
 +
</br>
 +
 +
</br>
 +
31/07/13
 +
 +
</br>
 +
We started by purifying our 6 succesful PCR extraction from the 29/07 and 30/07. So we managed to extract EntA, EntB, EntC, EntD and EntF from E. coli's genomic DNA and sfGFP in the golden gate format with a RBS (plasmid 3 construction) from plasmidic DNA.
 +
</br>
 +
 +
</br>
 +
Additionnaly, we optimized our PCR to extract our missing gene, EntE. We obtained a smear +/- a double band. As a consequence, we adjusted the annealing temperature with a range from 54°C to 66°C. This will allow us to try extracting the missing gene (EntE) in the appropriate conditions.
 +
</br>
 +
 +
</br>
 +
01/08/13
 +
<p>
 +
</br>
 +
We made a PCR for the EntE gene with a gradient of temperature to know at which temperature the PCR result were the best. <br>
 +
We made 8 (ou mettre 6 ?) tubes with the same composition:
 +
  <ul>
 +
    <li> Water </li>
 +
    <li> Taq Buffer 5 </li>
 +
    <li> dNTPs </li>
 +
    <li> Rev EntE 2,5 </li>
 +
    <li> For EntE 2,5 </li>
 +
    <li> DNA 0,5 </li>
 +
    <li> Taq polymerase 0,5 </li>
 +
  </ul>
 +
We also made positive and negative controles (with Rev EntC and For EntC instead of EntE).<br>
 +
After electrophoresis, we obtain the best band at .. °C.<br>
 +
However our experiment have been made with Taq polymerase (and Taq Buffer) instead of Q5 (and Q5 buffer) that we use usually. Then, we have to repeat this experiment with Q5 and Q5 buffer. <br>
 +
The did a gradient PCR and migrated on a gel our samples. On the gel, we notice that both the smear and the double band disappear when the annealing temperature increases. So to conclude, we run tonight a PCR with Q5 polymerase with increasing temperature starting from 56 degrees. The absolute annealing temperature will be determined for the Q5 (not extrapolable from a OneTaq PCR).
 +
</br>
 +
 +
</br>
 +
Also, we made a golden gate for the construction of plasmid 3. For analysis purposes, only a GFP was added downstream of the PL LacO promoter. Also, the EntE gene is still to be extracted from the genome. Also, we runned 3 extras golden gate for the construction of plasmid 2. In fact, we corrected our calculations because we initially we used mass concentrations instead of molar concentrations.
 +
</br>
 +
</p>
 +
 +
</br>
 +
02/08/13
 +
<p>
 +
</br>
 +
The transformations of the golden gate 2 and 3 are successful and we managed to invert the rate of red colonies versus white colonies (red = false positive with mRFP, white = positive), thus meaning we have a lot of white ones! We conserved the plates for future isolation on coming monday.
 +
</br>
 +
 +
</br>
 +
Additionnaly, we migrated the PCR products of 01/08. The gel is empty, but the positive controls are there, meaning we made a mistake in the primers (the master mix is the one used for the positive control).
 +
</br>
 +
</p>
 +
</p>

Revision as of 14:16, 22 September 2013

Iron coli project

Week 1: 17th June - 23rd June

Tuesday, 19th June

As a start for the labwork, we first decided to make competent cells. We grew from glycerol samples three E. coli strains in 2 mL of LB medium.
The chemically competent strains chosen are as follows:

BL21
DH5α
TOP10


Protocol for pre-culture preparation

In a 15 mL tube, add 2 mL of LB medium and inoculate cells from glycerol

Thursday, 20th June


We started to make 200 mL of BL21 and DH5α E. coli strains and 400 mL of TOP10 to make competent cells.


Protocol for competent cells

First prepare the following solutions required to make competent cells:
Solution for 1M Cacl2:
Add 14,30g of CaCl2 into 100 ml desalted water

Solution for 0,1M Cacl2:
Add 50 mL of CaCl2 1M solution into 450 ml of desalted water

Solution for 0,1M Cacl2 + 15% glycerol:
Add 50 mL of CaCl2 1M solution and 75 mL of glycerol 100% into 450 ml of desalted water


For 200 ml LB medium, add 400 µL of strain sample.
Let the bacteria grow until it reaches an OD between 0,3 and 0,35.
Once it reached the right OD, put the medium on ice for 30 minutes to slow down growth.
Split the 200 mL into 4x50 mL tubes then centrifuge at 3000 rpm for 5 minutes at 4°C and suppress supernatant afterwards.
Resuspend the cells with 5 mL of Cacl2 at 0,1M for each 50 mL tube.
Again, put the medium on ice for 30 minutes.
Centrifuge at 3000 rpm for 5 minutes at 4°C then suppress supernatant.
Resuspend the cells with 1 mL of Cacl2 at 0,1M + 15% glycerol for each 50 mL tube.
Split the total 4 mL into 40 tubes containing each 100 µL of concentrated cell solution. This step should be executed fast enough and on ice.



Friday, 21st June


To check the quality of our work, each strain has been plated on LB medium with ampicillin, kanamycin or chloramphenicol in order to evaluate if it contaminated or not. Furthermore, to evaluate wether our strains are competent or not, we also transformed our bacteria with a pSB1A3 plasmid (red colonies) and plated them on LB medium with ampicillin only.

Protocol for transformation

For 100 µL of chemical competent cells, add 1 µL of plasmidic DNA.
Let 30 minutes on ice.
Let the cells at 42°C for exactly 50 seconds.
Let 5 minutes on ice.
Resuspend the cells with 1 mL of LB spread on a petri dish then let it at 37°C.

Week 2: 24th June - 30th June

Monday, June 24th

Our competents cells made on friday the 21st were plated and incubated the whole week-end at 30°C. Today, we analyzed the plates and came to the conclusion that the medium was contaminated due to poor protocol execution. As a consequence, we decided to make new LB medium and recalculate the antibiotic concentrations.


Protocol for LB medium preparation

Add 20 g of LB broth into 1000 mL of desalted water and autoclave.


Protocol for antibiotic solutions preparation

Kanamycin is used as an effective concentration of 10-50 µg/ml and at a storage concentration of 10 mg/ml
Carbenicillin is used as an effective concentration of 20-60 µg/ml and at a storage concentration of 50 mg/ml
Chloramphenicol is used as an effective concentration of 25-170 µg/ml and at a storage concentration of 34 mg/ml (ethanol)
Tetracyclin is used as an effective concentration of 10-50 and at a storage concentration of 5 mg/ml (ethanol)


Tuesday, June 25th

New competent cells have been made with the same bacterial strains (BL21, DH5α and TOP10). We thought that the contamination of our cells was due to the CaCl2 solution that may not have been sterile and/or incorrect manipulated. As a consequence, we prepared new CaCl2 solutions made sure to autoclave them before usage. We ran the same tests as monday the 24th to evaluate the quality of our work and check for potential contamination.

Wednesday, June 26th

The results of the competent cells protocol is as follows:
DH5α strain was not contaminated but competent
TOP10 strain was contaminated only on the Kanamycin plate but competent
BL21 strain was completely contaminated and, thus, not usable.

Thurdsay, June 27th

We finished designing are first two plasmids and constructed the primers to extract the natural promoters from the genomic DNA from E. coli and the different parts for Golden Gate assembly.

Friday, June 28th

We want to obtain reasonnable competent cells for our oncoming transformation for newt week. Thus, we decided to plate the three strains on LB-Agar medium in order to isolate one and only one colony for monday when we'll start over the whole competent process.

Week 3: 1st July - 7th July

Tuesday, July 2nd

We prepared the BL21 and BG1655 strains to be competent.

Wednesday, July 3rd

The two strains have been contaminated and are not usable.

Thursday, July 4th

Genomic DNA extraction of the BL21 and BG1655 strains have been done using two different methods. We received the primers we have ordered. They have been prepared to do a PCR. In order to get our genes of interest we did a PCR using as the DNA template the genomic DNA extracted from BL21 and BG1655.

Friday, July 5th

To ensure the amplification of our genes by PCR, we did a agarose gel electrophoresis. To do so, we first prepared a TAE 50X stock solution. By diluting it, we use a 1X TAE solution to do a 1% agarose gel. We obtain the following gel. That showed that only two of our genes have been amplificated by PCR. A new PCR will be made.

Week 4: 8th July - 14th July

Monday, July 8th

We prepared the BL21 and BG1655 strains to be competent.

Tuesday, July 9th

Petri box Petri box

Both strain are not contaminated.

Petri box Petri box

BL21 are highly competent, BG1655 are little competent.

Wednesday, July 10th

PCR procedure optimization

Aim: Define the number of matrix of optimal DNA to amplify a gene by PCR.

Data:

  • [primer] = --- ng/µl
  • size(primer) = --- nt

For PCR we chose to use … ng of primer (V=…µL). As we know the size of our primers, we could define the number of primers that we have to use to begin the reaction. We define the number of recquired matrix DNA in terms of concentration of sample with the following formula:

AJOUTER LA FORMULE

Analysis of Agarose gel's results:

AJOUTER LA PHOTO LEGENDEE

Extraction of natural promoter sequence

Aim: We want to have sequences genes' promoters' sequences under the control of FUR (Ferric Uptake Regulation)transcription factor.

We remake PCR samples that failed the previous week.

  • Fec A
  • Ent C
  • Fec C
  • Ace B

PCR products are placed on gel and purificate. After, PCR products are tested with nanodrop.

Fec A (BG1655)
[c] = 23.7 ng/µl
260/280 = 1.90

Fec A (BL21)
[c] = 53.1 ng/µl
260/280 = 1.73

Ent C (BG1655)
[c] = 52.4 ng/µl
260/280 = 1.71

Ent C (BL21)
[c] = 50.0 ng/µl
260/280 = 1.94

Ace B (BG1655)
[c] = 46.5 ng/µl
260/280 = 1.89

Fec C (BG1655)
[c] = 34.2 ng/µl
260/280 = 1.91
pcr 10-07

Tris-HCl solution preparation(1M) : Stock solution 100X

Aim: Tris-HCl solution will be use in a final concentration of 10 mM to resuspend our primers.

Preparation of the stock solution (1 M, pH 7.5) for a volume of 50 mL:


  1. Disolve 4,6g of Trisbase in 30 mL of distilled water.
  2. Adjust pH with concentrated HCl(~4 mL) until pH=7,5.
  3. Add distilled water until 50 mL then put in autoclave.

Note: If the solution has a yellow coloration, do the preparation again with better Trisbase.

Preparation of the solution (10mM, pH 7.5) for a volume of 50 mL.
Take 50µL of the stock solution at 1M and add 49.5 mL of water for the dilution.

Kanamycin preparation

Preparation of 3 tubes of 1.5 mL of Kanamycin.

Thursday, July 11

Preparation of primers solution:

  1. Centrifugate tubes at 8000 rpm, begin 30 seconds.
  2. Add 250 µL of Tris HCl (10 mM); [Primer] = 100µM

We prepare a diluted solution at 5 µM from to the stock solution, for PCR reactions.
Dilution at 1/20: We take 5 µL of the stock solution (100 µM) and we diluate in 95 µL of tris-HCl (10 mM).

Transformation of BL21:

Chemical transformation of BL21 with Cyrille's samples:

  • Terminator (T)
  • Promotor (P)
  • Plasmid 1K3
  • Plasmid 1C3
  • sfGFP

These transformations allow us to do glycerols of these constrctions.

PCR on E.coli genom (BG1655 strain)

  • Fep A (Primers P021 and P022)
  • Fes (Primers P023 and P024)
  • sdh C (Primers P025 and P026)
  • ybi L (Primers P027 and P028)
  • ync E (Primers P029 and P030)
pcr 11-07

Friday, July 12

Transformations test

  • Terminator (T) = OK
  • Promotor (P) = OK
  • Plasmid 1K3 = OK
  • Plasmid 1C3 = OK
  • sfGFP = OK

Note : The negative control was suspect.

Petri dishes are le on the bench at room temperature during the week-end, colonies will be reisolate next week.

Migration of samples of PCR extraction.

pcr 12-07

The promoter sequence of genes Fep A, Fes, Sdh C, ybi L and ync E were extracted successfully.
Samples were purified and, after, tested with nanodrop.

Fep A (BG1655)
[c] = 38.3 ng/µl
260/280 = 1.93

Fes (BG1655)
[c] = 45.5 ng/µl
260/280 = 1.89

Sdh C (BG1655)
[c] = 28.5 ng/µl
260/280 = 1.86

ybi L (BG1655)
[c] = 31.0 ng/µl
260/280 = 2.09

ync E (BG1655)
[c] = 25.1 ng/µl
260/280 = 1.93

Voir s'il ne faut pas faire le tableau recap des extractions

Week 5: 15th July - 21st July

Week 5: 15th July - 21st July

Monday, July 15th

Tuesday, July 16th

PCR Optimisation

Wednesday, July 17th

We prepared the TOP10 to be competent.


We launch our first Golden Gates.

Thursday, July 18th

Friday, July 19th

Week 6: 22nd July - 28th July

Monday, July 22nd

We did a PCR on the seven products of golden gate 1 using the VR and VF2 primers. We mixed :

  • 10 uL of One Taq Buffer 10X
  • 1 uL of 10 mM dNTPs
  • 1 uL of each VF2 and VR primers
  • 35,5 uL of H2O
  • 1 uL of the One Taq enzyme
  • 1 uL of the golden gate products

We used the following PCR program :

  1. 95°C for 3 min
  2. 95°C for 15 sec
  3. 55°C for 30 sec
  4. 68°C for 2 min
  5. 68°C for 10 min

Note: We repeated step 2, 3 and 4 29 times.

The gel migration didn't reveal any band. We supposed that it hadn't worked due to the annealing temperature we used. Indeed, with the help of the New England BioLabs Tm Calculator website, we have found that the optimal annealing temperature for those primers was 53°C.

Tuesday, July 23rd

In order to test the best annealing temperature for our following PCRs, we did a PCR on our 5 first golden gate products with two different annealing temperature : 53 or 51°C. We also prepared two positiv controls for each conditions with either bacteria transformed by empty PSB1A3 plasmid or 1 uL of the miniprep plasmid. This was realised in order to verify if the problem of our first PCR was due to a bad lysis of our bacteria. What is more, we prepared one negativ control with an annealing temperature of 55°C.


(Mettre photo gel)

According to our results, we can assume that our first PCR was badly realised due to a handling error and not because of the annealing temperature we used.


In the meantime, we did the Golden Gate 1 again in order to obtain better ligation results. We transformed TOP 10 E. coli with the golden gate products and plated them on LB-Agar with carbenicillin antibiotic. The petri dishes have been let overnight at 37°C.

Wednesday, July 24th

We did a colony PCR from colony obtained on the petri dishes plated with golden gate 2 products transformed bacteria. We chose one white colony of each of the first ten different FUR BS constructions. The positiv control was prepared by using the PSB1A3 plasmid. The same PCR program was used but we were out of One Taq so we utilize Dream Taq.

We re isolate colony of bacteria transformed with our golden gate 1 products.

Thursday, July 25th

We did a 1% gel in order to migrate our PCR products. It revealed that our PCR hadn't work. We then realised that we hadn't changed the elongation temperature of our PCR program. Indeed, the dream taq functions at 72°C.

We did a PCR colony with the white colony obtained after we plated our golden gate 1 products transformed bacteria.

We used the following PCR program :

  1. 95°C for 3 min
  2. 95°C for 15 sec
  3. 55°C for 30 sec
  4. 72°C for 2 min
  5. 72°C for 10 min

(mettre photo gel)

Considering our gel, we can reasonably think that our golden gate had worked properly. We then did miniprep.

Clone 1-4 FecA AceB FepA Clone 5-8 FecA AceB FepA
Clone 1-4 FecA AceB FepA Clone 5-8 FecA AceB FepA

Friday, July 26th

We used nanodrop to measure the concentration of plasmid of our minipreps. The concentrations obtained were extremely low. We decided to do it again, but we got the same low results.

Week 7: 29th July - 4th August

Construction of plasmid N°1

We make an electrophoresis with 5 µL of plamsid to check the plasmid purification made on the last friday.

Mettre l'image légendée ici

There is not the 3 bandes that we sould see, so to check another time, we make a digestion with Pst I and EcoR I:

  • NEB Buffer 10X 5 µL
  • BSA 10X 5 µL
  • DNA 2 µg
  • PstI 2 µL
  • EcoRI 2 µL
  • Water 50 µL
After another electrophoresis, we obtain no band.

Sequencing preparation

Name Clone Concentration 260/280 260/230 Sequencing

AceB Fur Binding Site
(with sfGFP)

 1 166.1 ng/µL 1.86 1.76 Good
2 192.0 ng/µL 1.62 1.02 Good
3 123.5 ng/µL 1.87 1.75 Good
4 128.0 ng/µL 1.82 1.66 Good

FepA Fur Binding Site
(with sfGFP)

 1 21.1 ng/µL 2.06 - Good
2 22.4 ng/µL 1.96 - Good
3 55.4 ng/µL 1.87 1.58 Good
4 68.5 ng/µL 1.89 1.67 Good

Fes Fur Binding Site
(with sfGFP)

 1 39.9 ng/µL 1.87 1.80 Good
2 40.6 ng/µL 1.74 - No sequencing
3 68.4 ng/µL 1.87 1.63 Good
4 20.8 ng/µL 2.05 - No sequencing

ybiL Fur Binding Site
(with sfGFP)

 1 58.2 ng/µL 1.87 1.73 Good
2 16.3 ng/µL 1.95 - No sequencing
3 18.8 ng/µL 1.95 - No sequencing
4 45.5 ng/µL 1.90 1.81 Good

FecA Fur Binding Site
(with sfGFP)

 1 20.0 ng/µL 1.96 - Good
2 27.5 ng/µL 1.77 - No sequencing
3 31.5 ng/µL 1.94 1.81 Good
4 57.0 ng/µL 1.89 1.64 Good

yncE Fur Binding Site
(with sfGFP)

 1 92.4 ng/µL 1.90 1.75 Good
2 64.7 ng/µL 1.89 1.66 Good
3 38.6 ng/µL 1.91 1.80 Good

Construction of plasmid N°2

Our plasmid N°2 is building with a synthetic promote sequence which is composed of:

  • Andersen's promotor
  • Fur Binding Site (15 different)
  • RBS
  • sfGFP
  • Terminator

Golden Gate

In order to associate these sequences we performed a golden gate, using for each sample :

  • 80 ng of Andersen's promotor
  • 80 ng of Fur Binding Site
  • 80 ng of RBS
  • 80 ng of sfGFP
  • 80 ng of Terminator
  • 1.5 µL of T4 buffer (10X)
  • 15 Unit of T4 ligase
  • 2.5 Unit of Bsa I

The Golden Gate products are chemically transformed into E. coli Top 10 strains. After the transformation process, bacteria are plated into LB medium with carbenicillin and icubated overnigth at 37°C.

Golden Gates plates are composed at 98% of red colonies. The problem was probably on the equimolarity ratio between our different part which were not totally respected. We isolated white colonies (4 maximum by each sample) on LB medium with carbenicillin and we incubated them overnight at 37°c.



We made pre culture (V = 10 mL), using bacterial colonies isolated from our plates of Golden Gate transformation. Then minipreps and glycerol conservation were made using these pre cultures.

Fur Binding Site Sequence Clone Concentration

Fur BS 1

ATTATTGATAACTATTTG

Clone n°1

80.8 ng/µL

Clone n°2

83.8 ng/µL

Clone n°4

90.3 ng/µL

Fur BS 2

GATAACTATTTGCATTTGCA

Clone n°1

68.6 ng/µL

Clone n°2

77.4 ng/µL

Clone n°3

68.0 ng/µL

Fur BS 3

CAAATGCAAATAGTTATCCA

Clone n°1

79.4 ng/µL

Clone n°2

110.5 ng/µL

Clone n°3

457.4 ng/µL

Fur BS 4

CAAATAGTTATCAATAATCA

Clone n°1

73.2 ng/µL

Clone n°2

66.2 ng/µL

Fur BS 5

GTAAATTAATATTATTTACA

Clone n°1

57.2 ng/µL

Fur BS 6

AATAATGCTTCTCATTTTCA

Clone n°1

63.8 ng/µL

Fur BS 7

ATAAATGATAATCATTATCA

Clone n°1

70.6 ng/µL

Fur BS 8

GAAAATAATTCTTATTTCCA

Clone n°1

75.6 ng/µL

Clone n°2

87.2 ng/µL

Clone n°3

48.8 ng/µL

Clone n°4

57.8 ng/µL

Fur BS 9

AGCACTTATTATTATTTTCA

Clone n°1

58.6 ng/µL

Fur BS 10

GATAATTGTTATCGTTTGCA

No Clone

-

Fur BS 11

GATAATGCTTATCAAAATCA

Clone n°2

75.6 ng/µL

Clone n°3

398.0 ng/µL

Clone n°4

57.4 ng/µL

Fur BS 12

CAAAATTATTATCACTTTCA

Clone n°1

50.0 ng/µL

Fur BS 13

AATAATGATTACCATTCCCA

Clone n°2

70.7 ng/µL

Fur BS 14

GAAATTGTTTTTGATTTTCA

Clone n°1

42.7 ng/µL

Clone n°2

32.8 ng/µL

Clone n°3

37.8 ng/µL

Clone n°4

45.7 ng/µL

Fur BS 15

GTTAATTGTAATGATTTTCA

Clone n°1

37.0 ng/µL

Clone n°2

53.2 ng/µL

Clone n°3

196.0 ng/µL

Construction of plasmid N°3

29/07/13
We received the primers ordered on friday the 26th of July and started to dilute them into TrisCL at 10 mM. Secondly, we diluted the previous stock solution at a rate of 1/20th to obtain a final concentration of 5 µM for our intermediate solution.

Then, for our plasmid three construction, we need to first extract the 6 enterobactin gene (EntA, EntB, EntC, EntD, EntE and EntF). The ordered primers from friday will theoretically extract them in the appropriate Golden Gate format with their own RBS upstream (designed from Salis RBS). This will allow us to construct our two N°3 plasmids, one containing EntA, EntD and EntF, and the other one EntB, EntC and EntE. The genes are spread like this to obtain two equivalent plasmids xxx

We proceeded to a genomic extraction of the 6 genes of interest and migrated to PCR products on a 1% gel. We successfully extracted 5 out of the 6.

30/07/13
We annealled the oligonucleotides of the PL-LacO part in the golden gate format. Also, we extracted the sfGFP with the RBS upstream which will allow us to construct the control positive plasmid 3 for future TECAN experiments.

31/07/13
We started by purifying our 6 succesful PCR extraction from the 29/07 and 30/07. So we managed to extract EntA, EntB, EntC, EntD and EntF from E. coli's genomic DNA and sfGFP in the golden gate format with a RBS (plasmid 3 construction) from plasmidic DNA.

Additionnaly, we optimized our PCR to extract our missing gene, EntE. We obtained a smear +/- a double band. As a consequence, we adjusted the annealing temperature with a range from 54°C to 66°C. This will allow us to try extracting the missing gene (EntE) in the appropriate conditions.

01/08/13


We made a PCR for the EntE gene with a gradient of temperature to know at which temperature the PCR result were the best.
We made 8 (ou mettre 6 ?) tubes with the same composition:

  • Water
  • Taq Buffer 5
  • dNTPs
  • Rev EntE 2,5
  • For EntE 2,5
  • DNA 0,5
  • Taq polymerase 0,5
We also made positive and negative controles (with Rev EntC and For EntC instead of EntE).
After electrophoresis, we obtain the best band at .. °C.
However our experiment have been made with Taq polymerase (and Taq Buffer) instead of Q5 (and Q5 buffer) that we use usually. Then, we have to repeat this experiment with Q5 and Q5 buffer.
The did a gradient PCR and migrated on a gel our samples. On the gel, we notice that both the smear and the double band disappear when the annealing temperature increases. So to conclude, we run tonight a PCR with Q5 polymerase with increasing temperature starting from 56 degrees. The absolute annealing temperature will be determined for the Q5 (not extrapolable from a OneTaq PCR).

Also, we made a golden gate for the construction of plasmid 3. For analysis purposes, only a GFP was added downstream of the PL LacO promoter. Also, the EntE gene is still to be extracted from the genome. Also, we runned 3 extras golden gate for the construction of plasmid 2. In fact, we corrected our calculations because we initially we used mass concentrations instead of molar concentrations.


02/08/13


The transformations of the golden gate 2 and 3 are successful and we managed to invert the rate of red colonies versus white colonies (red = false positive with mRFP, white = positive), thus meaning we have a lot of white ones! We conserved the plates for future isolation on coming monday.

Additionnaly, we migrated the PCR products of 01/08. The gel is empty, but the positive controls are there, meaning we made a mistake in the primers (the master mix is the one used for the positive control).

Week 8: 5th August - 11th August

Week 9: 12th August - 18th August

Week 10: 19th August - 25th August

Week 11: 26th August - 1st September

Week 12: 2nd September - 8th September

Week 13: 9th September - 15th September

Week 14: 16th September - 22nd September

Week 15: 23rd September - 29th September

Week 16: 30th September - 6th October

Week 17: 7th October - 13th October

Week 18: 14th October - 20th October

Week 19: 21st October - 27th October

Week 20: 28th October - 3rd November

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