Team:Grenoble/Biology/Notebook/July/week 28

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(Difference between revisions)
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<a href="https://2012.igem.org/Team:Grenoble/Biology/Notebook/July/week_29">Week 29</a> •  
<a href="https://2012.igem.org/Team:Grenoble/Biology/Notebook/July/week_29">Week 29</a> •  
<a href="https://2012.igem.org/Team:Grenoble/Biology/Notebook/July/week_30">Week 30</a>
<a href="https://2012.igem.org/Team:Grenoble/Biology/Notebook/July/week_30">Week 30</a>
-
</section><br/>
+
</section>
 +
<br/>
<section>
<section>
-
<h1> Week 28: July 09<span class="exposant">th</span> to 15<span class="exposant">th</span> </h1>
+
<h1> Week 29: July 16<span class="exposant">th</span> to 22<span class="exposant">nd</span> </h1>
<h2> Goal of the week: </h2>
<h2> Goal of the week: </h2>
-
We wanted to recover and amplify the biobricks involved in our genetic networks:
+
We wanted to test the Gibson Assembly and the transformation protocols, recover and amplify some biobricks involved in our genetic networks:
<ul>
<ul>
<li>pAra/Bad_RBS_GFP (1300bp)</li>
<li>pAra/Bad_RBS_GFP (1300bp)</li>
<li>RBS_Cya (2600bp)</li>
<li>RBS_Cya (2600bp)</li>
-
<li>pLAC (100bp)</li>
+
<li>fha1 (80bp)</li>
-
<li>fha (80bp)</li>
+
<li>eCFP (800bp)</li>
<li>eCFP (800bp)</li>
-
<li>pLAC_RBS (120bp)</li>
 
-
<li>RsmA (200bp)</li>
 
-
<li>rsmY (170bp)</li>
 
-
<li>pSB1A3 (2400bp)</li>
 
<li>pSB4K5 (2400bp)</li>
<li>pSB4K5 (2400bp)</li>
-
<li>pSB3C5 (2400bp)</li>
 
</ul>
</ul>
-
<br/>We also planned to realise the gibson assemblies for the first constructions.
 
</section>
</section>
<section>
<section>
-
<h2> Monday, July 09<span class="exposant">th</span>:</h2>
+
<h2> Monday, July 16<span class="exposant">th</span>:</h2>
Precultured cells are prepared:
Precultured cells are prepared:
<ul>
<ul>
-
<li>Strains = BW25113 WT, BW25113 cya<span class="exposant">-</span>, BW25113 cya<span class="exposant">-</span>pAra/Bad and the strain transformed with pSB4K5 </li>
+
<li>Strains = BW25113 WT and BW25113 cya- pAra/Bad </li>
-
<li>Conditions = LB liquid medium, 37°C, 200rpm, overnight </li>
+
<li>Conditions = Conditions = LB liquid medium, 37°C, 200rpm, overnight </li>
</ul>
</ul>
</section>
</section>
<section>
<section>
-
<h2>Tuesday, July 10<span class="exposant">th</span>:</h2>
+
<h2>Tuesday, July 17<span class="exposant">th</span>:</h2>
-
For the PCRs, we changed the enzyme from GoTaq to High Fidelity (HF) Phusion.<br/>
+
Using iGEM 2012 biobricks and the Gibson Assembly product we transformed (protocol) BW25113 WT cells. We obtained four transformed strains with:
-
We did some colony PCR with a HF Phusion enzyme (protocol) to amplify:
+
<ul>
<ul>
-
<li>fha1, RsmA, rsmY and pSB1A3 from iGEM Grenoble 2011 glycerol stock.</li>
+
<li>BBa_I13601: pLAC_RBS</li>
-
<li>pAra/Bad_RBS_GFP from BW25113 cya<span class="exposant">-</span> pAra/Bad precultured cells.</li>
+
<li>BBa_E0422: eCFP</li>
-
<li>RBS_Cya from BW25113 WT precultured cells.</li>
+
<li>GA: pLAC_rsmY (pSB1A3) </li>
 +
<li>GA: pLAC_RBS_RsmA (pSB3C5) </li>
</ul>
</ul>
-
We did the PCR both with and without DMSO (Amplification of difficult targets, such as those with GC-rich sequences or secondary structure, may be improved by the presence of additives such as DMSO).<br/>
 
<br/>
<br/>
-
To separate the PCR products, we prepared two gels:
+
We did a miniprep (protocol kit: Nucleospin plasmid Quick Pure) on 2 strains, on which we wanted to recover pSB4K5 and the plasmid with pAra/Bad_RBS_GFP:
<ul>
<ul>
-
<li>a 1.3% TAE agarose gel, for the small fragments: RsmA, fha1 and rsmY (length < 1000bp)</li>
+
<li>transformed strain with pSB4K5</li>
-
<li>a 0.8% TAE agarose gel, for the big fragments: pSB1A3, RBS_Cya, and pAra/Bad_RBS_GFP (length > 1000bp)</li>
+
<li>BW25113 cya- pAra/Bad</li>
</ul>
</ul>
-
Migration conditions = 50V during 1h15.<br/>
+
<br/>We did PCRs with HF Phusion enzyme (protocol) on this miniprep in order to amplify pSB4K5, and from colony to amplify pLAC (fha), pLAC (rsmY), pLAC_RBS, fha and pSB4K5. We did the PCR both with and without DMSO.<br/>
-
We used EtBr to reveal the DNA fragments.<br/>
+
<br/>
<br/>
-
<center><img src="https://static.igem.org/mediawiki/2012/b/b6/120710.jpg" alt="photo_gel_4"/></center>
+
To separate the PCR products and the miniprep products, we prepared a 1.8% TAE agarose gel.<br/>
-
<div class="legend"><u>Migration result for the 1.3% TAE agarose gel (small fragments)</u><br/>
+
Migration conditions = 100V during 30 min.<br/>
-
      <i>(the DNA ladder scale is in kb)</i><br/>
+
In order to reveal the DNA fragments, we used EtBr.<br/>
-
Lane 1: DNA ladder 100bp (biolabs)<br/>
+
-
Lanes 2 and 3: fha1 PCR product<br/>
+
-
Lanes 4 and 5: RsmA PCR product<br/>
+
-
Lanes 6 and 7: rsmY PCR product<br/>
+
-
Lanes 8 and 9: fha1 PCR product (DMSO)<br/>
+
-
Lanes 10 and 11: RsmA PCR product (DMSO)<br/>
+
-
Lane 12: rsmY PCR product (DMSO)<br/>
+
-
Lane 13: DNA ladder 100bp (biolabs)<br/></div>
+
<br/>
<br/>
-
<center><img src="https://static.igem.org/mediawiki/2012/4/4e/120710_%282%29.jpg" alt="photo_gel_5"/></center>
+
<center><img src="https://static.igem.org/mediawiki/2012/3/33/120717.jpg" alt="photo_gel_11"/></center>
-
<div class="legend"><u>Migration result for the 0.8% TAE agarose gel (big fragments)</u><br/>
+
<div class="legend"><u>Migration result for a 1.8% TAE agarose gel</u><br/>
-
            <i> (the DNA ladder scale is in kb)</i><br/>
+
  <i>(the DNA ladder scale is in kb)</i><br/>
Lane 1: DNA ladder 1kb (biolabs)<br/>
Lane 1: DNA ladder 1kb (biolabs)<br/>
-
Lanes 2 and 3: RBS_Cya PCR product<br/>
+
Lanes 2 and 3: the deposits have failed<br/>
-
Lanes 4 and 5: pSB1A3 PCR product<br/>
+
Lane 4: pSB4K5 colony PCR product (DMSO)<br/>
-
Lanes 6 and 7: pAra/Bad_RBS_GFP PCR product<br/>
+
Lane 5: pSB4K5 colony PCR product<br/>
-
Lanes 8 and 9: RBS_Cya PCR product (DMSO)<br/>
+
Lane 6: pSB4K5 PCR (miniprep) product (DMSO)<br/>
-
Lanes 10 and 11: pSBA13 PCR product (DMSO)<br/>
+
Lane 7: pSB4K5 PCR (miniprep) product<br/>
-
Lane 12: pAra/Bad_GFP PCR product (DMSO)<br/>
+
Lane 8: DNA ladder 1kb (biolabs)<br/>
-
Lane 13: DNA ladder 1kb (biolabs)<br/></div>
+
Lane 9: DNA ladder 100bp (biolabs)<br/>
 +
Lane 10: pLAC (fha1) PCR product (DMSO)<br/>
 +
Lane 11: pLAC (fha1) PCR product<br/>
 +
Lane 12: pLAC_RBS PCR product (DMSO)<br/>
 +
Lane 13: pLAC_RBS PCR product<br/>
 +
Lane 14: pLAC (rsmY) PCR product (DMSO)<br/>
 +
Lane 15: pLAC (rsmY) PCR product<br/>
 +
Lane 16: fha1 PCR product (DMSO)<br/>
 +
Lane 17: fha1 PCR product<br/>
 +
Lane 18: empty<br/>
 +
Lane 19: pSB4K5 miniprep product<br/>
 +
Lane 20: plasmid with pAra/Bad_RBS_GFP miniprep product<br/></div>
<br/>
<br/>
-
There was a migration problem on the second gel (DNA ladder migration was not right) and we only saw primer dimer bands. There was a PCR condition problem.<br/>
+
We achieved to amplify pLAC (fha1), pLAC (rsmY) and pLAC_RBS. We didn’t achieve to amplify pSB4K5 and fha1.<br/>
-
<br/>
+
-
Precultured cells were prepared:
+
-
<ul>
+
-
<li>Strains = BW25113 WT.</li>
+
-
<li>Conditions = LB liquid medium, 37°C, 200rpm, overnight.</li>
+
-
</ul>
+
</section>
</section>
<section>
<section>
-
<h2> Wednesday, July 11<span class="exposant">th</span>:</h2>
+
<h2> Wednesday, July 18<span class="exposant">th</span>:</h2>
-
We did a miniprep (protocol kit: Nucleospin plasmid Quick Pure) on three iGEM Grenoble 2011 strains:
+
We did PCRs with HF Phusion enzyme (protocol) on a miniprep (12/07/17) in order to amplify pAra/Bad_RBS_GFP, on colony (iGEM Grenoble 2011) to amplify fha1, and on glycerol stock (BW25113 WT) to amplify RBS_Cya. We did the PCR both with and without DMSO.<br/>
-
<ul>
+
-
<li>fha1</li>
+
-
<li>RsmA</li>
+
-
<li>rsmY</li>
+
-
</ul>
+
<br/>
<br/>
-
We did a 15min digestion (protocol) by some restriction enzymes (XbaI and PstI) in order to check if there were the right plasmids in the strains.<br/>
+
To separate the PCR products, we prepared a 1.8% TAE agarose gel.<br/>
-
<br/>
+
Migration conditions = 100V during 30 min.<br/>
-
To separate the digestion products, we prepared a 1.3% TAE agarose gel.<br/>
+
-
Migration conditions = 50V during 1h15.<br/>
+
In order to reveal the DNA fragments, we used EtBr.<br/>
In order to reveal the DNA fragments, we used EtBr.<br/>
<br/>
<br/>
-
<center><img src="https://static.igem.org/mediawiki/2012/3/3f/120711.jpg" alt="photo_gel_6"/></center>
+
<center><img src="https://static.igem.org/mediawiki/2012/8/83/120718.jpg" alt="photo_gel_12"/></center>
-
<center><div class="legend"><u>Migration result for a 1.3% TAE agarose gel</u><br/>
+
<div class="legend"><u>Migration result for a 1.8% TAE agarose gel</u> <br/>
-
            <i>(the DNA ladder scale is in kb)</i><br/>
+
  <i>(the DNA ladder scale is in kb)</i> <br/>
-
Lane 1: DNA ladder 100pb (biolabs)<br/>
+
Lane 1: DNA ladder 100bp (biolabs)<br/>
-
Lanes 2 and 3: fha1 digestion product (XbaI)<br/>
+
Lane 2: pAra/Bad_RBS_GFP PCR product (1μL/DMSO)<br/>
-
Lanes 4 and 5: RsmA digestion product (XbaI)<br/>
+
Lane 3: pAra/Bad_RBS_GFP PCR product (2μL/DMSO)<br/>
-
Lanes 6 and 7: rsmY digestion product (XbaI)<br/>
+
Lane 4: pAra/Bad_RBS_GFP PCR product (1μL)<br/>
-
Lanes 8 and 9: fha digestion product (pSTI)<br/>
+
Lane 5: pAra/Bad_RBS_GFP PCR product (2μL)<br/>
-
Lanes 10 and 11: RsmA digestion product (pSTI)<br/>
+
Lane 6: RBS_Cya PCR product (DMSO)<br/>
-
Lanes 12 and 13: rsmY digestion product (pSTI)<br/>
+
Lane 7: RBS_Cya PCR product<br/>
-
Lanes 14 and 15: fha1 digestion product (XbaI-pSTI)<br/>
+
Lane 8: fha1 PCR product (DMSO)<br/>
-
Lanes 16 and 17: RsmA digestion product (XbaI-pSTI)<br/>
+
Lane 9: fha1 PCR product<br/>
-
Lanes 18 and 19: rsmY digestion product (XbaI-pSTI)<br/>
+
Lane 10: DNA ladder 100bp (biolabs)<br/></div>
-
Lane 20: DNA ladder 100pb (biolabs)<br/></div></center>
+
<br/>
<br/>
-
We seen the DNA bands were at the wrong position, like there was no digestion. We thought the digestion problem occured during the heating step (we had a problem using the thermoblock).<br/>
+
We didn’t see anything. The PCR didn’t work.<br/>
<br/>
<br/>
-
Using iGEM 2012 biobricks we transformed (protocol) BW25113 WT cells. We obtained five transformed strains with five different biobricks:
+
We did PCRs with HF Phusion enzyme (protocol) on miniprep (12/07/17) in order to amplify pSB4K5, and on 2 colonies (iGEM Grenoble 2011) to amplify fha1. We did the PCR both with and without DMSO.<br/>
-
<ul>
+
-
<li>BBa_I13601: pLAC_RBS</li>
+
-
<li>BBa_E0422: eCFP </li>
+
-
<li>pSB3C5 plasmid </li>
+
-
<li>psB4K5 plasmid </li>
+
-
<li>psB1A3 plasmid</li>
+
-
</ul>
+
<br/>
<br/>
-
Precultured cells were prepared:
+
To separate the PCR products, we prepared a 1.8% TAE agarose gel.<br/>
 +
Migration conditions = 100V during 30 min.<br/>
 +
In order to reveal the DNA fragments, we used EtBr.<br/>
 +
<br/>
 +
No result. (data not shown)<br/>
 +
<br/>
 +
Using biobricks from the 2012 iGEM kit and our Gibson Assemblies product we transformed (new protocol) BW25113 WT competent cells (protocol). We obtained eight transformed strains with:
<ul>
<ul>
-
<li>Strains = BW25113 cya<span class="exposant">-</span> pAra/Bad.</li>
+
<li>lux pR (BBa_R0062)</li>
-
<li>Conditions = LB liquid medium, 37°C, 200rpm, overnight.</li>
+
<li>LuxI (BBa_C0061)</li>
 +
<li>LuxR (BBa_I0462)</li>
 +
<li>eCFP (BBa_E0022)</li>
 +
<li>eCFP (BBa_E0422)</li>
 +
<li>pLAC_RBS (BBa_I13601)</li>
 +
<li>GA: pLAC_rsmY (pSB1A3) </li>
 +
<li>GA: pLAC_RBS_RsmA (pSB3C5) </li>
</ul>
</ul>
</section>
</section>
<section>
<section>
-
<h2> Thursday, July 12<span class="exposant">th</span>:</h2>
+
<h2> Thursday, July 19<span class="exposant">th</span>:</h2>
-
The transformation using pSB4K5 is the only one out of five transformations that showed results.<br/>
+
Six transformations (12/07/18) out of eight showed results: RsmA, lux pR, LuxI, LuxR, E0422, E0022.<br/>
<br/>
<br/>
-
We did some PCRs with a HF Phusion enzyme (protocol) from miniprep (12/07/11) to amplify rsmY and fha1. We did the PCR both with and without DMSO.<br/>
+
We did a miniprep (protocol kit: Nucleospin plasmid Quick Pure) on these transformed strains (12/07/18): RsmA, lux pR, LuxI, LuxR, E0422, E0022.<br/>
<br/>
<br/>
-
To separate the PCR products, we prepared a 1.3% TAE agarose gel.<br/>
+
We also did some digestions (protocol) in order to check if the Gibson Assembly product (pLAC_RBS_RsmA) is the right one. The digestions were realised with two restriction enzymes : XbaI and SpeI during 10 minutes.<br/>
-
Migration conditions = 50V during 1h.<br/>
+
<br/>
 +
To separate the digestion products, we prepared a 1.8% TAE agarose gel.<br/>
 +
Migration conditions = 100V during 30 min.<br/>
In order to reveal the DNA fragments, we used EtBr.<br/>
In order to reveal the DNA fragments, we used EtBr.<br/>
<br/>
<br/>
-
<center><img src="https://static.igem.org/mediawiki/2012/f/f6/120712.jpg" alt="photo_gel_7"/></center>
+
<center><img src="https://static.igem.org/mediawiki/2012/5/51/120719.jpg" alt="photo_gel_13"/></center>
-
<div class="legend"><u>Migration result for a 1.3% TAE agarose gel</u><br/>
+
<div class="legend"><u>Migration result for a 1.8% TAE agarose gel</u><br/>
-
    <i> (the DNA ladder scale is in kb)</i><br/>
+
  <i>(the DNA ladder scale is in kb)</i><br/>
Lane 1: DNA ladder 100bp (biolabs)<br/>
Lane 1: DNA ladder 100bp (biolabs)<br/>
-
Lane 2: fha1 PCR product<br/>
+
Lane 2: GA (pLAC_RBS_RsmA) digestion product (XbaI)<br/>
-
Lane 3: rsmY PCR product<br/>
+
Lane 3: GA (pLAC_RBS_RsmA) digestion product (SpeI)<br/>
-
Lane 4: fha1 PCR product (DMSO)<br/>
+
Lane 4: GA (pLAC_RBS_RsmA) digestion product (XbaI & SpeI)<br/>
-
Lane 5: rsmY PCR product (DMSO)<br/>
+
Lane 5: DNA ladder 100bp (biolabs)<br/></div>
-
Lane 6: DNA ladder 100bp (biolabs)<br/>
+
-
Lane 7 : empty<br/></div>
+
<br/>
<br/>
-
We saw no DNA bands at the right position, we thought there was still a PCR condition problem.<br/>
+
We concluded that the Gibson Assembly (pLAC_RBS_RsmA on pSB1A3) worked. The digestion result is the expected one.<br/>
<br/>
<br/>
-
We did a miniprep (protocol kit: Nucleospin plasmid Quick Pure) on 2 strains, on which we wanted to recover pSB4K5 and the plasmid with pAra/Bad_RBS_GFP:
+
We did PCRs with HF Phusion enzyme (protocol) from minipreps in order to amplify pAra/Bad_RBS_GFP (12/07/17), eCFP (E0022 et E0422) (12/07/19). And a colony PCR to amplify RBS_Cya. We did the PCR both with and without DMSO.<br/>
-
<ul>
+
-
<li>transformed strain with pSB4K5</li>
+
-
<li>BW25113 cya<span class="exposant">-</span> pAra/Bad</li>
+
-
</ul>
+
<br/>
<br/>
-
We did some colony PCR with a HF Phusion enzyme (protocol) from iGEM Grenoble 2011 glycerol stock, to amplify: pLAC (fha1), pLAC (rsmY), pLAC_RBS, eCFP and RsmA.<br/>
+
To separate the PCR products, we prepared a 1.3% TAE agarose gel.<br/>
-
<br/>
+
-
To separate the PCR products and the miniprep products, we prepared a 1.3% TAE agarose gel.<br/>
+
Migration conditions = 100V during 30 min.<br/>
Migration conditions = 100V during 30 min.<br/>
In order to reveal the DNA fragments, we used EtBr.<br/>
In order to reveal the DNA fragments, we used EtBr.<br/>
<br/>
<br/>
-
<center><img src="https://static.igem.org/mediawiki/2012/7/76/120712_%282%29.jpg" alt="photo_gel_8"/></center>
+
<center><img src="https://static.igem.org/mediawiki/2012/7/77/120719_%282%29.jpg" alt="photo_gel_14"/></center>
-
<div class="legend"><u>Migration result for a 1.3% TAE agarose gel</u><br/>
+
<div class="legend"><u>Migration result for a 1.3% TAE agarose gel</u></br>
-
    <i> (the DNA ladder scale is in kb)</i><br/>
+
  <i>(the DNA ladder scale is in kb)</i></br>
-
Lane 2: DNA ladder 1kb (biolabs)<br/>
+
Lane 1: DNA ladder 1kb (biolabs)</br>
-
Lane 3: pSB4K5 miniprep product<br/>
+
Lane 2: eCFP (E0022) PCR product</br>
-
Lane 4: plasmid with pAra/Bad miniprep product<br/>
+
Lane 3: eCFP (E0022) PCR product (DMSO)</br>
-
Lane 5: empty<br/>
+
Lane 4: RBS_Cya PCR product</br>
-
Lane 7: eCFP PCR product<br/>
+
Lane 5: RBS_Cya PCR product (DMSO)</br>
-
Lane 8: pLAC (fha1) PCR product<br/>
+
Lane 6: pAra/Bad_GFP PCR product 1</br>
-
Lane 9: pLAC_RBS PCR product<br/>
+
Lane 7: pAra/Bad_GFP PCR product 1 (DMSO)</br>
-
Lane 10: pLAC (rsmY) PCR product<br/>
+
Lane 8: pAra/Bad_GFP PCR product 2</br>
-
Lane 11: RsmA PCR product<br/>
+
Lane 9: pAra/Bad_GFP PCR product 2 (DMSO)</br>
-
Lane 12: DNA ladder 100bp (biolabs)<br/>
+
Lane 10: eCFP (E0422) PCR product </br>
-
Lane 13 : empty<br/></div>
+
Lane 11: eCFP (E0422) PCR product (DMSO)</br>
 +
Lane 12: DNA ladder 1kb (biolabs)</br></div>
 +
</br>
 +
We saw primer dimer bands and the bands corresponding to the amplified plasmids which brought eCFP (E0022 & E0422). There was a PCR condition problem.<br/>
<br/>
<br/>
-
The DNA bands corresponding to pLAC(fha1), pLAC_RBS pLAC(rsmY) and RsmA were at the expected positions. We thus decided to do the migration again in order to purify these PCR products.<br/>
+
One transformation (12/07/18) showed result during the day: rsmY. We decided to relaunch it in fresh LB medium + antibiotics.<br/>
 +
</section>
 +
<section>
 +
<h2>Friday, July 20<span class="exposant">th</span>:</h2>
 +
We did a miniprep (protocol kit: Nucleospin plasmid Quick Pure) on the transformed strains (12/07/18) with Gibson Assembly product (RsmA and rsmY).<br/>
<br/>
<br/>
-
We decided to fix the agarose percentage in our gels at 1.8% to separe the small fragments (length < 1000bp) and at 1.3% for biggest fragments (length > 1000bp).<br/>
+
In order to check the products of Gibson Assembly (RsmA and rsmY) we launched digestion experiments on these miniprep products, using restriction enzymes (EcoRI, BamHI, XbaI and SpeI) during 10 minutes. We also launched digestion experiments on the eCFP miniprep products (12/07/19), using the same restriction enzymes, in order to recover the eCFP coding sequence.<br/>
<br/>
<br/>
-
Precultured cells were prepared:
+
To separate these digestion products, we prepared a 1.8% TAE agarose gel.<br/>
-
<ul>
+
-
<li>Strains = BW25113 WT, pSB1A3 (iGEM Grenoble 2011) and pSB3C5 (iGEM Grenoble 2011).</li>
+
-
<li>Conditions = LB liquid medium, 37°C, 200rpm, overnight.</li>
+
-
</ul>
+
-
</section>
+
-
<section>
+
-
<h2> Friday, July 13<span class="exposant">th</span>:</h2>
+
-
To separate the PCR products (PCRs 07/12), we prepared a 1.8% TAE agarose gel.<br/>
+
Migration conditions = 100V during 30 min.<br/>
Migration conditions = 100V during 30 min.<br/>
In order to reveal the DNA fragments, we used EtBr.<br/>
In order to reveal the DNA fragments, we used EtBr.<br/>
<br/>
<br/>
-
<center><img src="https://static.igem.org/mediawiki/2012/6/60/120713.jpg" alt="photo_gel_9"/></center>
+
<center><img src="https://static.igem.org/mediawiki/2012/0/00/120720.jpg" alt="photo_gel_15"/></center>
<div class="legend"><u>Migration result for a 1.8% TAE agarose gel</u><br/>
<div class="legend"><u>Migration result for a 1.8% TAE agarose gel</u><br/>
-
              <i> (the DNA ladder scale is in kb)</i><br/>
+
  <i>(the DNA ladder scale is in kb)</i><br/>
-
Lane 1: DNA ladder 100bp (biolabs)<br/>
+
Lane 1: DNA ladder 1kb (biolabs)<br/>
-
Lane 2: eCFP PCR product<br/>
+
Lane 2: E0422 digestion product<br/>
-
Lane 3: pLAC (fha1) PCR product<br/>
+
Lane 3: E0022 digestion product<br/>
-
Lane 4: pLAC_RBS PCR product<br/>
+
Lane 4: GA (RsmA) miniprep product<br/>
-
Lane 5: pLAC (rsmY) PCR product<br/>
+
Lane 5: GA (rsmY) miniprep product<br/>
-
Lane 6: RsmA PCR product<br/>
+
Lane 6: DNA ladder 1kb (biolabs)<br/>
-
Lane 7: empty<br/>
+
Lane 7: DNA ladder 100bp (biolabs)<br/>
-
Lane 8: rsmY PCR product<br/>
+
Lane 8: GA (RsmA) digestion product<br/>
-
Lane 9: fha1 PCR product<br/>
+
Lane 9: GA (rsmY) digestion product<br/>
-
Lane 10: empty<br/>
+
Lane 10: DNA ladder 100bp (biolabs)<br/></div>
-
Lane 11: rsmY PCR product<br/>
+
-
Lane 12: fha PCR product<br/>
+
-
Lane 13: DNA ladder 100bp (biolabs)<br/></div>
+
<br/>
<br/>
-
We realised a DNA extraction (protocol kit: Nucleospin extract II) from all the fragments except the fha1 PCR product (we didn’t see anything) and the eCFP PCR product (because its size did not correspond to the expected one: 800bp):
+
We realised a DNA extraction (protocol kit: Nucleospin extract II) on the bands corresponding to the eCFP coding sequence (E0422 and E0022) from digestion products <span class="code">120720AM_DIG_018 // 120720AM_DIG_19</span>.<br/>
-
<ul>
+
On these extractions, we did PCRs with HF Phusion enzyme (protocol) in order to amplify eCFP. We did the PCR both with and without DMSO.<br/>
-
<li>pLAC (fha1) <span class="code">120713PP_PCR_009</span></li>
+
-
<li>pLAC_RBS <span class="code">120713PP_PCR_010</span></li>
+
-
<li>pLAC (rsmY) <span class="code">120713PP_PCR_011</span></li>
+
-
<li>RsmA <span class="code">120713PP_PCR_012</span></li>
+
-
<li>rsmY <span class="code">120713PP_PCR_013</span></li>
+
-
</ul>
+
<br/>
<br/>
-
We did a miniprep (protocol kit: Nucleospin plasmid Quick Pure) on 2 strains (12/07/12) to recover pSB1A3 and pSB3C5.<br/>
+
To separate the PCR products and the digestion products, we prepared a 1.3% TAE agarose gel.<br/>
-
<br/>
+
-
We did some PCR with HF Phusion enzyme (protocol) on this miniprep in order to amplify pSB1A3, pSB3C5 and on another miniprep (12/07/12) to amplify pSB4K5. We did the PCR both with and without DMSO.<br/>
+
-
<br/>
+
-
To separate the PCR products, we prepared a 1.3% TAE agarose gel.<br/>
+
Migration conditions = 100V during 30 min.<br/>
Migration conditions = 100V during 30 min.<br/>
In order to reveal the DNA fragments, we used EtBr.<br/>
In order to reveal the DNA fragments, we used EtBr.<br/>
<br/>
<br/>
-
<center><img src="https://static.igem.org/mediawiki/2012/4/4e/120713_%282%29.jpg" alt="photo_gel_10"/></center>
+
<center><img src="https://static.igem.org/mediawiki/2012/4/4c/120720_%282%29.jpg" alt="photo_gel_15"/></center>
<div class="legend"><u>Migration result for a 1.3% TAE agarose gel</u><br/>
<div class="legend"><u>Migration result for a 1.3% TAE agarose gel</u><br/>
-
              <i> (the DNA ladder scale is in kb)</i><br/>
+
  <i>(the DNA ladder scale is in kb)</i><br/>
Lane 1: DNA ladder 1kb (biolabs)<br/>
Lane 1: DNA ladder 1kb (biolabs)<br/>
-
Lane 2: pSB1A3 PCR product<br/>
+
Lane 2: E0022 digestion product<br/>
-
Lane 3: pSB3C5 (cya) PCR product<br/>
+
Lane 3: E0422 digestion product<br/>
-
Lane 4: pSB3C5 (RsmA) PCR product<br/>
+
Lane 4: E0022 PCR product<br/>
-
Lane 5: pSB4K5 PCR product<br/>
+
Lane 5: E0022 PCR product (DMSO)<br/>
-
Lane 6: pSB1A3 PCR product (DMSO)<br/>
+
Lane 6: E0422 PCR product<br/>
-
Lane 7: pB3C5 (cya) PCR product (DMSO)<br/>
+
Lane 7: E0422 PCR product (DMSO)<br/>
-
Lane 8: pSB3C5 (RsmA) PCR product (DMSO)<br/>
+
Lane 8: DNA ladder 1kb (biolabs)<br/></div>
-
Lane 9: pSB4K5 PCR product (DMSO)<br/>
+
-
Lane 10: DNA ladder 1kb (biolabs)<br/></div>
+
<br/>
<br/>
-
We realised a DNA extraction (protocol kit: Nucleospin extract II) for all the fragments except the pSB4K5 PCR product (we didn’t see anything):
+
We saw primer dimer bands. There was a PCR condition problem.<br/>
-
<ul>
+
-
<li>pSB1A3 <span class="code">120713PP_PCR_014</span></li>
+
-
<li>pSB3C5 (cya) <span class="code">120713PP_PCR_015</span></li>
+
-
<li>pSB3C5 (RsmA) <span class="code">120713PP_PCR_016</span></li>
+
-
</ul>
+
<br/>
<br/>
-
We realised two Gibson Assembly (protocol) to build two plasmids:
+
We did PCRs with HF Phusion enzyme (protocol) in order to amplify pSB4K5 and fha from miniprep. We did the same PCR twice: one with DMSO and one without.<br/>
-
<ul style="text-align:left";>
+
-
<li>pSB1A3 <span class="code">120713PP_PCR_014</span> with pLAC (rsmY) <span class="code">120713PP_PCR_011</span> and rsmY <span class="code">120713PP_PCR_013</span></li>
+
-
<li>pSB3C5 (RsmA) <span class="code">120713PP_PCR_016</span> with pLAC_RBS <span class="code">120713PP_PCR_010</span> and RsmA <span class="code">120713PP_PCR_012</span></li>
+
-
 
+
-
</ul>
+
</section>
</section>
<section>
<section>
<h2>Conclusion of the week:</h2>
<h2>Conclusion of the week:</h2>
-
We have achieved to amplify :
+
We have achieved our first Gibson Assembly : pLAC_RBS_RsmA on pSB3C5 and we began the experiments in order to recover luxpR, LuxI and LuxR (for the 1<span class="exposant">st</span> network).<br/>
-
pLAC (fha1) // pLAC (rsmY) // pLAC_RBS // RsmA // rsmY // pSB1A3 // pSB3C5 (Cya) // pSB3C5 (RsmA)<br/>
+
-
<br/>
+
-
We tried our first Gibson Assemblies.
+
</section>
</section>
</div>
</div>

Revision as of 13:56, 20 August 2012

iGEM Grenoble 2012

iGEM Grenoble iGEM
iGEM 2012
main page
Project

July

Week 27 • Week 28 • Week 29 • Week 30

Week 29: July 16th to 22nd

Goal of the week:

We wanted to test the Gibson Assembly and the transformation protocols, recover and amplify some biobricks involved in our genetic networks:
  • pAra/Bad_RBS_GFP (1300bp)
  • RBS_Cya (2600bp)
  • fha1 (80bp)
  • eCFP (800bp)
  • pSB4K5 (2400bp)

Monday, July 16th:

Precultured cells are prepared:
  • Strains = BW25113 WT and BW25113 cya- pAra/Bad
  • Conditions = Conditions = LB liquid medium, 37°C, 200rpm, overnight

Tuesday, July 17th:

Using iGEM 2012 biobricks and the Gibson Assembly product we transformed (protocol) BW25113 WT cells. We obtained four transformed strains with:
  • BBa_I13601: pLAC_RBS
  • BBa_E0422: eCFP
  • GA: pLAC_rsmY (pSB1A3)
  • GA: pLAC_RBS_RsmA (pSB3C5)

We did a miniprep (protocol kit: Nucleospin plasmid Quick Pure) on 2 strains, on which we wanted to recover pSB4K5 and the plasmid with pAra/Bad_RBS_GFP:
  • transformed strain with pSB4K5
  • BW25113 cya- pAra/Bad

We did PCRs with HF Phusion enzyme (protocol) on this miniprep in order to amplify pSB4K5, and from colony to amplify pLAC (fha), pLAC (rsmY), pLAC_RBS, fha and pSB4K5. We did the PCR both with and without DMSO.

To separate the PCR products and the miniprep products, we prepared a 1.8% TAE agarose gel.
Migration conditions = 100V during 30 min.
In order to reveal the DNA fragments, we used EtBr.

photo_gel_11
Migration result for a 1.8% TAE agarose gel
(the DNA ladder scale is in kb)
Lane 1: DNA ladder 1kb (biolabs)
Lanes 2 and 3: the deposits have failed
Lane 4: pSB4K5 colony PCR product (DMSO)
Lane 5: pSB4K5 colony PCR product
Lane 6: pSB4K5 PCR (miniprep) product (DMSO)
Lane 7: pSB4K5 PCR (miniprep) product
Lane 8: DNA ladder 1kb (biolabs)
Lane 9: DNA ladder 100bp (biolabs)
Lane 10: pLAC (fha1) PCR product (DMSO)
Lane 11: pLAC (fha1) PCR product
Lane 12: pLAC_RBS PCR product (DMSO)
Lane 13: pLAC_RBS PCR product
Lane 14: pLAC (rsmY) PCR product (DMSO)
Lane 15: pLAC (rsmY) PCR product
Lane 16: fha1 PCR product (DMSO)
Lane 17: fha1 PCR product
Lane 18: empty
Lane 19: pSB4K5 miniprep product
Lane 20: plasmid with pAra/Bad_RBS_GFP miniprep product

We achieved to amplify pLAC (fha1), pLAC (rsmY) and pLAC_RBS. We didn’t achieve to amplify pSB4K5 and fha1.

Wednesday, July 18th:

We did PCRs with HF Phusion enzyme (protocol) on a miniprep (12/07/17) in order to amplify pAra/Bad_RBS_GFP, on colony (iGEM Grenoble 2011) to amplify fha1, and on glycerol stock (BW25113 WT) to amplify RBS_Cya. We did the PCR both with and without DMSO.

To separate the PCR products, we prepared a 1.8% TAE agarose gel.
Migration conditions = 100V during 30 min.
In order to reveal the DNA fragments, we used EtBr.

photo_gel_12
Migration result for a 1.8% TAE agarose gel
(the DNA ladder scale is in kb)
Lane 1: DNA ladder 100bp (biolabs)
Lane 2: pAra/Bad_RBS_GFP PCR product (1μL/DMSO)
Lane 3: pAra/Bad_RBS_GFP PCR product (2μL/DMSO)
Lane 4: pAra/Bad_RBS_GFP PCR product (1μL)
Lane 5: pAra/Bad_RBS_GFP PCR product (2μL)
Lane 6: RBS_Cya PCR product (DMSO)
Lane 7: RBS_Cya PCR product
Lane 8: fha1 PCR product (DMSO)
Lane 9: fha1 PCR product
Lane 10: DNA ladder 100bp (biolabs)

We didn’t see anything. The PCR didn’t work.

We did PCRs with HF Phusion enzyme (protocol) on miniprep (12/07/17) in order to amplify pSB4K5, and on 2 colonies (iGEM Grenoble 2011) to amplify fha1. We did the PCR both with and without DMSO.

To separate the PCR products, we prepared a 1.8% TAE agarose gel.
Migration conditions = 100V during 30 min.
In order to reveal the DNA fragments, we used EtBr.

No result. (data not shown)

Using biobricks from the 2012 iGEM kit and our Gibson Assemblies product we transformed (new protocol) BW25113 WT competent cells (protocol). We obtained eight transformed strains with:
  • lux pR (BBa_R0062)
  • LuxI (BBa_C0061)
  • LuxR (BBa_I0462)
  • eCFP (BBa_E0022)
  • eCFP (BBa_E0422)
  • pLAC_RBS (BBa_I13601)
  • GA: pLAC_rsmY (pSB1A3)
  • GA: pLAC_RBS_RsmA (pSB3C5)

Thursday, July 19th:

Six transformations (12/07/18) out of eight showed results: RsmA, lux pR, LuxI, LuxR, E0422, E0022.

We did a miniprep (protocol kit: Nucleospin plasmid Quick Pure) on these transformed strains (12/07/18): RsmA, lux pR, LuxI, LuxR, E0422, E0022.

We also did some digestions (protocol) in order to check if the Gibson Assembly product (pLAC_RBS_RsmA) is the right one. The digestions were realised with two restriction enzymes : XbaI and SpeI during 10 minutes.

To separate the digestion products, we prepared a 1.8% TAE agarose gel.
Migration conditions = 100V during 30 min.
In order to reveal the DNA fragments, we used EtBr.

photo_gel_13
Migration result for a 1.8% TAE agarose gel
(the DNA ladder scale is in kb)
Lane 1: DNA ladder 100bp (biolabs)
Lane 2: GA (pLAC_RBS_RsmA) digestion product (XbaI)
Lane 3: GA (pLAC_RBS_RsmA) digestion product (SpeI)
Lane 4: GA (pLAC_RBS_RsmA) digestion product (XbaI & SpeI)
Lane 5: DNA ladder 100bp (biolabs)

We concluded that the Gibson Assembly (pLAC_RBS_RsmA on pSB1A3) worked. The digestion result is the expected one.

We did PCRs with HF Phusion enzyme (protocol) from minipreps in order to amplify pAra/Bad_RBS_GFP (12/07/17), eCFP (E0022 et E0422) (12/07/19). And a colony PCR to amplify RBS_Cya. We did the PCR both with and without DMSO.

To separate the PCR products, we prepared a 1.3% TAE agarose gel.
Migration conditions = 100V during 30 min.
In order to reveal the DNA fragments, we used EtBr.

photo_gel_14
Migration result for a 1.3% TAE agarose gel
(the DNA ladder scale is in kb)
Lane 1: DNA ladder 1kb (biolabs)
Lane 2: eCFP (E0022) PCR product
Lane 3: eCFP (E0022) PCR product (DMSO)
Lane 4: RBS_Cya PCR product
Lane 5: RBS_Cya PCR product (DMSO)
Lane 6: pAra/Bad_GFP PCR product 1
Lane 7: pAra/Bad_GFP PCR product 1 (DMSO)
Lane 8: pAra/Bad_GFP PCR product 2
Lane 9: pAra/Bad_GFP PCR product 2 (DMSO)
Lane 10: eCFP (E0422) PCR product
Lane 11: eCFP (E0422) PCR product (DMSO)
Lane 12: DNA ladder 1kb (biolabs)

We saw primer dimer bands and the bands corresponding to the amplified plasmids which brought eCFP (E0022 & E0422). There was a PCR condition problem.

One transformation (12/07/18) showed result during the day: rsmY. We decided to relaunch it in fresh LB medium + antibiotics.

Friday, July 20th:

We did a miniprep (protocol kit: Nucleospin plasmid Quick Pure) on the transformed strains (12/07/18) with Gibson Assembly product (RsmA and rsmY).

In order to check the products of Gibson Assembly (RsmA and rsmY) we launched digestion experiments on these miniprep products, using restriction enzymes (EcoRI, BamHI, XbaI and SpeI) during 10 minutes. We also launched digestion experiments on the eCFP miniprep products (12/07/19), using the same restriction enzymes, in order to recover the eCFP coding sequence.

To separate these digestion products, we prepared a 1.8% TAE agarose gel.
Migration conditions = 100V during 30 min.
In order to reveal the DNA fragments, we used EtBr.

photo_gel_15
Migration result for a 1.8% TAE agarose gel
(the DNA ladder scale is in kb)
Lane 1: DNA ladder 1kb (biolabs)
Lane 2: E0422 digestion product
Lane 3: E0022 digestion product
Lane 4: GA (RsmA) miniprep product
Lane 5: GA (rsmY) miniprep product
Lane 6: DNA ladder 1kb (biolabs)
Lane 7: DNA ladder 100bp (biolabs)
Lane 8: GA (RsmA) digestion product
Lane 9: GA (rsmY) digestion product
Lane 10: DNA ladder 100bp (biolabs)

We realised a DNA extraction (protocol kit: Nucleospin extract II) on the bands corresponding to the eCFP coding sequence (E0422 and E0022) from digestion products 120720AM_DIG_018 // 120720AM_DIG_19.
On these extractions, we did PCRs with HF Phusion enzyme (protocol) in order to amplify eCFP. We did the PCR both with and without DMSO.

To separate the PCR products and the digestion products, we prepared a 1.3% TAE agarose gel.
Migration conditions = 100V during 30 min.
In order to reveal the DNA fragments, we used EtBr.

photo_gel_15
Migration result for a 1.3% TAE agarose gel
(the DNA ladder scale is in kb)
Lane 1: DNA ladder 1kb (biolabs)
Lane 2: E0022 digestion product
Lane 3: E0422 digestion product
Lane 4: E0022 PCR product
Lane 5: E0022 PCR product (DMSO)
Lane 6: E0422 PCR product
Lane 7: E0422 PCR product (DMSO)
Lane 8: DNA ladder 1kb (biolabs)

We saw primer dimer bands. There was a PCR condition problem.

We did PCRs with HF Phusion enzyme (protocol) in order to amplify pSB4K5 and fha from miniprep. We did the same PCR twice: one with DMSO and one without.

Conclusion of the week:

We have achieved our first Gibson Assembly : pLAC_RBS_RsmA on pSB3C5 and we began the experiments in order to recover luxpR, LuxI and LuxR (for the 1st network).

Retrieved from "http://2012.igem.org/Team:Grenoble/Biology/Notebook/July/week_28"