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

From 2012.igem.org

(Difference between revisions)
Line 10: Line 10:
<a href="http://2012.igem.org/Team:Grenoble/Biology/Notebook/July/week_29">Week 29</a> •  
<a href="http://2012.igem.org/Team:Grenoble/Biology/Notebook/July/week_29">Week 29</a> •  
<a href="http://2012.igem.org/Team:Grenoble/Biology/Notebook/July/week_30">Week 30</a>
<a href="http://2012.igem.org/Team:Grenoble/Biology/Notebook/July/week_30">Week 30</a>
-
</section>
+
</section><br/>
-
<br/>
+
<section>
<section>
-
<h1> Week 29: July 16<span class="exposant">th</span> to 22<span class="exposant">nd</span> </h1>
+
<h1> Week 28: July 09<span class="exposant">th</span> to 15<span class="exposant">th</span> </h1>
<h2> Goal of the week: </h2>
<h2> Goal of the week: </h2>
-
We wanted to test the Gibson Assembly and the transformation protocols, recover and amplify some biobricks involved in our genetic networks:
+
We wanted to recover and amplify the 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>fha1 (80bp)</li>
+
<li>pLAC (100bp)</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 16<span class="exposant">th</span>:</h2>
+
<h2> Monday, July 09<span class="exposant">th</span>:</h2>
Precultured cells are prepared:
Precultured cells are prepared:
<ul>
<ul>
-
<li>Strains = BW25113 WT and BW25113 cya- pAra/Bad </li>
+
<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>Conditions = Conditions = LB liquid medium, 37°C, 200rpm, overnight </li>
+
<li>Conditions = LB liquid medium, 37°C, 200rpm, overnight </li>
</ul>
</ul>
</section>
</section>
<section>
<section>
-
<h2>Tuesday, July 17<span class="exposant">th</span>:</h2>
+
<h2>Tuesday, July 10<span class="exposant">th</span>:</h2>
-
Using iGEM 2012 biobricks and the Gibson Assembly product we transformed (protocol) BW25113 WT cells. We obtained four transformed strains with:
+
For the PCRs, we changed the enzyme from GoTaq to High Fidelity (HF) Phusion.<br/>
 +
We did some colony PCR with a HF Phusion enzyme (protocol) to amplify:
<ul>
<ul>
-
<li>BBa_I13601: pLAC_RBS</li>
+
<li>fha1, RsmA, rsmY and pSB1A3 from iGEM Grenoble 2011 glycerol stock.</li>
-
<li>BBa_E0422: eCFP</li>
+
<li>pAra/Bad_RBS_GFP from BW25113 cya<span class="exposant">-</span> pAra/Bad precultured cells.</li>
-
<li>GA: pLAC_rsmY (pSB1A3) </li>
+
<li>RBS_Cya from BW25113 WT precultured cells.</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/>
-
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:
+
To separate the PCR products, we prepared two gels:
<ul>
<ul>
-
<li>transformed strain with pSB4K5</li>
+
<li>a 1.3% TAE agarose gel, for the small fragments: RsmA, fha1 and rsmY (length < 1000bp)</li>
-
<li>BW25113 cya- pAra/Bad</li>
+
<li>a 0.8% TAE agarose gel, for the big fragments: pSB1A3, RBS_Cya, and pAra/Bad_RBS_GFP (length > 1000bp)</li>
</ul>
</ul>
-
<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/>
+
Migration conditions = 50V during 1h15.<br/>
 +
We used EtBr to reveal the DNA fragments.<br/>
<br/>
<br/>
-
To separate the PCR products and the miniprep products, we prepared a 1.8% TAE agarose gel.<br/>
+
<center><img src="http://2012.igem.org/wiki/images/b/b6/120710.jpg" alt="photo_gel_4"/></center>
-
Migration conditions = 100V during 30 min.<br/>
+
<div class="legend"><u>Migration result for the 1.3% TAE agarose gel (small fragments)</u><br/>
-
In order to reveal the DNA fragments, we used EtBr.<br/>
+
      <i>(the DNA ladder scale is in kb)</i><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="http://2012.igem.org/wiki/images/3/33/120717.jpg" alt="photo_gel_11"/></center>
+
<center><img src="http://2012.igem.org/wiki/images/4/4e/120710_%282%29.jpg" alt="photo_gel_5"/></center>
-
<div class="legend"><u>Migration result for a 1.8% TAE agarose gel</u><br/>
+
<div class="legend"><u>Migration result for the 0.8% TAE agarose gel (big fragments)</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: the deposits have failed<br/>
+
Lanes 2 and 3: RBS_Cya PCR product<br/>
-
Lane 4: pSB4K5 colony PCR product (DMSO)<br/>
+
Lanes 4 and 5: pSB1A3 PCR product<br/>
-
Lane 5: pSB4K5 colony PCR product<br/>
+
Lanes 6 and 7: pAra/Bad_RBS_GFP PCR product<br/>
-
Lane 6: pSB4K5 PCR (miniprep) product (DMSO)<br/>
+
Lanes 8 and 9: RBS_Cya PCR product (DMSO)<br/>
-
Lane 7: pSB4K5 PCR (miniprep) product<br/>
+
Lanes 10 and 11: pSBA13 PCR product (DMSO)<br/>
-
Lane 8: DNA ladder 1kb (biolabs)<br/>
+
Lane 12: pAra/Bad_GFP PCR product (DMSO)<br/>
-
Lane 9: DNA ladder 100bp (biolabs)<br/>
+
Lane 13: DNA ladder 1kb (biolabs)<br/></div>
-
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/>
-
We achieved to amplify pLAC (fha1), pLAC (rsmY) and pLAC_RBS. We didn’t achieve to amplify pSB4K5 and fha1.<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/>
 +
<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 18<span class="exposant">th</span>:</h2>
+
<h2> Wednesday, July 11<span class="exposant">th</span>:</h2>
-
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/>
+
We did a miniprep (protocol kit: Nucleospin plasmid Quick Pure) on three iGEM Grenoble 2011 strains:
 +
<ul>
 +
<li>fha1</li>
 +
<li>RsmA</li>
 +
<li>rsmY</li>
 +
</ul>
<br/>
<br/>
-
To separate the PCR products, we prepared a 1.8% TAE agarose gel.<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/>
-
Migration conditions = 100V during 30 min.<br/>
+
<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="http://2012.igem.org/wiki/images/8/83/120718.jpg" alt="photo_gel_12"/></center>
+
<center><img src="http://2012.igem.org/wiki/images/3/3f/120711.jpg" alt="photo_gel_6"/></center>
-
<div class="legend"><u>Migration result for a 1.8% TAE agarose gel</u> <br/>
+
<center><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 100bp (biolabs)<br/>
+
Lane 1: DNA ladder 100pb (biolabs)<br/>
-
Lane 2: pAra/Bad_RBS_GFP PCR product (1μL/DMSO)<br/>
+
Lanes 2 and 3: fha1 digestion product (XbaI)<br/>
-
Lane 3: pAra/Bad_RBS_GFP PCR product (2μL/DMSO)<br/>
+
Lanes 4 and 5: RsmA digestion product (XbaI)<br/>
-
Lane 4: pAra/Bad_RBS_GFP PCR product (1μL)<br/>
+
Lanes 6 and 7: rsmY digestion product (XbaI)<br/>
-
Lane 5: pAra/Bad_RBS_GFP PCR product (2μL)<br/>
+
Lanes 8 and 9: fha digestion product (pSTI)<br/>
-
Lane 6: RBS_Cya PCR product (DMSO)<br/>
+
Lanes 10 and 11: RsmA digestion product (pSTI)<br/>
-
Lane 7: RBS_Cya PCR product<br/>
+
Lanes 12 and 13: rsmY digestion product (pSTI)<br/>
-
Lane 8: fha1 PCR product (DMSO)<br/>
+
Lanes 14 and 15: fha1 digestion product (XbaI-pSTI)<br/>
-
Lane 9: fha1 PCR product<br/>
+
Lanes 16 and 17: RsmA digestion product (XbaI-pSTI)<br/>
-
Lane 10: DNA ladder 100bp (biolabs)<br/></div>
+
Lanes 18 and 19: rsmY digestion product (XbaI-pSTI)<br/>
 +
Lane 20: DNA ladder 100pb (biolabs)<br/></div></center>
<br/>
<br/>
-
We didn’t see anything. The PCR didn’t work.<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/>
<br/>
<br/>
-
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/>
+
Using iGEM 2012 biobricks we transformed (protocol) BW25113 WT cells. We obtained five transformed strains with five different biobricks:
 +
<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/>
-
To separate the PCR products, we prepared a 1.8% TAE agarose gel.<br/>
+
Precultured cells were prepared:
-
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>lux pR (BBa_R0062)</li>
+
<li>Strains = BW25113 cya<span class="exposant">-</span> pAra/Bad.</li>
-
<li>LuxI (BBa_C0061)</li>
+
<li>Conditions = LB liquid medium, 37°C, 200rpm, overnight.</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 19<span class="exposant">th</span>:</h2>
+
<h2> Thursday, July 12<span class="exposant">th</span>:</h2>
-
Six transformations (12/07/18) out of eight showed results: RsmA, lux pR, LuxI, LuxR, E0422, E0022.<br/>
+
The transformation using pSB4K5 is the only one out of five transformations that showed results.<br/>
<br/>
<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/>
+
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/>
<br/>
<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/>
+
To separate the PCR products, we prepared a 1.3% TAE agarose gel.<br/>
-
<br/>
+
Migration conditions = 50V during 1h.<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="http://2012.igem.org/wiki/images/5/51/120719.jpg" alt="photo_gel_13"/></center>
+
<center><img src="http://2012.igem.org/wiki/images/f/f6/120712.jpg" alt="photo_gel_7"/></center>
-
<div class="legend"><u>Migration result for a 1.8% TAE agarose gel</u><br/>
+
<div class="legend"><u>Migration result for a 1.3% 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 100bp (biolabs)<br/>
Lane 1: DNA ladder 100bp (biolabs)<br/>
-
Lane 2: GA (pLAC_RBS_RsmA) digestion product (XbaI)<br/>
+
Lane 2: fha1 PCR product<br/>
-
Lane 3: GA (pLAC_RBS_RsmA) digestion product (SpeI)<br/>
+
Lane 3: rsmY PCR product<br/>
-
Lane 4: GA (pLAC_RBS_RsmA) digestion product (XbaI & SpeI)<br/>
+
Lane 4: fha1 PCR product (DMSO)<br/>
-
Lane 5: DNA ladder 100bp (biolabs)<br/></div>
+
Lane 5: rsmY PCR product (DMSO)<br/>
 +
Lane 6: DNA ladder 100bp (biolabs)<br/>
 +
Lane 7 : empty<br/></div>
<br/>
<br/>
-
We concluded that the Gibson Assembly (pLAC_RBS_RsmA on pSB1A3) worked. The digestion result is the expected one.<br/>
+
We saw no DNA bands at the right position, we thought there was still a PCR condition problem.<br/>
<br/>
<br/>
-
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/>
+
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>
 +
<li>transformed strain with pSB4K5</li>
 +
<li>BW25113 cya<span class="exposant">-</span> pAra/Bad</li>
 +
</ul>
<br/>
<br/>
-
To separate the PCR products, we prepared a 1.3% TAE agarose gel.<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/>
 +
<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="http://2012.igem.org/wiki/images/7/77/120719_%282%29.jpg" alt="photo_gel_14"/></center>
+
<center><img src="http://2012.igem.org/wiki/images/7/76/120712_%282%29.jpg" alt="photo_gel_8"/></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 2: DNA ladder 1kb (biolabs)<br/>
-
Lane 2: eCFP (E0022) PCR product</br>
+
Lane 3: pSB4K5 miniprep product<br/>
-
Lane 3: eCFP (E0022) PCR product (DMSO)</br>
+
Lane 4: plasmid with pAra/Bad miniprep product<br/>
-
Lane 4: RBS_Cya PCR product</br>
+
Lane 5: empty<br/>
-
Lane 5: RBS_Cya PCR product (DMSO)</br>
+
Lane 7: eCFP PCR product<br/>
-
Lane 6: pAra/Bad_GFP PCR product 1</br>
+
Lane 8: pLAC (fha1) PCR product<br/>
-
Lane 7: pAra/Bad_GFP PCR product 1 (DMSO)</br>
+
Lane 9: pLAC_RBS PCR product<br/>
-
Lane 8: pAra/Bad_GFP PCR product 2</br>
+
Lane 10: pLAC (rsmY) PCR product<br/>
-
Lane 9: pAra/Bad_GFP PCR product 2 (DMSO)</br>
+
Lane 11: RsmA PCR product<br/>
-
Lane 10: eCFP (E0422) PCR product </br>
+
Lane 12: DNA ladder 100bp (biolabs)<br/>
-
Lane 11: eCFP (E0422) PCR product (DMSO)</br>
+
Lane 13 : empty<br/></div>
-
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/>
-
One transformation (12/07/18) showed result during the day: rsmY. We decided to relaunch it in fresh LB medium + antibiotics.<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/>
-
</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/>
-
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/>
+
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/>
<br/>
<br/>
-
To separate these digestion products, we prepared a 1.8% TAE agarose gel.<br/>
+
Precultured cells were prepared:
 +
<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="http://2012.igem.org/wiki/images/0/00/120720.jpg" alt="photo_gel_15"/></center>
+
<center><img src="http://2012.igem.org/wiki/images/6/60/120713.jpg" alt="photo_gel_9"/></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 1kb (biolabs)<br/>
+
Lane 1: DNA ladder 100bp (biolabs)<br/>
-
Lane 2: E0422 digestion product<br/>
+
Lane 2: eCFP PCR product<br/>
-
Lane 3: E0022 digestion product<br/>
+
Lane 3: pLAC (fha1) PCR product<br/>
-
Lane 4: GA (RsmA) miniprep product<br/>
+
Lane 4: pLAC_RBS PCR product<br/>
-
Lane 5: GA (rsmY) miniprep product<br/>
+
Lane 5: pLAC (rsmY) PCR product<br/>
-
Lane 6: DNA ladder 1kb (biolabs)<br/>
+
Lane 6: RsmA PCR product<br/>
-
Lane 7: DNA ladder 100bp (biolabs)<br/>
+
Lane 7: empty<br/>
-
Lane 8: GA (RsmA) digestion product<br/>
+
Lane 8: rsmY PCR product<br/>
-
Lane 9: GA (rsmY) digestion product<br/>
+
Lane 9: fha1 PCR product<br/>
-
Lane 10: DNA ladder 100bp (biolabs)<br/></div>
+
Lane 10: empty<br/>
 +
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) 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/>
+
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):
-
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/>
+
<ul>
 +
<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/>
-
To separate the PCR products and the digestion products, we prepared a 1.3% TAE agarose gel.<br/>
+
We did a miniprep (protocol kit: Nucleospin plasmid Quick Pure) on 2 strains (12/07/12) to recover pSB1A3 and pSB3C5.<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="http://2012.igem.org/wiki/images/4/4c/120720_%282%29.jpg" alt="photo_gel_15"/></center>
+
<center><img src="http://2012.igem.org/wiki/images/4/4e/120713_%282%29.jpg" alt="photo_gel_10"/></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: E0022 digestion product<br/>
+
Lane 2: pSB1A3 PCR product<br/>
-
Lane 3: E0422 digestion product<br/>
+
Lane 3: pSB3C5 (cya) PCR product<br/>
-
Lane 4: E0022 PCR product<br/>
+
Lane 4: pSB3C5 (RsmA) PCR product<br/>
-
Lane 5: E0022 PCR product (DMSO)<br/>
+
Lane 5: pSB4K5 PCR product<br/>
-
Lane 6: E0422 PCR product<br/>
+
Lane 6: pSB1A3 PCR product (DMSO)<br/>
-
Lane 7: E0422 PCR product (DMSO)<br/>
+
Lane 7: pB3C5 (cya) PCR product (DMSO)<br/>
-
Lane 8: DNA ladder 1kb (biolabs)<br/></div>
+
Lane 8: pSB3C5 (RsmA) PCR product (DMSO)<br/>
 +
Lane 9: pSB4K5 PCR product (DMSO)<br/>
 +
Lane 10: DNA ladder 1kb (biolabs)<br/></div>
<br/>
<br/>
-
We saw primer dimer bands. There was a PCR condition problem.<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):
 +
<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 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/>
+
We realised two Gibson Assembly (protocol) to build two plasmids:
 +
<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 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/>
+
We have achieved to amplify :
 +
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 09:40, 24 September 2012

iGEM Grenoble 2012

Project

July

Week 27Week 28Week 29Week 30

Week 28: July 09th to 15th

Goal of the week:

We wanted to recover and amplify the biobricks involved in our genetic networks:
  • pAra/Bad_RBS_GFP (1300bp)
  • RBS_Cya (2600bp)
  • pLAC (100bp)
  • fha (80bp)
  • eCFP (800bp)
  • pLAC_RBS (120bp)
  • RsmA (200bp)
  • rsmY (170bp)
  • pSB1A3 (2400bp)
  • pSB4K5 (2400bp)
  • pSB3C5 (2400bp)

We also planned to realise the gibson assemblies for the first constructions.

Monday, July 09th:

Precultured cells are prepared:
  • Strains = BW25113 WT, BW25113 cya-, BW25113 cya-pAra/Bad and the strain transformed with pSB4K5
  • Conditions = LB liquid medium, 37°C, 200rpm, overnight

Tuesday, July 10th:

For the PCRs, we changed the enzyme from GoTaq to High Fidelity (HF) Phusion.
We did some colony PCR with a HF Phusion enzyme (protocol) to amplify:
  • fha1, RsmA, rsmY and pSB1A3 from iGEM Grenoble 2011 glycerol stock.
  • pAra/Bad_RBS_GFP from BW25113 cya- pAra/Bad precultured cells.
  • RBS_Cya from BW25113 WT precultured cells.
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).

To separate the PCR products, we prepared two gels:
  • a 1.3% TAE agarose gel, for the small fragments: RsmA, fha1 and rsmY (length < 1000bp)
  • a 0.8% TAE agarose gel, for the big fragments: pSB1A3, RBS_Cya, and pAra/Bad_RBS_GFP (length > 1000bp)
Migration conditions = 50V during 1h15.
We used EtBr to reveal the DNA fragments.

photo_gel_4
Migration result for the 1.3% TAE agarose gel (small fragments)
(the DNA ladder scale is in kb)
Lane 1: DNA ladder 100bp (biolabs)
Lanes 2 and 3: fha1 PCR product
Lanes 4 and 5: RsmA PCR product
Lanes 6 and 7: rsmY PCR product
Lanes 8 and 9: fha1 PCR product (DMSO)
Lanes 10 and 11: RsmA PCR product (DMSO)
Lane 12: rsmY PCR product (DMSO)
Lane 13: DNA ladder 100bp (biolabs)

photo_gel_5
Migration result for the 0.8% TAE agarose gel (big fragments)
(the DNA ladder scale is in kb)
Lane 1: DNA ladder 1kb (biolabs)
Lanes 2 and 3: RBS_Cya PCR product
Lanes 4 and 5: pSB1A3 PCR product
Lanes 6 and 7: pAra/Bad_RBS_GFP PCR product
Lanes 8 and 9: RBS_Cya PCR product (DMSO)
Lanes 10 and 11: pSBA13 PCR product (DMSO)
Lane 12: pAra/Bad_GFP PCR product (DMSO)
Lane 13: DNA ladder 1kb (biolabs)

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.

Precultured cells were prepared:
  • Strains = BW25113 WT.
  • Conditions = LB liquid medium, 37°C, 200rpm, overnight.

Wednesday, July 11th:

We did a miniprep (protocol kit: Nucleospin plasmid Quick Pure) on three iGEM Grenoble 2011 strains:
  • fha1
  • RsmA
  • rsmY

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.

To separate the digestion products, we prepared a 1.3% TAE agarose gel.
Migration conditions = 50V during 1h15.
In order to reveal the DNA fragments, we used EtBr.

photo_gel_6
Migration result for a 1.3% TAE agarose gel
(the DNA ladder scale is in kb)
Lane 1: DNA ladder 100pb (biolabs)
Lanes 2 and 3: fha1 digestion product (XbaI)
Lanes 4 and 5: RsmA digestion product (XbaI)
Lanes 6 and 7: rsmY digestion product (XbaI)
Lanes 8 and 9: fha digestion product (pSTI)
Lanes 10 and 11: RsmA digestion product (pSTI)
Lanes 12 and 13: rsmY digestion product (pSTI)
Lanes 14 and 15: fha1 digestion product (XbaI-pSTI)
Lanes 16 and 17: RsmA digestion product (XbaI-pSTI)
Lanes 18 and 19: rsmY digestion product (XbaI-pSTI)
Lane 20: DNA ladder 100pb (biolabs)

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).

Using iGEM 2012 biobricks we transformed (protocol) BW25113 WT cells. We obtained five transformed strains with five different biobricks:
  • BBa_I13601: pLAC_RBS
  • BBa_E0422: eCFP
  • pSB3C5 plasmid
  • psB4K5 plasmid
  • psB1A3 plasmid

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

Thursday, July 12th:

The transformation using pSB4K5 is the only one out of five transformations that showed results.

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.

To separate the PCR products, we prepared a 1.3% TAE agarose gel.
Migration conditions = 50V during 1h.
In order to reveal the DNA fragments, we used EtBr.

photo_gel_7
Migration result for a 1.3% TAE agarose gel
(the DNA ladder scale is in kb)
Lane 1: DNA ladder 100bp (biolabs)
Lane 2: fha1 PCR product
Lane 3: rsmY PCR product
Lane 4: fha1 PCR product (DMSO)
Lane 5: rsmY PCR product (DMSO)
Lane 6: DNA ladder 100bp (biolabs)
Lane 7 : empty

We saw no DNA bands at the right position, we thought there was still a PCR condition problem.

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 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.

To separate the PCR products and the miniprep 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_8
Migration result for a 1.3% TAE agarose gel
(the DNA ladder scale is in kb)
Lane 2: DNA ladder 1kb (biolabs)
Lane 3: pSB4K5 miniprep product
Lane 4: plasmid with pAra/Bad miniprep product
Lane 5: empty
Lane 7: eCFP PCR product
Lane 8: pLAC (fha1) PCR product
Lane 9: pLAC_RBS PCR product
Lane 10: pLAC (rsmY) PCR product
Lane 11: RsmA PCR product
Lane 12: DNA ladder 100bp (biolabs)
Lane 13 : empty

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.

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).

Precultured cells were prepared:
  • Strains = BW25113 WT, pSB1A3 (iGEM Grenoble 2011) and pSB3C5 (iGEM Grenoble 2011).
  • Conditions = LB liquid medium, 37°C, 200rpm, overnight.

Friday, July 13th:

To separate the PCR products (PCRs 07/12), 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_9
Migration result for a 1.8% TAE agarose gel
(the DNA ladder scale is in kb)
Lane 1: DNA ladder 100bp (biolabs)
Lane 2: eCFP PCR product
Lane 3: pLAC (fha1) PCR product
Lane 4: pLAC_RBS PCR product
Lane 5: pLAC (rsmY) PCR product
Lane 6: RsmA PCR product
Lane 7: empty
Lane 8: rsmY PCR product
Lane 9: fha1 PCR product
Lane 10: empty
Lane 11: rsmY PCR product
Lane 12: fha PCR product
Lane 13: DNA ladder 100bp (biolabs)

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):
  • pLAC (fha1) 120713PP_PCR_009
  • pLAC_RBS 120713PP_PCR_010
  • pLAC (rsmY) 120713PP_PCR_011
  • RsmA 120713PP_PCR_012
  • rsmY 120713PP_PCR_013

We did a miniprep (protocol kit: Nucleospin plasmid Quick Pure) on 2 strains (12/07/12) to recover pSB1A3 and pSB3C5.

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.

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_10
Migration result for a 1.3% TAE agarose gel
(the DNA ladder scale is in kb)
Lane 1: DNA ladder 1kb (biolabs)
Lane 2: pSB1A3 PCR product
Lane 3: pSB3C5 (cya) PCR product
Lane 4: pSB3C5 (RsmA) PCR product
Lane 5: pSB4K5 PCR product
Lane 6: pSB1A3 PCR product (DMSO)
Lane 7: pB3C5 (cya) PCR product (DMSO)
Lane 8: pSB3C5 (RsmA) PCR product (DMSO)
Lane 9: pSB4K5 PCR product (DMSO)
Lane 10: DNA ladder 1kb (biolabs)

We realised a DNA extraction (protocol kit: Nucleospin extract II) for all the fragments except the pSB4K5 PCR product (we didn’t see anything):
  • pSB1A3 120713PP_PCR_014
  • pSB3C5 (cya) 120713PP_PCR_015
  • pSB3C5 (RsmA) 120713PP_PCR_016

We realised two Gibson Assembly (protocol) to build two plasmids:
  • pSB1A3 120713PP_PCR_014 with pLAC (rsmY) 120713PP_PCR_011 and rsmY 120713PP_PCR_013
  • pSB3C5 (RsmA) 120713PP_PCR_016 with pLAC_RBS 120713PP_PCR_010 and RsmA 120713PP_PCR_012

Conclusion of the week:

We have achieved to amplify : pLAC (fha1) // pLAC (rsmY) // pLAC_RBS // RsmA // rsmY // pSB1A3 // pSB3C5 (Cya) // pSB3C5 (RsmA)

We tried our first Gibson Assemblies.