Team:Grenoble/Biology/Notebook/July/week 30

From 2012.igem.org

(Difference between revisions)
Line 5: Line 5:
<div id="cadre">
<div id="cadre">
<section>
<section>
-
<h1>July</h1>
+
<h1>August</h1>
-
<a href="https://2012.igem.org/Team:Grenoble/Biology/Notebook/July">Week 27</a> •  
+
<a href="https://2012.igem.org/Team:Grenoble/Biology/Notebook/August">Week 31</a> •
-
<a href="https://2012.igem.org/Team:Grenoble/Biology/Notebook/July/week_28">Week 28</a> •  
+
<a href="https://2012.igem.org/Team:Grenoble/Biology/Notebook/July/week_32">Week 32</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_33">Week 33</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_34">Week 34</a> •  
 +
<a href="https://2012.igem.org/Team:Grenoble/Biology/Notebook/July/week_35">Week 35</a>
</section>
</section>
<br/>
<br/>
<section>
<section>
-
<h1> Week 30: July 23<span class="exposant">rd</span> to 29<span class="exposant">th</span> </h1>
+
<h1> Week 31: July 30<span class="exposant">th</span> to August 05<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 some biobricks involved in our genetic networks:
<ul>
<ul>
-
<li>pAra/Bad_RBS_GFP (1300bp)</li>
+
<li>pSB4C5 (2400bp)</li>
-
<li>RBS_Cya (2600bp)</li>
+
<li>pompC (100bp)</li>
-
<li>fha (80bp)</li>
+
<li>mcherry (900bp)</li>
<li>eCFP (800bp)</li>
<li>eCFP (800bp)</li>
-
<li>pSB4K5 (2400bp)</li>
+
<li>GFP (100bp)</li>
</ul>
</ul>
-
<br/>
 
-
We also wanted to test the "AND" gate (CRP°cAMP - araC) without the amplifier module.<br/>
 
</section>
</section>
<section>
<section>
-
<h2> Monday, July 23<span class="exposant">rd</span>:</h2>
+
<h2> Tuesday, July 31<span class="exposant">th</span>:</h2>
-
We did an experiment (protocol) in order to test the "AND" gate (CRP°cAMP - araC) without the amplifier module. <br/>
+
We did a miniprep (protocol kit: Nucleospin plasmid Quick Pure) on the transformed strain with pSB4C5 (12/07/25), on which we wanted to recover pSB4C5.<br/>
<br/>
<br/>
-
<center><img src="https://static.igem.org/mediawiki/2012/2/2d/AND-gate.png" alt="results_AND_Gate"/></center>
+
We did some colony PCRs (on 12/07/25 colony) and PCRs on miniprep with HF Phusion enzyme (protocol) in order to amplify pSB4C5. <br/>Annealing temperature = 60°C.<br/>
-
<div class="legend">strain used = BW25113 cya<span class="exposant">-</span> transformed with pAra/Bad_RBS_GFP<br/></div>
+
-
<br/>The experiments worked well, the "AND" gate worked as expected.<br/>
+
<br/>
<br/>
-
We did PCRs with HF Phusion enzyme (protocol) on miniprep in order to amplify pAra/Bad_RBS_GFP, and on colony to amplify RBS_Cya. We did the PCR both with and without DMSO.<br/>
+
To separate the PCR products, we prepared a 1.8% TAE agarose gel.<br/>
-
<br/>
+
-
To separate the PCR products (12/07/23 and 12/07/20), 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/c/c1/120723.jpg" alt="photo_gel_16"/></center>
+
<center><img src="https://static.igem.org/mediawiki/2012/a/ac/120731.jpg" alt="photo_gel_22"/></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 80pb-10kb (fermentas)<br/>
+
Lane 1: DNA ladder 1kb (biolabs)<br/>
-
Lane 2: pSB4K5 PCR product (DMSO)<br/>
+
Lane 2: pSB4C5 (miniprep) PCR product (DMSO)<br/>
-
Lane 3: fha1 PCR product (DMSO)<br/>
+
Lane 3: pSB4C5 (miniprep) PCR product (DMSO)<br/>
-
Lane 4: fha1 PCR product<br/>
+
Lane 4: pSB4C5 (miniprep) PCR product<br/>
-
Lane 5: pSB4K5 PCR product<br/>
+
Lane 5: pSB4C5 (miniprep) PCR product<br/>
-
Lane 6: pAra/Bad_RBS_GFP PCR product (DMSO)<br/>
+
Lane 6: pSB4C5 (colony) PCR product (DMSO)<br/>
-
Lane 7: pAra/Bad_RBS_GFP PCR product (DMSO)<br/>
+
Lane 7: pSB4C5 (colony) PCR product (DMSO)<br/>
-
Lane 8: RBS_Cya PCR product (DMSO)<br/>
+
Lane 8: pSB4C5 (colony) PCR product <br/>
-
Lane 9: RBS_Cya PCR product<br/>
+
Lane 9: pSB4C5 (colony) PCR product <br/>
-
Lane 10: pAra/Bad_RBS_GFP PCR product<br/>
+
Lane 10: DNA ladder 1kb (biolabs)<br/></div>
-
Lane 11: pAra/Bad_RBS_GFP PCR product<br/>
+
-
Lane 12: DNA ladder 80bp-10kb (fermentas)<br/></div>
+
<br/>
<br/>
-
We saw primer dimer bands and the bands corresponding to the amplified fha1 sequence (lanes 3 & 4). For the other, there was a PCR condition problem.<br/>
+
We only saw primer dimer bands, there was a PCR condition problem.<br/>
<br/>
<br/>
-
We realised a DNA extraction (protocol kit: Nucleospin extract II) from the two fha PCR products <span class="code">120723AM_PCR_020</span>.<br/>
+
We did some PCRs on miniprep with HF Phusion enzyme (protocol) in order to amplify pSB4C5. <br/>Annealing temperature = 65°C.<br/>
<br/>
<br/>
-
We did touchdown PCRs with HF Phusion enzyme (protocol) on digestion product (eCFP E0022 and E0422) (12/07/20) in order to amplify eCFP. We did the PCR both with and without DMSO.<br/>
+
We did some digestions (protocol) on miniprep (pSB4C5) in order to do the construction: pAra/Bad_RBS_GFP_RBS_Cya. The digestions were achieved with two restriction enzymes : XbaI and SpeI during 10 minutes.<br/>
<br/>
<br/>
-
Using iGEM 2012 biobricks we transformed (new protocol) BW25113 WT competent cells (protocol) with pSB4K5.<br/>
+
To separate the PCR products and the digestion products, we prepared a 1.8% TAE agarose gel.<br/>
-
</section>
+
-
<section>
+
-
<h2> Tuesday, July 24<span class="exposant">th</span>:</h2>
+
-
To separate the PCR products (12/07/23), 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/e/e0/120724_%282%29.jpg" alt="photo_gel_17"/></center>
+
<center><img src="https://static.igem.org/mediawiki/2012/9/9e/120731_%282%29.jpg" alt="photo_gel_23"/></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 80bp-10kb (fermentas)<br/>
+
Lane 1: DNA ladder 1kb (biolabs)<br/>
-
Lane 2: E0022 PCR product<br/>
+
Lane 2: pSB4C5 digestion product <br/>
-
Lane 3: E0022 PCR product (DMSO)<br/>
+
Lane 3: pSB4C5 digestion product <br/>
-
Lane 4: E0422 PCR product<br/>
+
Lane 4: pSB4C5 (miniprep) PCR product (DMSO)<br/>
-
Lane 5: E0422 PCR product (DMSO)<br/>
+
Lane 5: pSB4C5 (miniprep) PCR product (DMSO)<br/>
-
Lane 6: DNA ladder 80bp-10kb (fermentas)<br/></div>
+
Lane 6: pSB4C5 (miniprep) PCR product<br/>
 +
Lane 7: pSB4C5 (miniprep) PCR product<br/>
 +
Lane 8: pSB4C5 (colony) PCR product (DMSO)<br/>
 +
Lane 9: pSB4C5 (colony) PCR product (DMSO)<br/>
 +
Lane 10: pSB4C5 (colony) PCR product <br/>
 +
Lane 11: pSB4C5 (colony) PCR product <br/>
 +
Lane 12: DNA ladder 1kb (biolabs)<br/></div>
<br/>
<br/>
-
We only saw primer dimer bands, there was a PCR condition problem.<br/>
+
For the PCR results, we only saw primer dimer bands, there was a PCR condition problem.<br/>
-
<br/>
+
The digestion worked well, we realised a DNA extraction (protocol kit: Nucleospin extract II) from the two digestion products <span class="code">120731AM_DIG_027</span>.<br/>
-
The transformation with pSB4K5 (12/07/23) showed result (growth on plates).<br/>
+
</section>
-
<br/>
+
<section>
-
We did some digestions (protocol) on miniprep (12/07/19) to recover eCFP (E0022). The digestions were achieved with two restriction enzymes : XbaI and SpeI during 10 minutes.<br/>
+
<h2> Wednesday, August 01<span class="exposant">st</span>:</h2>
 +
We did a touchdown PCR (from 70°C to 60°C) from miniprep (12/07/31) with HF Phusion enzyme (protocol) in order to amplify pSB4C5.<br/>
<br/>
<br/>
-
To separate the digestion products, we prepared a 1.3% TAE agarose gel.<br/>
+
To separate the PCR products, 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/2012/6/6b/120724.jpg" alt="photo_gel_18"/></center>
+
<center><img src="https://static.igem.org/mediawiki/2012/6/6f/120801.jpg" alt="photo_gel_24"/></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 1kb (biolabs)<br/>
Lane 1: DNA ladder 1kb (biolabs)<br/>
-
Lane 2: E0022 digestion product<br/>
+
Lane 2: pSBAC5 (digestion) PCR product <br/>
-
Lane 3: pSB4K5 digestion product<br/>
+
Lane 3: pSB4C5 (digestion) PCR product (DMSO)<br/>
-
Lane 4: DNA ladder 80bp-1kb (fermentas)<br/></div>
+
Lane 4: pSB4C5 (miniprep) PCR product<br/>
 +
Lane 5: pSB4C5 (miniprep) PCR product (DMSO)<br/>
 +
Lane 6: DNA ladder 1kb (biolabs)<br/></div>
<br/>
<br/>
-
The digestion worked, we saw DNA bands at the expected positions.<br/>
+
We only saw primer dimer bands, there was a PCR condition problem.<br/>
<br/>
<br/>
-
We did  PCR with HF Phusion enzyme (protocol) from one of these digestion product (eCFP E0022) in order to amplify eCFP and a colony PCR to amplify pSB4K5.<br/>
+
We realised eight Gibson Assemblies (protocol) to build four plasmids:<br/>
 +
<ul>
 +
<li>pSB4C5 (fha1) <span class="code">120726AM_PCR_021</span> with pLAC (fha1) <span class="code">120713PP_PCR_009</span>, fha1 <span class="code">120723AM_PCR_020</span> and eCFP <span class="code">120720_DIG_019</span></li>
 +
<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 (Cya) <span class="code">120713PP_PCR_015</span> with pAra/Bad_RBS_GFP <span class="code">120727AM_PCR_023</span> and RBS_Cya <span class="code">120727AM_PCR_022</span></li>
 +
<li>pSB4C5 <span class="code">120731AM_DIG_027</span> with pAra/Bad_RBS_GFP <span class="code">120727AM_PCR_023</span> and RBS_Cya <span class="code">120727AM_PCR_022</span></li>
 +
</ul>
<br/>
<br/>
-
To separate the PCR products, we prepared a 1.3% TAE agarose gel.<br/>
+
For each plasmid construct, we did two Gibson Assemblies (protocol).  
-
Migration conditions = 100V during 30 min.<br/>
+
-
In order to reveal the DNA fragments, we used EtBr.<br/>
+
-
<br/>
+
-
There was no result (data not shown).<br/>
+
-
<br/>
+
-
We didn’t achieve to amplify pSB4K5. We decided to change it, we chose pSB4C5 instead. So we had to change pSB3C5, we chose pSB3T5. <br/>
+
-
<br/>
+
-
With iGEM 2012 biobricks we transformed (new protocol) BW25113 WT competent cells (protocol) with pSB4C5.<br/>
+
-
<br/>
+
-
We did an overlapping PCR with HF Phusion enzyme (protocol) on the digestion product (eCFP E0022) in order to amplify eCFP and on miniprep to amplify fha1.<br/>
+
-
</section>
+
-
<section>
+
-
<h2> Wednesday, July 25<span class="exposant">th</span>:</h2>
+
-
To separate the PCR products (12/07/24), 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/>
<br/>
-
There was a migration problem (data not shown).<br/>
+
We transformed (new protocol) BW25113 WT competent cells (protocol) with the GA products:<br/> pLAC_fha1_eCFP and pLAC_rsmY. <br/>
 +
We transformed (new protocol) BW25113 Cya- competent cells (protocol) with the GA products:<br/> pAra/Bad_RBS_GFP_RBS_Cya.<br/>
<br/>
<br/>
-
The transformation with pSB4C5 (12/07/24) was successful (growth on plates). We decided to relaunch it in fresh liquid LB.<br/>
+
We did some minipreps (protocol kit: Nucleospin plasmid Quick Pure) on the transformed strains (12/07/25) with pOmpC, mcherry, eCFP and pSB4C5 ; in order to set up the test on the receptor with a reporter.<br/>
<br/>
<br/>
-
We did some glycerol stocks :
+
Then, we did some digestions (protocol) on these minipreps. The digestions were achieved with two restriction enzymes:  
<ul>
<ul>
-
<li>GA: pLAC_RBS_RsmA <span class="code">120725GH_001</span></li>
+
<li> pOmpC with EcoRI and SpeI during 10 minutes </li>
-
<li>pSB4K5 <span class="code">120725GH_002</span></li>
+
<li> mcherry and eCFP with XbaI and SpeI during 10 minutes</li>
-
<li>E0422 (eCFP) <span class="code">120725GH_003</span></li>
+
<li> pSB4C5 with EcoRI and PstI during 10 minutes</li>
-
<li>LuxI <span class="code">120725GH_004</span></li>
+
-
<li>E0022 (eCFP) <span class="code">120725GH_005</span></li>
+
-
<li>LuxR <span class="code">120725GH_006</span></li>
+
-
<li>pLux <span class="code">120725GH_007</span></li>
+
-
<li>I13601 <span class="code">120725GH_008</span></li>
+
</ul>
</ul>
<br/>
<br/>
-
We did a colony PCR with GoTaq enzyme (protocol) in order to amplify pSB4C5.<br/>
+
To separate the digestion products, we prepared a 1.8% TAE agarose gel.<br/>
-
<br/>
+
-
To separate the PCR products (07/24), 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/2012/3/3a/120725_%28PCR_colonie_pSB4C5%29.jpg" alt="photo_gel_19"/></center>
+
<center><img src="https://static.igem.org/mediawiki/2012/0/05/120801_%282%29.jpg" alt="photo_gel_25"/></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 80pb-10kb (fermentas)<br/>
-
Lane 2: pSB4C5 PCR product<br/>
+
Lane 2: pOmpC digestion product<br/>
-
Lane 3: DNA ladder 1kb (biolabs)<br/></div>
+
Lane 3: pOmpC digestion product<br/>
 +
Lane 4: DNA ladder 80pb-10kb (fermentas)<br/>
 +
Lane 5: eCFP digestion product<br/>
 +
Lane 6: eCFP digestion product<br/>
 +
Lane 7: mcherry digestion product<br/>
 +
Lane 8: mcherry digestion product<br/>
 +
Lane 9: pSB4C5 digestion product<br/>
 +
Lane 10: pSB4C5 digestion product<br/>
 +
Lane 11: DNA ladder 1kb (biolabs)<br/></div>
<br/>
<br/>
-
We concluded that the pSB4C5 amplification worked well, so we decided to do the PCR again in order to do a DNA extraction.<br/>
+
The digestion worked well, we realised a DNA extraction (protocol kit: Nucleospin extract II) from the eCFP, mcherry and pSB4C5 digestion products.<br/>
</section>
</section>
<section>
<section>
-
<h2> Thursday, July 26<span class="exposant">th</span>:</h2>
+
<h2> Thursday, August 02<span class="exposant">nd</span>:</h2>
-
We did glycerol stock of the strain transformed with pSB4C5 <span class="code">120726AM_009</span>.<br/>
+
We did a miniprep (protocol kit: Nucleospin plasmid Quick Pure) on the transformed strain (12/07/25) with pOmpC.<br/>
<br/>
<br/>
-
We did a colony PCR withs HF Phusion Enzyme (protocol) in order to amplify pSB4C5.<br/>
+
Then, we did a digestion (protocol) on this miniprep. The digestions were achieved with two restriction enzymes: EcoRI and SpeI during 10 minutes.<br/>
<br/>
<br/>
-
We did a miniprep (protocol kit: Nucleospin plasmid Quick Pure) on the transformed strain with pSB4K5, on which we wanted to recover pSB4K5.<br/>
+
To separate the digestion products, we prepared a 1.8% TAE agarose gel.<br/>
-
<br/>
+
-
We did some digestions (protocol) on this miniprep, in order to check if pSB4K5 was ok. The digestion was realised with two restriction enzymes : XbaI and PstI during 10 minutes.<br/>
+
-
<br/>
+
-
To separate the PCR products (12/07/24), 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/2012/6/66/120726.jpg" alt="photo_gel_20"/></center>
+
<center><img src="https://static.igem.org/mediawiki/2012/1/1f/120802.jpg" alt="photo_gel_26"/></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 80pb-10kb (fermentas)<br/>
-
Lanes 2 and 3: pSB4K5 without digestion<br/>
+
Lane 2: pOmpC miniprep product<br/>
-
Lanes 4 and 5: pSB4K5 digestion product <br/>
+
Lane 3: pOmpC miniprep product<br/>
-
Lane 6: empty lane<br/>
+
Lane 4: pOmpC digestion product<br/>
-
Lanes 7 and 8: pSB4C5 PCR product<br/>
+
Lane 5: pOmpC digestion product<br/>
-
Lane 9: DNA ladder 1kb (biolabs)<br/></div>
+
Lane 6: DNA ladder 80pb-10kb (fermentas)<br/></div>
<br/>
<br/>
-
We concluded that the digestion worked well (we seen the expected DNA bands), and so was the PCR on pSB4C5.<br/>
+
There was a problem with the miniprep or the transformation, we didn't see any DNA bands.<br/>
<br/>
<br/>
-
We realised a DNA extraction (protocol kit: Nucleospin extract II) on the two pSB4C5 PCR products <span class="code">120726AM_PCR_021</span>.<br/>
+
With iGEM 2012 biobricks we transformed (new protocol) BW25113 WT competent cells (protocol) with pOmpC.<br/>
<br/>
<br/>
-
We didn’t achieve to amplify eCFP, we decided to change it. We chose mcherry and GFP.<br/>
+
We decided to relaunch the transformed strains with the GA products (12/08/01) in fresh liquid LB.<br/>
-
<br/>
+
-
We transformed (new protocol) BW25113 WT competent cells (protocol) with biobricks from iGEM 2012 kit :
+
-
<ul>
+
-
<li>BBa_I763011 (GFP) </li>
+
-
<li>BBa_J06702 (mcherry) (in duplicate : in LB with Ampicillin and in LB with Ampicillin and Kanamycin)</li>
+
-
<li>pSB1A3</li>
+
-
<li>BBa_R0082 (pompC)</li>
+
-
<li>BBa_J23119 (pConst)</li>
+
-
</ul>
+
</section>
</section>
<section>
<section>
-
<h2> Friday, July 27<span class="exposant">th</span>:</h2>
+
<h2> Friday, August 03<span class="exposant">rd</span>:</h2>
-
The five transformations (12/07/26) were successful (growth on the expected plates). We did glycerol stocks of:
+
We did some minipreps (protocol kit: Nucleospin plasmid Quick Pure) on the transformed strains with the GA products.<br/>
 +
<br/>
 +
We did some glycerol stocks:
<ul>
<ul>
-
<li>BBa_J06702 (mcherry in LB with Ampicillin and Kanamycin) <span class="code">120727GH_010</span></li>
+
<li>GA: pLAC_rsmY (pSB1A3) <b>1</b>                    <span class="code">120803GH_016</span></li>
-
<li>pSB1A3 <span class="code">120727GH_011</span></li>
+
<li>GA: pLAC_fha1_eCFP (pSB4C5) <b>1</b> <span class="code">120803GH_017</span></li>
-
<li>BBa_J23119 (pConst) <span class="code">120727GH_012</span></li>
+
<li>GA: pLAC_fha1_eCFP (pSB4C5) <b>2</b>                <span class="code">120803GH_018</span></li>
-
<li>BBa_R0082 (pOmpC) <span class="code">120727GH_013</span></li>
+
<li>GA: pAra/Bad_RBS_GFP_RBS_Cya (pSB3C5) <b>1</b> <span class="code">120803GH_019</span></li>
-
<li>BBa_J06702 (mcherry in LB with Ampicillin) <span class="code">120727GH_014</span></li>
+
<li>GA: pAra/Bad_RBS_GFP_RBS_Cya (pSB4C5) <b>1</b> <span class="code">120803GH_020</span></li>
-
<li>BBa_I763011 (GFP) <span class="code">120727GH_015</span></li>
+
<li>GA: pAra/Bad_RBS_GFP_RBS_Cya (pSB3C5) <b>2</b> <span class="code">120803GH_021</span></li>
 +
<li>GA: pAra/Bad_RBS_GFP_RBS_Cya (pSB4C5) <b>2</b> <span class="code">120803GH_022</span></li>
 +
<li>GA: pLAC_rsmY (pSB1A3) <b>2</b>    <span class="code">120803GH_023</span></li>
 +
<li>GA: pAra/Bad_RBS_GFP_RBS_Cya (pSB4C5) <b>3</b> <span class="code">120803GH_024</span></li>
 +
<li>GA: pLAC_rsmY (pSB1A3) <b>3</b>    <span class="code">120803GH_025</span></li>
 +
<li>GA: pAra/Bad_RBS_GFP_RBS_Cya (pSB4C5) <b>4</b> <span class="code">120803GH_026</span></li>
 +
<li>GA: pAra/Bad_RBS_GFP_RBS_Cya (pSB4C5) <b>5</b> <span class="code">120803GH_027</span></li>
</ul>
</ul>
-
<br/>
 
-
We did some colony PCRs with HF Phusion enzyme (protocol) in order to amplify RBS_Cya (on BW25113 WT strain), pAra/Bad_RBS_GFP (on BW25113 Cya- pAra/Bad) and pSB4C5 (on 12/07/25 colony).<br/>
 
-
<br/>
 
-
To separate the PCR products, we prepared a 1.3% TAE agarose gel.<br/>
 
-
Migration conditions = 100V during 30 min.<br/>
 
-
In order to reveal the DNA fragments, we used EtBr.<br/>
 
-
<br/>
 
-
<center><img src="https://static.igem.org/mediawiki/2012/b/b1/120727.jpg" alt="photo_gel_21"/></center>
 
-
<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/>
 
-
Lane 1: DNA ladder 1kb (biolabs)<br/>
 
-
Lanes 2 and 3: pAra/Bad_RBS_GFP PCR product<br/>
 
-
Lanes 4 and 5: RBS_Cya PCR product<br/>
 
-
Lanes 6 and 7: pSB4C5 PCR product<br/>
 
-
Lane 8: DNA ladder 1kb (biolabs)<br/></div>
 
-
 
-
 
-
<br/>
 
-
The pSB4C5 amplification didn’t work. However, the RBS_Cya and pAra/Bad_RBS_GFP amplications worked.<br/>
 
-
<br/>
 
-
We realised a DNA extraction (protocol kit: Nucleospin extract II) from the two pAra/Bad_RBS_GFP PCR products <span class="code">120727AM_PCR_023</span> and the two Cya PCR products <span class="code">120727AM_PCR_022</span>.<br/>
 
</section>
</section>
<section>
<section>
<h2> Conclusion of the week:</h2>
<h2> Conclusion of the week:</h2>
-
We replaced pSB4K5 with pSB4C5 and pSB3C5 with pSB3T5. We also replaced eCFP with mcherry or GFP.<br/>
 
-
<br/>We have achieved to amplify pAra/Bad_RBS_GFP // RBS_Cya // fha // pSB4C5 (fha) and we began the experiments in order to recover pOmpC (for the detection module) and pConst.<br/>
 
-
<br/>The "AND" gate worked as expected.
 
</section>
</section>
</div>
</div>

Revision as of 13:58, 20 August 2012

iGEM Grenoble 2012

Project

August

Week 31Week 32Week 33Week 34Week 35

Week 31: July 30th to August 05th

Goal of the week:

We wanted to recover and amplify some biobricks involved in our genetic networks:
  • pSB4C5 (2400bp)
  • pompC (100bp)
  • mcherry (900bp)
  • eCFP (800bp)
  • GFP (100bp)

Tuesday, July 31th:

We did a miniprep (protocol kit: Nucleospin plasmid Quick Pure) on the transformed strain with pSB4C5 (12/07/25), on which we wanted to recover pSB4C5.

We did some colony PCRs (on 12/07/25 colony) and PCRs on miniprep with HF Phusion enzyme (protocol) in order to amplify pSB4C5.
Annealing temperature = 60°C.

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_22
Migration result for a 1.8% TAE agarose gel
(the DNA ladder scale is in kb)
Lane 1: DNA ladder 1kb (biolabs)
Lane 2: pSB4C5 (miniprep) PCR product (DMSO)
Lane 3: pSB4C5 (miniprep) PCR product (DMSO)
Lane 4: pSB4C5 (miniprep) PCR product
Lane 5: pSB4C5 (miniprep) PCR product
Lane 6: pSB4C5 (colony) PCR product (DMSO)
Lane 7: pSB4C5 (colony) PCR product (DMSO)
Lane 8: pSB4C5 (colony) PCR product
Lane 9: pSB4C5 (colony) PCR product
Lane 10: DNA ladder 1kb (biolabs)

We only saw primer dimer bands, there was a PCR condition problem.

We did some PCRs on miniprep with HF Phusion enzyme (protocol) in order to amplify pSB4C5.
Annealing temperature = 65°C.

We did some digestions (protocol) on miniprep (pSB4C5) in order to do the construction: pAra/Bad_RBS_GFP_RBS_Cya. The digestions were achieved with two restriction enzymes : XbaI and SpeI during 10 minutes.

To separate the PCR products and 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_23
Migration result for a 1.8% TAE agarose gel
(the DNA ladder scale is in kb)
Lane 1: DNA ladder 1kb (biolabs)
Lane 2: pSB4C5 digestion product
Lane 3: pSB4C5 digestion product
Lane 4: pSB4C5 (miniprep) PCR product (DMSO)
Lane 5: pSB4C5 (miniprep) PCR product (DMSO)
Lane 6: pSB4C5 (miniprep) PCR product
Lane 7: pSB4C5 (miniprep) PCR product
Lane 8: pSB4C5 (colony) PCR product (DMSO)
Lane 9: pSB4C5 (colony) PCR product (DMSO)
Lane 10: pSB4C5 (colony) PCR product
Lane 11: pSB4C5 (colony) PCR product
Lane 12: DNA ladder 1kb (biolabs)

For the PCR results, we only saw primer dimer bands, there was a PCR condition problem.
The digestion worked well, we realised a DNA extraction (protocol kit: Nucleospin extract II) from the two digestion products 120731AM_DIG_027.

Wednesday, August 01st:

We did a touchdown PCR (from 70°C to 60°C) from miniprep (12/07/31) with HF Phusion enzyme (protocol) in order to amplify pSB4C5.

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_24
Migration result for a 1.8% TAE agarose gel
(the DNA ladder scale is in kb)
Lane 1: DNA ladder 1kb (biolabs)
Lane 2: pSBAC5 (digestion) PCR product
Lane 3: pSB4C5 (digestion) PCR product (DMSO)
Lane 4: pSB4C5 (miniprep) PCR product
Lane 5: pSB4C5 (miniprep) PCR product (DMSO)
Lane 6: DNA ladder 1kb (biolabs)

We only saw primer dimer bands, there was a PCR condition problem.

We realised eight Gibson Assemblies (protocol) to build four plasmids:
  • pSB4C5 (fha1) 120726AM_PCR_021 with pLAC (fha1) 120713PP_PCR_009, fha1 120723AM_PCR_020 and eCFP 120720_DIG_019
  • pSB1A3 120713PP_PCR_014 with pLAC (rsmY) 120713PP_PCR_011 and rsmY 120713PP_PCR_013
  • pSB3C5 (Cya) 120713PP_PCR_015 with pAra/Bad_RBS_GFP 120727AM_PCR_023 and RBS_Cya 120727AM_PCR_022
  • pSB4C5 120731AM_DIG_027 with pAra/Bad_RBS_GFP 120727AM_PCR_023 and RBS_Cya 120727AM_PCR_022

For each plasmid construct, we did two Gibson Assemblies (protocol).
We transformed (new protocol) BW25113 WT competent cells (protocol) with the GA products:
pLAC_fha1_eCFP and pLAC_rsmY.
We transformed (new protocol) BW25113 Cya- competent cells (protocol) with the GA products:
pAra/Bad_RBS_GFP_RBS_Cya.

We did some minipreps (protocol kit: Nucleospin plasmid Quick Pure) on the transformed strains (12/07/25) with pOmpC, mcherry, eCFP and pSB4C5 ; in order to set up the test on the receptor with a reporter.

Then, we did some digestions (protocol) on these minipreps. The digestions were achieved with two restriction enzymes:
  • pOmpC with EcoRI and SpeI during 10 minutes
  • mcherry and eCFP with XbaI and SpeI during 10 minutes
  • pSB4C5 with EcoRI and PstI 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_25
Migration result for a 1.8% TAE agarose gel
(the DNA ladder scale is in kb)
Lane 1: DNA ladder 80pb-10kb (fermentas)
Lane 2: pOmpC digestion product
Lane 3: pOmpC digestion product
Lane 4: DNA ladder 80pb-10kb (fermentas)
Lane 5: eCFP digestion product
Lane 6: eCFP digestion product
Lane 7: mcherry digestion product
Lane 8: mcherry digestion product
Lane 9: pSB4C5 digestion product
Lane 10: pSB4C5 digestion product
Lane 11: DNA ladder 1kb (biolabs)

The digestion worked well, we realised a DNA extraction (protocol kit: Nucleospin extract II) from the eCFP, mcherry and pSB4C5 digestion products.

Thursday, August 02nd:

We did a miniprep (protocol kit: Nucleospin plasmid Quick Pure) on the transformed strain (12/07/25) with pOmpC.

Then, we did a digestion (protocol) on this miniprep. The digestions were achieved with two restriction enzymes: EcoRI 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_26
Migration result for a 1.8% TAE agarose gel
(the DNA ladder scale is in kb)
Lane 1: DNA ladder 80pb-10kb (fermentas)
Lane 2: pOmpC miniprep product
Lane 3: pOmpC miniprep product
Lane 4: pOmpC digestion product
Lane 5: pOmpC digestion product
Lane 6: DNA ladder 80pb-10kb (fermentas)

There was a problem with the miniprep or the transformation, we didn't see any DNA bands.

With iGEM 2012 biobricks we transformed (new protocol) BW25113 WT competent cells (protocol) with pOmpC.

We decided to relaunch the transformed strains with the GA products (12/08/01) in fresh liquid LB.

Friday, August 03rd:

We did some minipreps (protocol kit: Nucleospin plasmid Quick Pure) on the transformed strains with the GA products.

We did some glycerol stocks:
  • GA: pLAC_rsmY (pSB1A3) 1 120803GH_016
  • GA: pLAC_fha1_eCFP (pSB4C5) 1 120803GH_017
  • GA: pLAC_fha1_eCFP (pSB4C5) 2 120803GH_018
  • GA: pAra/Bad_RBS_GFP_RBS_Cya (pSB3C5) 1 120803GH_019
  • GA: pAra/Bad_RBS_GFP_RBS_Cya (pSB4C5) 1 120803GH_020
  • GA: pAra/Bad_RBS_GFP_RBS_Cya (pSB3C5) 2 120803GH_021
  • GA: pAra/Bad_RBS_GFP_RBS_Cya (pSB4C5) 2 120803GH_022
  • GA: pLAC_rsmY (pSB1A3) 2 120803GH_023
  • GA: pAra/Bad_RBS_GFP_RBS_Cya (pSB4C5) 3 120803GH_024
  • GA: pLAC_rsmY (pSB1A3) 3 120803GH_025
  • GA: pAra/Bad_RBS_GFP_RBS_Cya (pSB4C5) 4 120803GH_026
  • GA: pAra/Bad_RBS_GFP_RBS_Cya (pSB4C5) 5 120803GH_027

Conclusion of the week: