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iGEM Grenoble 2012

iGEM 2012
main page

July

Week 27 • Week 28 • Week 29 • Week 30

Week 30: July 23rd to 29th

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)
fha (80bp)
eCFP (800bp)
pSB4K5 (2400bp)

We also wanted to test the "AND" gate (CRP-cAMP & araC) without the amplifier module.

Monday, July 23rd:

We did an experiment (protocol) in order to test the "AND" gate (CRP-cAMP & araC) without the amplifier module.

results_AND_Gate

strain used = BW25113 cya- transformed with pAra/Bad_RBS_GFP. The medium was complemented: 0.03% glucose & 0.03% acetate.

The experiments worked well, the "AND" gate worked as expected.

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.

To separate (protocol) the PCR products (12/07/23 and 12/07/20), 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_16

Migration result for a 1.3% TAE agarose gel
(the DNA ladder scale is in kb)

Lane 1: DNA ladder 80pb-10kb (fermentas)
Lane 2: pSB4K5 PCR product (DMSO)
Lane 3: fha1 PCR product (DMSO)
Lane 4: fha1 PCR product
Lane 5: pSB4K5 PCR product
Lane 6: pAra/Bad_RBS_GFP PCR product (DMSO)
Lane 7: pAra/Bad_RBS_GFP PCR product (DMSO)
Lane 8: RBS_Cya PCR product (DMSO)
Lane 9: RBS_Cya PCR product
Lane 10: pAra/Bad_RBS_GFP PCR product
Lane 11:pAra/Bad_RBS_GFP PCR product
Lane 12: DNA ladder 80bp-10kb (fermentas)

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.

We realised a DNA extraction (protocol) from the two fha PCR products 120723AM_PCR_020.

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.

Using iGEM 2012 biobricks we transformed (new protocol) BW25113 WT competent cells (protocol) with pSB4K5.

Tuesday, July 24th:

To separate (protocol) the PCR products (12/07/23), 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_17

Migration result for a 1.3% TAE agarose gel
(the DNA ladder scale is in kb)

Lane 1: DNA ladder 80bp-10kb (fermentas)
Lane 2: E0022 PCR product
Lane 3: E0022 PCR product (DMSO)
Lane 4: E0422 PCR product
Lane 5: E0422 PCR product (DMSO)
Lane 6: DNA ladder 80bp-10kb (fermentas)

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

The transformation with pSB4K5 (12/07/23) showed result (growth on plates).

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.

To separate (protocol) 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_18

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: pSB4K5 digestion product
Lane 4: DNA ladder 80bp-1kb (fermentas)

The digestion worked, we saw DNA bands at the expected positions.

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.

To separate (protocol) 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.

There was no result (data not shown).

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.

With iGEM 2012 biobricks we transformed (new protocol) BW25113 WT competent cells (protocol) with pSB4C5.

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.

Wednesday, July 25th:

To separate (protocol) the PCR products (12/07/24), we prepared a 1.8% TAE agarose gel.
Migration conditions = 100V during 30 min.
In order to reveal the DNA fragments, we used EtBr.

There was a migration problem (data not shown).

The transformation with pSB4C5 (12/07/24) was successful (growth on plates). We decided to relaunch it in fresh liquid LB.

We did some glycerol stocks:

GA: pLAC_RBS_RsmA 120725GH_001
pSB4K5 120725GH_002
E0422 (eCFP) 120725GH_003
LuxI 120725GH_004
E0022 (eCFP) 120725GH_005
LuxR 120725GH_006
pLux 120725GH_007
I13601 120725GH_008

We did a colony PCR with GoTaq enzyme (protocol) in order to amplify pSB4C5.

To separate (protocol) the PCR products (07/24), 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_19

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 PCR product
Lane 3: DNA ladder 1kb (biolabs)

We concluded that the pSB4C5 amplification worked well, so we decided to do the PCR again in order to do a DNA extraction.

Thursday, July 26th:

We did a glycerol stock of the strain transformed with pSB4C5 120726AM_009.

We did a colony PCR withs HF Phusion Enzyme (protocol) in order to amplify pSB4C5.

We did a miniprep (protocol) on the transformed strain with pSB4K5, on which we wanted to recover pSB4K5.

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.

To separate (protocol) the PCR products (12/07/24), 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_20

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: pSB4K5 without digestion
Lanes 4 and 5: pSB4K5 digestion product
Lane 6: empty lane
Lanes 7 and 8: pSB4C5 PCR product
Lane 9: DNA ladder 1kb (biolabs)

We concluded that the digestion worked well (we seen the expected DNA bands), and so was the PCR on pSB4C5.

We realised a DNA extraction (protocol) on the two pSB4C5 PCR products 120726AM_PCR_021.

We didn’t achieve to amplify eCFP, we decided to change it. We chose mcherry and GFP.

We transformed (new protocol) BW25113 WT competent cells (protocol) with biobricks from iGEM 2012 kit :

BBa_I763011 (GFP)
BBa_J06702 (mcherry) (in duplicate : in LB with Ampicillin and in LB with Ampicillin and Kanamycin)
pSB1A3
BBa_R0082 (pompC)
BBa_J23119 (pConst)

Friday, July 27th:

The five transformations (12/07/26) were successful (growth on the expected plates). We did glycerol stocks of:

BBa_J06702 (mcherry in LB with Ampicillin and Kanamycin) 120727GH_010
pSB1A3 120727GH_011
BBa_J23119 (pConst) 120727GH_012
BBa_R0082 (pOmpC) 120727GH_013
BBa_J06702 (mcherry in LB with Ampicillin) 120727GH_014
BBa_I763011 (GFP) 120727GH_015

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).

To separate (protocol) 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_21

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: pAra/Bad_RBS_GFP PCR product
Lanes 4 and 5: RBS_Cya PCR product
Lanes 6 and 7: pSB4C5 PCR product
Lane 8: DNA ladder 1kb (biolabs)

The pSB4C5 amplification didn’t work. However, the RBS_Cya and pAra/Bad_RBS_GFP amplications worked.

We realised a DNA extraction (protocol) from the two pAra/Bad_RBS_GFP PCR products 120727AM_PCR_023 and the two Cya PCR products 120727AM_PCR_022.

Conclusion of the week:

We replaced pSB4K5 with pSB4C5 and pSB3C5 with pSB3T5. We also replaced eCFP with mcherry or GFP.

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.

The "AND" gate worked as expected.

@@ Line 5: / Line 5: @@
 <div id="cadre">
 <section>
-<h1>August</h1>
+<h1>July</h1>
-<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 27</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_28">Week 28</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_29">Week 29</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_30">Week 30</a>
-<a href="https://2012.igem.org/Team:Grenoble/Biology/Notebook/July/week_35">Week 35</a>
 </section>
 <br/>
 <section>
-<h1> Week 31: July 30<span class="exposant">th</span> to August 05<span class="exposant">th</span> </h1>
+<h1> Week 30: July 23<span class="exposant">rd</span> to 29<span class="exposant">th</span> </h1>
 <h2> Goal of the week: </h2>
-We wanted to recover and amplify some 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>
-<li>pSB4C5 (2400bp)</li>
+<li>pAra/Bad_RBS_GFP (1300bp)</li>
-<li>pompC (100bp)</li>
+<li>RBS_Cya (2600bp)</li>
-<li>mcherry (900bp)</li>
+<li>fha (80bp)</li>
 <li>eCFP (800bp)</li>
-<li>GFP (100bp)</li>
+<li>pSB4K5 (2400bp)</li>
 </ul>
+<br/>
+We also wanted to test the "AND" gate (CRP-cAMP & araC) without the amplifier module.<br/>
 </section>
 <section>
-<h2> Tuesday, July 31<span class="exposant">th</span>:</h2>
+<h2> Monday, July 23<span class="exposant">rd</span>:</h2>
-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/>
+We did an experiment (<a href="https://2012.igem.org/Team:Grenoble/Biology/Protocols/AND_test">protocol</a>) in order to test the "AND" gate (CRP-cAMP & araC) without the amplifier module. <br/>
 <br/>
-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/>
+<center><img src="https://static.igem.org/mediawiki/2012/2/2d/AND-gate.png" alt="results_AND_Gate"/></center>
+<div class="legend">strain used = BW25113 cya<span class="exposant">-</span> transformed with pAra/Bad_RBS_GFP. The medium was complemented: 0.03% glucose & 0.03% acetate.
+<br/></div>
+<br/>The experiments worked well, the "AND" gate worked as expected.<br/>
 <br/>
-To separate the PCR products, we prepared a 1.8% TAE agarose gel.<br/>
+We did PCRs with HF Phusion enzyme (<a href="https://2012.igem.org/Team:Grenoble/Biology/Protocols/PCR_2">protocol</a>) 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/>
+ <br/>
+To separate (<a href="https://2012.igem.org/Team:Grenoble/Biology/Protocols/Gel">protocol</a>) 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/>
-In order to reveal the DNA fragments, we used EtBr.<br/>
+In order to reveal the DNA fragments, we used <a href="https://2012.igem.org/Team:Grenoble/Biology/Protocols/EtBr">EtBr</a>.<br/>
 <br/>
-<center><img src="https://static.igem.org/mediawiki/2012/a/ac/120731.jpg" alt="photo_gel_22"/></center>
+<center><img src="https://static.igem.org/mediawiki/2012/c/c1/120723.jpg" alt="photo_gel_16"/></center>
-<div class="legend"><u>Migration result for a 1.8% TAE agarose gel</u><br/>
+<div class="legend"><p><center><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></center></p>
-Lane 1: DNA ladder 1kb (biolabs)<br/>
+<ul><li><b>Lane 1:</b> DNA ladder 80pb-10kb (fermentas)</li>
-Lane 2: pSB4C5 (miniprep) PCR product (DMSO)<br/>
+<li><b>Lane 2:</b> pSB4K5 PCR product (DMSO)</li>
-Lane 3: pSB4C5 (miniprep) PCR product (DMSO)<br/>
+<li><b>Lane 3:</b> fha1 PCR product (DMSO)</li>
-Lane 4: pSB4C5 (miniprep) PCR product<br/>
+<li><b>Lane 4:</b> fha1 PCR product</li>
-Lane 5: pSB4C5 (miniprep) PCR product<br/>
+<li><b>Lane 5:</b> pSB4K5 PCR product</li>
-Lane 6: pSB4C5 (colony) PCR product (DMSO)<br/>
+<li><b>Lane 6:</b> pAra/Bad_RBS_GFP PCR product (DMSO)</li>
-Lane 7: pSB4C5 (colony) PCR product (DMSO)<br/>
+<li><b>Lane 7:</b> pAra/Bad_RBS_GFP PCR product (DMSO)</li>
-Lane 8: pSB4C5 (colony) PCR product <br/>
+<li><b>Lane 8:</b> RBS_Cya PCR product (DMSO)</li>
-Lane 9: pSB4C5 (colony) PCR product <br/>
+<li><b>Lane 9:</b> RBS_Cya PCR product</li>
-Lane 10: DNA ladder 1kb (biolabs)<br/></div>
+<li><b>Lane 10:</b> pAra/Bad_RBS_GFP PCR product</li>
+<li><b>Lane 11:</b>pAra/Bad_RBS_GFP PCR product</li>
+<li><b>Lane 12:</b> DNA ladder 80bp-10kb (fermentas)</li></ul></div>
+<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/>
+<br/>
+We realised a DNA extraction (<a href="https://2012.igem.org/Team:Grenoble/Biology/Protocols/DNA_extract">protocol</a>) from the two fha PCR products <span class="code">120723AM_PCR_020</span>.<br/>
+<br/>
+We did touchdown PCRs with HF Phusion enzyme (<a href="https://2012.igem.org/Team:Grenoble/Biology/Protocols/PCR_2">protocol</a>) 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/>
+<br/>
+Using iGEM 2012 biobricks we transformed (new <a href="https://2012.igem.org/Team:Grenoble/Biology/Protocols/Transformation_2">protocol</a>) BW25113 WT competent cells (<a href="https://2012.igem.org/Team:Grenoble/Biology/Protocols/Competence">protocol</a>) with pSB4K5.<br/>
+</section>
+<section>
+<h2> Tuesday, July 24<span class="exposant">th</span>:</h2>
+To separate (<a href="https://2012.igem.org/Team:Grenoble/Biology/Protocols/Gel">protocol</a>) the PCR products (12/07/23), 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 <a href="https://2012.igem.org/Team:Grenoble/Biology/Protocols/EtBr">EtBr</a>.<br/>
+<br/>
+<center><img src="https://static.igem.org/mediawiki/2012/e/e0/120724_%282%29.jpg" alt="photo_gel_17"/></center>
+<div class="legend"><p><center><u>Migration result for a 1.3% TAE agarose gel</u><br/>
+  <i>(the DNA ladder scale is in kb)</i></center></p>
+<ul><li><b>Lane 1:</b> DNA ladder 80bp-10kb (fermentas)</li>
+<li><b>Lane 2:</b> E0022 PCR product</li>
+<li><b>Lane 3:</b> E0022 PCR product (DMSO)</li>
+<li><b>Lane 4:</b> E0422 PCR product</li>
+<li><b>Lane 5:</b> E0422 PCR product (DMSO)</li>
+<li><b>Lane 6:</b> DNA ladder 80bp-10kb (fermentas)</li></ul></div>
 <br/>
 We only saw primer dimer bands, there was a PCR condition problem.<br/>
 <br/>
-We did some PCRs on miniprep with HF Phusion enzyme (protocol) in order to amplify pSB4C5. <br/>Annealing temperature = 65°C.<br/>
+The transformation with pSB4K5 (12/07/23) showed result (growth on plates).<br/>
 <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/>
+We did some digestions (<a href="https://2012.igem.org/Team:Grenoble/Biology/Protocols/Restriction">protocol</a>) on miniprep (12/07/19) to recover eCFP (E0022). The digestions were achieved with two restriction enzymes : XbaI and SpeI during 10 minutes.<br/>
 <br/>
-To separate the PCR products and the digestion products, we prepared a 1.8% TAE agarose gel.<br/>
+To separate (<a href="https://2012.igem.org/Team:Grenoble/Biology/Protocols/Gel">protocol</a>) the digestion 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/>
+In order to reveal the DNA fragments, we used <a href="https://2012.igem.org/Team:Grenoble/Biology/Protocols/EtBr">EtBr</a>.<br/>
 <br/>
-<center><img src="https://static.igem.org/mediawiki/2012/9/9e/120731_%282%29.jpg" alt="photo_gel_23"/></center>
+<center><img src="https://static.igem.org/mediawiki/2012/6/6b/120724.jpg" alt="photo_gel_18"/></center>
-<div class="legend"><u>Migration result for a 1.8% TAE agarose gel</u><br/>
+<div class="legend"><p><center><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></center></p>
-Lane 1: DNA ladder 1kb (biolabs)<br/>
+<ul><li><b>Lane 1:</b> DNA ladder 1kb (biolabs)</li>
-Lane 2: pSB4C5 digestion product <br/>
+<li><b>Lane 2:</b> E0022 digestion product</li>
-Lane 3: pSB4C5 digestion product <br/>
+<li><b>Lane 3:</b> pSB4K5 digestion product</li>
-Lane 4: pSB4C5 (miniprep) PCR product (DMSO)<br/>
+<li><b>Lane 4:</b> DNA ladder 80bp-1kb (fermentas)</li></ul></div>
-Lane 5: pSB4C5 (miniprep) PCR product (DMSO)<br/>
-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/>
-For the PCR results, we only saw primer dimer bands, there was a PCR condition problem.<br/>
+The digestion worked, we saw DNA bands at the expected positions.<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/>
+<br/>
-</section>
+We did  PCR with HF Phusion enzyme (<a href="https://2012.igem.org/Team:Grenoble/Biology/Protocols/PCR_2">protocol</a>) from one of these digestion product (eCFP E0022) in order to amplify eCFP and a colony PCR to amplify pSB4K5.<br/>
-<section>
-<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/>
-To separate the PCR products, we prepared a 1.8% TAE agarose gel.<br/>
+To separate (<a href="https://2012.igem.org/Team:Grenoble/Biology/Protocols/Gel">protocol</a>) 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/>
+In order to reveal the DNA fragments, we used <a href="https://2012.igem.org/Team:Grenoble/Biology/Protocols/EtBr">EtBr</a>.<br/>
 <br/>
-<center><img src="https://static.igem.org/mediawiki/2012/6/6f/120801.jpg" alt="photo_gel_24"/></center>
+There was no result (data not shown).<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/>
-Lane 1: DNA ladder 1kb (biolabs)<br/>
-Lane 2: pSBAC5 (digestion) PCR product <br/>
-Lane 3: pSB4C5 (digestion) PCR product (DMSO)<br/>
-Lane 4: pSB4C5 (miniprep) PCR product<br/>
-Lane 5: pSB4C5 (miniprep) PCR product (DMSO)<br/>
-Lane 6: DNA ladder 1kb (biolabs)<br/></div>
 <br/>
-We only saw primer dimer bands, there was a PCR condition problem.<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/>
-We realised eight Gibson Assemblies (protocol) to build four plasmids:<br/>
+With iGEM 2012 biobricks we transformed (new <a href="https://2012.igem.org/Team:Grenoble/Biology/Protocols/Transformation_2">protocol</a>) BW25113 WT competent cells (<a href="https://2012.igem.org/Team:Grenoble/Biology/Protocols/Competence">protocol</a>) with pSB4C5.<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/>
-For each plasmid construct, we did two Gibson Assemblies (protocol).
+We did an overlapping PCR with HF Phusion enzyme (<a href="https://2012.igem.org/Team:Grenoble/Biology/Protocols/PCR_2">protocol</a>) 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 (<a href="https://2012.igem.org/Team:Grenoble/Biology/Protocols/Gel">protocol</a>) 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 <a href="https://2012.igem.org/Team:Grenoble/Biology/Protocols/EtBr">EtBr</a>.<br/>
 <br/>
-We transformed (new protocol) BW25113 WT competent cells (protocol) with the GA products:<br/> pLAC_fha1_eCFP and pLAC_rsmY. <br/>
+There was a migration problem (data not shown).<br/>
-We transformed (new protocol) BW25113 Cya- competent cells (protocol) with the GA products:<br/> pAra/Bad_RBS_GFP_RBS_Cya.<br/>
 <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/>
+The transformation with pSB4C5 (12/07/24) was successful (growth on plates). We decided to relaunch it in fresh liquid LB.<br/>
 <br/>
-Then, we did some digestions (protocol) on these minipreps. The digestions were achieved with two restriction enzymes:
+We did some <a href="https://2012.igem.org/Team:Grenoble/Biology/Protocols/Glycerol">glycerol stocks</a>:
 <ul>
-<li> pOmpC with EcoRI and SpeI during 10 minutes </li>
+<li>GA: pLAC_RBS_RsmA 	<span class="code">120725GH_001</span></li>
-<li> mcherry and eCFP with XbaI and SpeI during 10 minutes</li>
+<li>pSB4K5 		<span class="code">120725GH_002</span></li>
-<li> pSB4C5 with EcoRI and PstI during 10 minutes</li>
+<li>E0422 (eCFP) 	<span class="code">120725GH_003</span></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>
 <br/>
-To separate the digestion products, we prepared a 1.8% TAE agarose gel.<br/>
+We did a colony PCR with GoTaq enzyme (<a href="https://2012.igem.org/Team:Grenoble/Biology/Protocols/PCR_1">protocol</a>) in order to amplify pSB4C5.<br/>
+<br/>
+To separate (<a href="https://2012.igem.org/Team:Grenoble/Biology/Protocols/Gel">protocol</a>) the PCR products (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/>
+In order to reveal the DNA fragments, we used <a href="https://2012.igem.org/Team:Grenoble/Biology/Protocols/EtBr">EtBr</a>.<br/>
 <br/>
-<center><img src="https://static.igem.org/mediawiki/2012/0/05/120801_%282%29.jpg" alt="photo_gel_25"/></center>
+<center><img src="https://static.igem.org/mediawiki/2012/3/3a/120725_%28PCR_colonie_pSB4C5%29.jpg" alt="photo_gel_19"/></center>
-<div class="legend"><u>Migration result for a 1.8% TAE agarose gel</u><br/>
+<div class="legend"><p><center><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></center></p>
-Lane 1: DNA ladder 80pb-10kb (fermentas)<br/>
+<ul><li><b>Lane 1:</b> DNA ladder 1kb (biolabs)</li>
-Lane 2: pOmpC digestion product<br/>
+<li><b>Lane 2:</b> pSB4C5 PCR product</li>
-Lane 3: pOmpC digestion product<br/>
+<li><b>Lane 3:</b> DNA ladder 1kb (biolabs)</li></ul></div>
-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/>
-The digestion worked well, we realised a DNA extraction (protocol kit: Nucleospin extract II) from the eCFP, mcherry and pSB4C5 digestion products.<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/>
 </section>
 <section>
-<h2> Thursday, August 02<span class="exposant">nd</span>:</h2>
+<h2> Thursday, July 26<span class="exposant">th</span>:</h2>
-We did a miniprep (protocol kit: Nucleospin plasmid Quick Pure) on the transformed strain (12/07/25) with pOmpC.<br/>
+We did a <a href="https://2012.igem.org/Team:Grenoble/Biology/Protocols/Glycerol">glycerol stock</a> of the strain transformed with pSB4C5 <span class="code">120726AM_009</span>.<br/>
 <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/>
+We did a colony PCR withs HF Phusion Enzyme (<a href="https://2012.igem.org/Team:Grenoble/Biology/Protocols/PCR_2">protocol</a>) in order to amplify pSB4C5.<br/>
 <br/>
-To separate the digestion products, we prepared a 1.8% TAE agarose gel.<br/>
+We did a miniprep (<a href="https://2012.igem.org/Team:Grenoble/Biology/Protocols/Miniprep">protocol</a>) on the transformed strain with pSB4K5, on which we wanted to recover pSB4K5.<br/>
+<br/>
+We did some digestions (<a href="https://2012.igem.org/Team:Grenoble/Biology/Protocols/Restriction">protocol</a>) 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 (<a href="https://2012.igem.org/Team:Grenoble/Biology/Protocols/Gel">protocol</a>) 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/>
+In order to reveal the DNA fragments, we used <a href="https://2012.igem.org/Team:Grenoble/Biology/Protocols/EtBr">EtBr</a>.<br/>
 <br/>
-<center><img src="https://static.igem.org/mediawiki/2012/1/1f/120802.jpg" alt="photo_gel_26"/></center>
+<center><img src="https://static.igem.org/mediawiki/2012/6/66/120726.jpg" alt="photo_gel_20"/></center>
-<div class="legend"><u>Migration result for a 1.8% TAE agarose gel</u><br/>
+<div class="legend"><p><center><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></center></p>
-Lane 1: DNA ladder 80pb-10kb (fermentas)<br/>
+<ul><li><b>Lane 1:</b> DNA ladder 1kb (biolabs)</li>
-Lane 2: pOmpC miniprep product<br/>
+<li><b>Lanes 2 and 3:</b> pSB4K5 without digestion</li>
-Lane 3: pOmpC miniprep product<br/>
+<li><b>Lanes 4 and 5:</b> pSB4K5 digestion product </li>
-Lane 4: pOmpC digestion product<br/>
+<li><b>Lane 6:</b> empty lane</li>
-Lane 5: pOmpC digestion product<br/>
+<li><b>Lanes 7 and 8:</b> pSB4C5 PCR product</li>
-Lane 6: DNA ladder 80pb-10kb (fermentas)<br/></div>
+<li><b>Lane 9:</b> DNA ladder 1kb (biolabs)</li></ul></div>
 <br/>
-There was a problem with the miniprep or the transformation, we didn't see any DNA bands.<br/>
+We concluded that the digestion worked well (we seen the expected DNA bands), and so was the PCR on pSB4C5.<br/>
 <br/>
-With iGEM 2012 biobricks we transformed (new protocol) BW25113 WT competent cells (protocol) with pOmpC.<br/>
+We realised a DNA extraction (<a href="https://2012.igem.org/Team:Grenoble/Biology/Protocols/DNA_extract">protocol</a>) on the two pSB4C5 PCR products <span class="code">120726AM_PCR_021</span>.<br/>
 <br/>
-We decided to relaunch the transformed strains with the GA products (12/08/01) in fresh liquid LB.<br/>
+We didn’t achieve to amplify eCFP, we decided to change it. We chose mcherry and GFP.<br/>
+<br/>
+We transformed  (new <a href="https://2012.igem.org/Team:Grenoble/Biology/Protocols/Transformation_2">protocol</a>) BW25113 WT competent cells (<a href="https://2012.igem.org/Team:Grenoble/Biology/Protocols/Competence">protocol</a>) 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>
-<h2> Friday, August 03<span class="exposant">rd</span>:</h2>
+<h2> Friday, July 27<span class="exposant">th</span>:</h2>
-We did some minipreps (protocol kit: Nucleospin plasmid Quick Pure) on the transformed strains with the GA products.<br/>
+The five transformations (12/07/26) were successful (growth on the expected plates). We did <a href="https://2012.igem.org/Team:Grenoble/Biology/Protocols/Glycerol">glycerol stocks</a> of:
-<br/>
-We did some glycerol stocks:
 <ul>
-<li>GA: pLAC_rsmY (pSB1A3) <b>1</b>                     <span class="code">120803GH_016</span></li>
+<li>BBa_J06702 (mcherry in LB with Ampicillin and Kanamycin)	<span class="code">120727GH_010</span></li>
-<li>GA: pLAC_fha1_eCFP (pSB4C5) <b>1</b>		<span class="code">120803GH_017</span></li>
+<li>pSB1A3 			<span class="code">120727GH_011</span></li>
-<li>GA: pLAC_fha1_eCFP (pSB4C5) <b>2</b>                <span class="code">120803GH_018</span></li>
+<li>BBa_J23119 (pConst)			<span class="code">120727GH_012</span></li>
-<li>GA: pAra/Bad_RBS_GFP_RBS_Cya (pSB3C5) <b>1</b> 	<span class="code">120803GH_019</span></li>
+<li>BBa_R0082 (pOmpC)			<span class="code">120727GH_013</span></li>
-<li>GA: pAra/Bad_RBS_GFP_RBS_Cya (pSB4C5) <b>1</b> 	<span class="code">120803GH_020</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 (pSB3C5) <b>2</b>	<span class="code">120803GH_021</span></li>
+<li>BBa_I763011 (GFP)	<span class="code">120727GH_015</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>
+<br/>
+We did some colony PCRs with HF Phusion enzyme (<a href="https://2012.igem.org/Team:Grenoble/Biology/Protocols/PCR_2">protocol</a>) 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 (<a href="https://2012.igem.org/Team:Grenoble/Biology/Protocols/Gel">protocol</a>) 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 <a href="https://2012.igem.org/Team:Grenoble/Biology/Protocols/EtBr">EtBr</a>.<br/>
+<br/>
+<center><img src="https://static.igem.org/mediawiki/2012/b/b1/120727.jpg" alt="photo_gel_21"/></center>
+<div class="legend"><p><center><u>Migration result for a 1.8% TAE agarose gel</u><br/>
+  <i>(the DNA ladder scale is in kb)</i></center></p>
+<ul><li><b>Lane 1:</b> DNA ladder 1kb (biolabs)</li>
+<li><b>Lanes 2 and 3:</b> pAra/Bad_RBS_GFP PCR product</li>
+<li><b>Lanes 4 and 5:</b> RBS_Cya PCR product</li>
+<li><b>Lanes 6 and 7:</b> pSB4C5 PCR product</li>
+<li><b>Lane 8:</b> DNA ladder 1kb (biolabs)</li></ul></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 (<a href="https://2012.igem.org/Team:Grenoble/Biology/Protocols/DNA_extract">protocol</a>) 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>
 <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>
 </div>

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

From 2012.igem.org

Latest revision as of 14:04, 26 September 2012

July

Week 30: July 23rd to 29th

Goal of the week:

Monday, July 23rd:

Tuesday, July 24th:

Wednesday, July 25th:

Thursday, July 26th:

Friday, July 27th:

Conclusion of the week: