Team:Grenoble/Biology/Notebook/August/week 34

From 2012.igem.org

(Difference between revisions)
 
Line 17: Line 17:
<h1> Week 34: August 20<span class="exposant">th</span> to 26<span class="exposant">th</span> </h1>
<h1> Week 34: August 20<span class="exposant">th</span> to 26<span class="exposant">th</span> </h1>
<h2> Goal of the week: </h2>
<h2> Goal of the week: </h2>
 +
Assembly of:
 +
<ul><li>pAra/Bad_RBS_GFP and RBS_Cya.</li>
 +
    <li>pompC and mcherry</li>
 +
    <li>Tap and EnvZ</li>
 +
</ul>
</section>
</section>
 +
<section>
 +
    <h2> Monday, August 20<span class="exposant">th</span>:</h2>
 +
We did some verification PCRs on miniprep with HF Phusion enzyme (<a href="http://2012.igem.org/Team:Grenoble/Biology/Protocols/PCR_2">protocol</a>) in order to check the
 +
Gibson Assembly products (12/09/09>.<br/>Annealing temperature = 55°C.<br/>
 +
<br/>
 +
To separate (<a href="http://2012.igem.org/Team:Grenoble/Biology/Protocols/Gel">protocol</a>) the PCR products, we prepared a 1.8% TAE agarose gel.<br/>
 +
Migration conditions = 100V during 30 min.<br/>
 +
In order to reveal the DNA fragments, we used <a href="http://2012.igem.org/Team:Grenoble/Biology/Protocols/EtBr">EtBr</a>.<br/>
 +
<br/>
 +
It showed no significant results (data not shown).
 +
</section>
 +
<section>
 +
    <h2> Tuesday, August 21<span class="exposant">th</span>:</h2>
 +
    We did an experiment (<a href="http://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 transformed (new <a href="http://2012.igem.org/Team:Grenoble/Biology/Protocols/Transformation_2">protocol</a>) BW25113 WT
 +
competent cells (<a href="http://2012.igem.org/Team:Grenoble/Biology/Protocols/Competence">protocol</a>) with pSB3C5.<br/>
 +
</section>
-
 
+
<section>
-
 
+
    <h2> Wednesday, August 22<span class="exposant">nd</span>:</h2>
 +
    We did an experiment (<a href="http://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. The M9 medium used was complemented: 0.1% acetate.
 +
</section>
<section>
<section>
-
     <h2> Conclusion of the week:</h2>
+
     <h2>Friday, August 24<span class="exposant">rd</span>:</h2>
 +
Then, we did some digestions (<a href="http://2012.igem.org/Team:Grenoble/Biology/Protocols/Restriction">protocol</a>). The digestions were achieved with two restriction enzymes:
 +
<ul>
 +
<li> pOmpC with EcoRI and SpeI during 10 minutes </li>
 +
<li> pOmpC with EcoRI during 10 minute</li>
 +
<li> pSB4K5 with EcoRI and SpeI during 10 minutes</li>
 +
<li> pSB4K5 with EcoRI during 10 minutes</li>
 +
</ul>
 +
<br/>
 +
To separate (<a href="http://2012.igem.org/Team:Grenoble/Biology/Protocols/Gel">protocol</a>) 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 <a href="http://2012.igem.org/Team:Grenoble/Biology/Protocols/EtBr">EtBr</a>.<br/>
 +
<br/>
 +
The PCR and the digestion worked well, we realised a DNA extraction (<a href="http://2012.igem.org/Team:Grenoble/Biology/Protocols/DNA_extract">protocol</a>) from the pSB1A3 (araC) and pSB4C5 (pAra/Bad_RBS_GFP_RBS_Cya) PCR products and from the pOmpC and pSB4K5 digestion products.<br/>
 +
<br/>
 +
We did some PCRs with HF Phusion enzyme (<a href="http://2012.igem.org/Team:Grenoble/Biology/Protocols/PCR_2">protocol</a>) in order to amplify pSB4C5 (mcherry),  pSB4C5 (pAra/Bad_RBS_GFP), RBS_Cya and pAra/Bad_RBS_GFP.<br/>
 +
<br/>
 +
Then, we did some digestions (<a href="http://2012.igem.org/Team:Grenoble/Biology/Protocols/Restriction">protocol</a>). The digestions were achieved with two restriction enzymes:
 +
<ul>
 +
<li> pSB4C5 with EcoRI and PstI during 10 minutes</li>
 +
<li> pSB4K5 with EcoRI and PstI during 10 minutes</li>
 +
<li> mcherry with PstI and XbaI during 10 minutes</li>
 +
<li> mcherry with PstI and XbaI during 10 minutes</li>
 +
</ul>
 +
<br/>
 +
To separate (<a href="http://2012.igem.org/Team:Grenoble/Biology/Protocols/Gel">protocol</a>) the PCR and 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 <a href="http://2012.igem.org/Team:Grenoble/Biology/Protocols/EtBr">EtBr</a>.<br/>
 +
<br/>
 +
The PCR and the digestion worked well, we realised a DNA extraction (<a href="http://2012.igem.org/Team:Grenoble/Biology/Protocols/DNA_extract">protocol</a>) from the Cya and pAra/Bad_RBS_GFP PCR products and from the mcherry digestion products.<br/>
 +
<br/>
 +
We realised a Gibson Assembly (<a href="http://2012.igem.org/Team:Grenoble/Biology/Protocols/GA">protocol</a>) to build: <ul><li>
 +
pSB4K5 with Tap-EnvZ</li>
 +
<li>pSB3C5 with pAra/Bad_RBS_GFP_RBS_Cya</li></ul><br/>
 +
We did some digestions (<a href="http://2012.igem.org/Team:Grenoble/Biology/Protocols/Restriction">protocol</a>) on pSB3C5, pSB4K5 and pSB1A3. The digestions were achieved with two restriction enzymes: EcoRI and PstI during 10 minutes.<br/>
 +
<br/>
 +
To separate (<a href="http://2012.igem.org/Team:Grenoble/Biology/Protocols/Gel">protocol</a>) the PCR and 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 <a href="http://2012.igem.org/Team:Grenoble/Biology/Protocols/EtBr">EtBr</a>.<br/>
 +
<br/>
 +
The digestion worked well, we realised a DNA extraction (<a href="http://2012.igem.org/Team:Grenoble/Biology/Protocols/DNA_extract">protocol</a>) from the pSB1A3 and pSB3C5 products.<br/>
</section>
</section>
   
   

Latest revision as of 01:59, 27 September 2012

iGEM Grenoble 2012

Project

August

Week 31Week 32Week 33Week 34Week 35

Week 34: August 20th to 26th

Goal of the week:

Assembly of:
  • pAra/Bad_RBS_GFP and RBS_Cya.
  • pompC and mcherry
  • Tap and EnvZ

Monday, August 20th:

We did some verification PCRs on miniprep with HF Phusion enzyme (protocol) in order to check the Gibson Assembly products (12/09/09>.
Annealing temperature = 55°C.

To separate (protocol) 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.

It showed no significant results (data not shown).

Tuesday, August 21th:

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

We transformed (new protocol) BW25113 WT competent cells (protocol) with pSB3C5.

Wednesday, August 22nd:

We did an experiment (protocol) in order to test the "AND" gate (CRP-cAMP & araC) without the amplifier module. The M9 medium used was complemented: 0.1% acetate.

Friday, August 24rd:

Then, we did some digestions (protocol). The digestions were achieved with two restriction enzymes:
  • pOmpC with EcoRI and SpeI during 10 minutes
  • pOmpC with EcoRI during 10 minute
  • pSB4K5 with EcoRI and SpeI during 10 minutes
  • pSB4K5 with EcoRI during 10 minutes

To separate (protocol) 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.

The PCR and the digestion worked well, we realised a DNA extraction (protocol) from the pSB1A3 (araC) and pSB4C5 (pAra/Bad_RBS_GFP_RBS_Cya) PCR products and from the pOmpC and pSB4K5 digestion products.

We did some PCRs with HF Phusion enzyme (protocol) in order to amplify pSB4C5 (mcherry), pSB4C5 (pAra/Bad_RBS_GFP), RBS_Cya and pAra/Bad_RBS_GFP.

Then, we did some digestions (protocol). The digestions were achieved with two restriction enzymes:
  • pSB4C5 with EcoRI and PstI during 10 minutes
  • pSB4K5 with EcoRI and PstI during 10 minutes
  • mcherry with PstI and XbaI during 10 minutes
  • mcherry with PstI and XbaI during 10 minutes

To separate (protocol) the PCR 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.

The PCR and the digestion worked well, we realised a DNA extraction (protocol) from the Cya and pAra/Bad_RBS_GFP PCR products and from the mcherry digestion products.

We realised a Gibson Assembly (protocol) to build:
  • pSB4K5 with Tap-EnvZ
  • pSB3C5 with pAra/Bad_RBS_GFP_RBS_Cya

We did some digestions (protocol) on pSB3C5, pSB4K5 and pSB1A3. The digestions were achieved with two restriction enzymes: EcoRI and PstI during 10 minutes.

To separate (protocol) the PCR 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.

The digestion worked well, we realised a DNA extraction (protocol) from the pSB1A3 and pSB3C5 products.