Team:Goettingen/week7-3

From 2012.igem.org

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Language: <img height="20", src="http://www.patrickreinke.de/igem/eng.jpg"> English, <img height="20", src="http://www.patrickreinke.de/igem/deu.jpg"> <a href="https://2012.igem.org/Team:Goettingen/main_deu">Deutsch</a> <br>
 
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<b>Insertion of TAR_QC into BioBricks plasmids</b><br>
<b>Insertion of TAR_QC into BioBricks plasmids</b><br>
<ul>
<ul>
-
<li>Experiment: <br>We selected 8 different promoters for our chemoreceptor TAR. We chose the following parts for this purpose: J23100, J23104, J23105, J23106, J23109, J23112, J23113, J23114; all of these in vector J61002 (for details click <a href="http://partsregistry.org/Main_Page">here</a>).
+
<li>Experiment: <br>We selected 8 different promoters for our chemoreceptor TAR. We chose the following parts for this purpose: <br>
 +
J23100 (18C) <br> J23104 (18K) <br> J23105 (18M) <br> J23106 (18O) <br> J23109 (2G) <br> J23112 (20E) <br> J23113 (20G) <br> J23114 (20I) <br> all of these in vector J61002 (for details click <a href="http://partsregistry.org/Main_Page">here</a>). These constructs were, prior to this experiment, transformed into <i>E. coli</i>, prepped and stored at -20 °C. <br>
 +
The vector J61002 was digested with <i>Spe</i>I and <i>Pst</i>I, the insert TAR_QC was digested with <i>Xba</i>I and <i>Pst</i>I and subsequently ligated downstream of our 8 promoters according to <a href="https://2012.igem.org/Team:Goettingen/Project/Methods">protocol</a>. We transformed <i>E. coli</i> &Delta; <i>tar</i> with the generated constructs. This is an <i>E. coli</i> DH10B strain where the <i>tar</i> gene is deleted. Consequently this strain cannot perceive Aspartate any longer. We use it to verify that our constructs are able to rescue this phenotype.
</li>
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</ul>
</ul>
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<td width="900" bordercolor="black" valign="top">
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<h2><b>V06_07-2 </b></h2><br>
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<h2><b>V06_13 </b></h2><br>
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<b>Miniprep of pUC18_TAR_QC</b><br>
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<b>Preparation of J61002_promoter_TAR_QC</b><br>
<ul>
<ul>
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<li>Experiment: <br>Minipreps were done with PeqGOLD MiniPrep Kit (Peqlab) according to the user manual. A test digestion was performed using <i>Xba</i>I and the expected fragment of 4342 bp was observed on the corresponding gel. Minipreps were stored at -20 °C.
+
<li>Experiment: <br>All 8 promoter constructs + TAR_QC were prepped using peqGOLD Plasmid Miniprep kit (PeqLab) following the user manual. We performed a test digestion using <i>Xba</i>I and <i>Pst</i>I to verify that the insert TAR_QC was successfully integrated into the plasmid. Glycerol stocks (30 %) of <i>E. coli</i> &Delta; <i>tar</i> J61002_promoter_TAR_QC were prepared and stored at -80 °C.
 +
</li>
 +
</ul>
 +
<ul>
 +
<li>Observations & Results: <br>All eight constructs were successfully generated!
</li>
</li>
</ul>
</ul>
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<a href="https://2012.igem.org/Team:Goettingen/Notebook">Back to overview</a><br>
<a href="https://2012.igem.org/Team:Goettingen/Notebook">Back to overview</a><br>
<br>
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<b>Important pages</b>:<br>
 
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<a href="https://2012.igem.org/Team:Goettingen">Home</a>;
 
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<a href="https://2012.igem.org/Team:Goettingen/Team">Team</a>;
 
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<a href="https://igem.org/Team.cgi?year=2012">Official Team Profile</a>;
 
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<a href="https://2012.igem.org/Team:Goettingen/Project">Project</a>;
 
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<a href="https://2012.igem.org/Team:Goettingen/Parts">Parts submitted to the Registry</a>;
 
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<a href="https://2012.igem.org/Team:Goettingen/Modeling">Modeling</a>;
 
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{{GoettingenFooter}}

Latest revision as of 13:10, 25 September 2012

Deutsch  / English 

#3 Chemoreceptor Library - 7th Week

Back to overview

V06_11


Insertion of TAR_QC into BioBricks plasmids
  • Experiment:
    We selected 8 different promoters for our chemoreceptor TAR. We chose the following parts for this purpose:
    J23100 (18C)
    J23104 (18K)
    J23105 (18M)
    J23106 (18O)
    J23109 (2G)
    J23112 (20E)
    J23113 (20G)
    J23114 (20I)
    all of these in vector J61002 (for details click here). These constructs were, prior to this experiment, transformed into E. coli, prepped and stored at -20 °C.
    The vector J61002 was digested with SpeI and PstI, the insert TAR_QC was digested with XbaI and PstI and subsequently ligated downstream of our 8 promoters according to protocol. We transformed E. coli Δ tar with the generated constructs. This is an E. coli DH10B strain where the tar gene is deleted. Consequently this strain cannot perceive Aspartate any longer. We use it to verify that our constructs are able to rescue this phenotype.


V06_13


Preparation of J61002_promoter_TAR_QC
  • Experiment:
    All 8 promoter constructs + TAR_QC were prepped using peqGOLD Plasmid Miniprep kit (PeqLab) following the user manual. We performed a test digestion using XbaI and PstI to verify that the insert TAR_QC was successfully integrated into the plasmid. Glycerol stocks (30 %) of E. coli Δ tar J61002_promoter_TAR_QC were prepared and stored at -80 °C.
  • Observations & Results:
    All eight constructs were successfully generated!


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