Team:Goettingen/week18-2

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                              <li><a href="https://2012.igem.org/Team:Goettingen/Human_Practice/Public_and_Media"><span><span>Public and Media</span></span></a></li>
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                              <!--</li><li><a href="https://2012.igem.org/Team:Goettingen/Newspaper"><span><span><div style="text-indent:20px;">&#8901; Newspaper</div></span></span></a></li>
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                              </li><li><a href="https://2012.igem.org/Team:Goettingen/Synthetic_Biology_Day"><span><span><div style="text-indent:20px;">&#8901; Synthetic Biology Day</div></span></span></a></li> -->
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                              <li><a href="https://2012.igem.org/Team:Goettingen/Human_Practice/Panel_Discussion"><span><span>Panel Discussion</span></span></a></li>-->
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                              <li><a href="https://2012.igem.org/Team:Goettingen/Human_Practice/Flash_coli"><span><span>Flash Coli</span></span></a></li>
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                                <!-- <li><a href="https://2012.igem.org/Team:Goettingen/Human_Practice/Sponsors"><span><span>Sponsors</span></span></a></li>
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                                <li><a href="https://2012.igem.org/Team:Goettingen/Human_Practice/Supporters"><span><span>Supporters</span></span></a></li>    -->
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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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<h2><b>V08_27 </b></h2><br>
<h2><b>V08_27 </b></h2><br>
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<b>Transformation of FlhDC-contructs into E.coli strain Mg </b><br>
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<b>V08_27_1 Miniprep of the <i>tar</i>-promoter constructs</b><br>
<ul>
<ul>
-
<li>Experiment: <br>Quick Change overlapping PCR was performed using designed primers and Pfu Turbo polymerase (see Quick Change protocol in "protocols"). Was not succesful. We Could not observed any PCR product.</li>
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<li>Experiment: <br>
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</ul>
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The Minipreps were conducted using the PeqGOLD MiniPrep Kit (Peqlab) according to the user manual.  <br></li>
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<br></td></tr>
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</ul><br>
 +
<b>V08_27_2 Chemical retransformation of the <i>tar</i>-promoter constructs into <i>E. coli</i> BL21 </b><br>
 +
<ul>
 +
<li>Experiment:  <br>
 +
The retransformation was performed as described in the standard <a href="https://2012.igem.org/Team:Goettingen/Project/Methods">protocol</a>.<br>
 +
<li>Observations & Results: <br>
 +
The retransformation was successful since numerous colonies could be obtained except for the empty negative control. </li>
 +
</ul><br>
 +
<b>V08_27_3 Chemical transformation of different motility increasing constructs into <i>E. coli</i> MG1655</b><br>
 +
<ul>
 +
<li>Experiment:  <br>
 +
We aimed to investigate the effect of our constructs on cells that are used to swim. Therefore competent MG1655 cells were transformed with the following constructs: <br>
 +
<br>
 +
<i>fliC</i> (DH10B) <br>
 +
<i>fliC</i> (<i>Salmonella</i>) <br>
 +
<i>motA</i> <br>
 +
<i>motB</i> <br>
 +
<i>yhjH</i> <br>
 +
<br>
 +
18M-<i>flhDC</i> <br>
 +
18O-<i>flhDC</i> <br>
 +
18C-<i>flhDC</i></li>
 +
<br>
 +
<li>Observations & Results: <br>
 +
The transformation was not successful. The next morning only one plate (the positive control) featured any visible colonies. However, after several additional hours of incubation also some very small colonies appeared at some other plates. Nevertheless, the efficiency was very low. We suggest that this is normal since MG1655 is no cloning strain and might have thus a much lower transformation efficiency. This idea was supported by our inquiries. </li>
 +
<br> </ul>
 +
<b>V08_27_4 Preparation of over night cultures</b><br>
 +
<ul>
 +
<li>Experiment: <br>
 +
In oder to perform a Real-Time Quantitative Reverse Transcription PCR, over night cultures of the <i>tar</i>-promoter constructs in BL21 were prepared.<br></li>
 +
</ul><br>  
 +
 
 +
</ul><br>
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</td></tr>
</table>
</table>
<br>
<br>
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<h2><b>V09_04 </b></h2><br>
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<h2><b>V08_28 </b></h2><br>
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<b>V09_04_1 Test digestion of Psb1c3(including FlHDC under the control of different promoters) with</b><br>
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<b>Preparative double digestion of the <i>flhDC</i>-promoter constructs and pSB1C3</b><br>
<ul>
<ul>
-
<li>Experiment: <br>Test digestion of Psb1c3 including FlHDC under the expression of 8 different promoters with the restriction enzymes EcoRI and PstI. Test digest worked for all clones.</li>
+
<li>Experiment: <br>
-
</ul>
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The flhDC-promoter constructs as well as the vector pSB1C3 still including <i>tar</i> were digested with <i>EcoRI</i> and <i>PstI</i> as described in the <a href="https://2012.igem.org/Team:Goettingen/Project/Methods">protocol</a>. Afterwards, the fragments of the desired size were cut out and frozen in order to extract the DNA from the gel slices the following day. </li>
-
<br>
+
<li>Observations & Results: <br>
-
<b>V09_04_2 Biobrick Standardization of motA, motB and yhjH</b><br>
+
The gel featured clearly visible bands of the expected length and thus the digestion was successful. </li>
-
<ul>
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-
<li>Experiment: <br> PCR of motA, motB and yhjH using primers containing the standard BioBrick sequences. Procedure was succesful and the PCR products were purified via PeqGOLD Gelextraction Kit (Peqlab).</li>
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</ul>
</ul>
<br></td></tr>
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<h2><b>V09_05 </b></h2><br>
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<b>V09_05_1 Double Digest of motA, motB, yhjH and Psb1c3</b><br>
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<h2><b>V08_29 </b></h2><br>
 +
<b>V08_19_1  Purification of the the <i>flhDC</i>-promoter constructs and pSB1C3 </b><br>
<ul>
<ul>
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<li>Experiment: <br> MotA, motB, yhjH and Psb1c3 were digested with EcoRI and PsT1. The prodcuts were seperated via gel electrophoresis and purified using PeqGOLD Gelextraction Kit (Peqlab).</li>
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<li>Experiment: <br>
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The restriction product was purified using peqGOLD Gel Extraction Kit (Peqlab) according to the manual. <br></li>
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<b>V09_05_2 Quickchange of FliC</b><br>
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<b>V08_29_2 Insertion of the <i>flhDC</i>-promoter constructs into pSB1C3</b><br>
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<li>Experiment: <br> Quick Change overlapping PCR was performed using designed primers and Pfu Turbo polymerase (see Quick Change protocol in "protocols"). Was not succesful. We Could not observed any PCR product.</li>
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<li>Experiment: <br>
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The ligation of the digested <i>flhDC</i>-promoter constructs with pSB1C3 was conducted as described in the <a href="https://2012.igem.org/Team:Goettingen/Project/Methods">protocol</a>. <br>
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<h2><b>V09_06 </b></h2><br>
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<h2><b>V08_30 </b></h2><br>
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<b>Ligation and Transformation of motA, motB and yhjH</b><br>
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<b>Miniprep of the <i>flhDC</i>-promoter constructs in pUC18</b><br>
<ul>
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<li>Experiment: <br> After the ligation of motA, motB and yhjH into the plasmid Psb1c3, the ligation products were transformed into the E. coli strain DH10B.</li>
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<li>Experiment: <br>
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In order to obtain further plasmid material Minipreps were conducted using the PeqGOLD MiniPrep Kit (Peqlab) according to the user manual. </li>
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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>
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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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<a href="https://2012.igem.org/Team:Goettingen/Notebook">Notebook</a>;
 
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<a href="https://2012.igem.org/Team:Goettingen/Saftey">Saftey</a>;
 
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<a href="https://2012.igem.org/Team:Goettingen/Attributions">Attributions</a>
 
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Latest revision as of 17:18, 22 September 2012

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#2 Speed Improvement - 18th week

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V08_27


V08_27_1 Miniprep of the tar-promoter constructs
  • Experiment:
    The Minipreps were conducted using the PeqGOLD MiniPrep Kit (Peqlab) according to the user manual.

V08_27_2 Chemical retransformation of the tar-promoter constructs into E. coli BL21
  • Experiment:
    The retransformation was performed as described in the standard protocol.
  • Observations & Results:
    The retransformation was successful since numerous colonies could be obtained except for the empty negative control.

V08_27_3 Chemical transformation of different motility increasing constructs into E. coli MG1655
  • Experiment:
    We aimed to investigate the effect of our constructs on cells that are used to swim. Therefore competent MG1655 cells were transformed with the following constructs:

    fliC (DH10B)
    fliC (Salmonella)
    motA
    motB
    yhjH

    18M-flhDC
    18O-flhDC
    18C-flhDC

  • Observations & Results:
    The transformation was not successful. The next morning only one plate (the positive control) featured any visible colonies. However, after several additional hours of incubation also some very small colonies appeared at some other plates. Nevertheless, the efficiency was very low. We suggest that this is normal since MG1655 is no cloning strain and might have thus a much lower transformation efficiency. This idea was supported by our inquiries.

V08_27_4 Preparation of over night cultures
  • Experiment:
    In oder to perform a Real-Time Quantitative Reverse Transcription PCR, over night cultures of the tar-promoter constructs in BL21 were prepared.



V08_28


Preparative double digestion of the flhDC-promoter constructs and pSB1C3
  • Experiment:
    The flhDC-promoter constructs as well as the vector pSB1C3 still including tar were digested with EcoRI and PstI as described in the protocol. Afterwards, the fragments of the desired size were cut out and frozen in order to extract the DNA from the gel slices the following day.
  • Observations & Results:
    The gel featured clearly visible bands of the expected length and thus the digestion was successful.


V08_29


V08_19_1 Purification of the the flhDC-promoter constructs and pSB1C3
  • Experiment:
    The restriction product was purified using peqGOLD Gel Extraction Kit (Peqlab) according to the manual.

V08_29_2 Insertion of the flhDC-promoter constructs into pSB1C3
  • Experiment:
    The ligation of the digested flhDC-promoter constructs with pSB1C3 was conducted as described in the protocol.


V08_30


Miniprep of the flhDC-promoter constructs in pUC18
  • Experiment:
    In order to obtain further plasmid material Minipreps were conducted using the PeqGOLD MiniPrep Kit (Peqlab) according to the user manual.


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