Team:Goettingen/week17-2

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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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<ul>
<ul>
<li>Experiment: <br>
<li>Experiment: <br>
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The vector pSB1C3 in its linearized as well as in its circularized form was 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. <br></li>
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The vector pSB1C3 in its linearized as well as in its circularized form was 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. <br></li>
<li>Observations & Results: <br>
<li>Observations & Results: <br>
The gel featured bands of the expected sizes when irradiated with UV rays.</li>
The gel featured bands of the expected sizes when irradiated with UV rays.</li>
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<li>Experiment:  <br>
<li>Experiment:  <br>
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For the chemical transformation the standard protocol was followed.<br></li>
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Since we aim to investigate the constructs using Real-Time Quantitative Reverse Transcription PCR, competent BL21 cells were chemically transformed with the ligation products according to the standard <a href="https://2012.igem.org/Team:Goettingen/Project/Methods">protocol</a>.<br></li>
<li>Observations & Results: <br>
<li>Observations & Results: <br>
The transformation was successful since numerous colonies were obtained except for the negative control.
The transformation was successful since numerous colonies were obtained except for the negative control.
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</ul><b>V08_24_3  Miniprep of 18C-<i>flhDC</i></b><br>
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<h2><b>V08_14 </b></h2><br>
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<b> Performance of motility assay  </b><br>
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<li>Experiment: <br>
<li>Experiment: <br>
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Here again methionine was added to the prepared M9 agar plates and the Whatman Filter Paper were imbued with an amino acid mixture solution as an attractant. The over night cultures were dropped onto the plates directly.  <br></li>
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In order to gain further plasmid material Minipreps were conducted using the PeqGOLD MiniPrep Kit (Peqlab) according to the user manual.
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<li>Observations & Results: <br>
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<br></li>
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Two days after motility assay preparation (16th of August) first stirrings of swimming were visible. In general BL21 shows the strongest motility followed by DH10B. XL1 Blue and  DH5&alpha; are rather poor. The strongest effect can be attributed to <i>fliC</i> (especially the gene originating from DH10B) and <i>motB</i>. However, the comparison to the reference pUC18 is for all strains rather disillusioning. Whereas pUC18 lead to rather small halos in comparison to <i>fliC</i> and <i>motB</i> at some plates, on other plates pUC18 caused the biggest ones. Thus, the results cannot unambiguously be evaluated.  
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<b>V08_24_4 Preparation of chemically competent cells of MG1655</b><br>
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<h2><b>V08_15 </b></h2><br>
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In order to reproduce the motility assay from the day before new over night cultures of <i>fliC</i> (DH10B), <i>fliC</i> (<i>Salmonella</i>), <i>motA</i>, <i>motB</i>, <i>yhjH</i> and pUC18 in <i>E. coli</i> DH10B, BL21, DH5&alpha; and XL1 Blue were prepared. <br></li>
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The preparation of the competent cells was performed according to the following <a href="https://2012.igem.org/Team:Goettingen/Project/Methods">protocol</a>.<br></li>
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The growing of the culture again posed a problem to us since some cultures grew quite nice, whereas others multiplied only poorly to not at all. Therefore, we were not able to conduct the assay.</li>
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<h2><b>V08_26 </b></h2><br>
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<b> Preparative double digestion of 18M-<i>flhDC</i>, 18O-<i>flhDC</i> and 18C-<i>flhDC</i> </b><br>
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<b> Preparation of overnight cultures </b><br>
<ul>
<ul>
<li>Experiment: <br>
<li>Experiment: <br>
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The <i>flhDC</i>-promoter constructs were digested with XbaI 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. <br></li>
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In order isolate the plasmids for a subsequent retransformation of correct vectors over night cultures of the eight <i>tar</i>-promoter constructs were prepared. <br></li>
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The gel featured clearly visible bands of the expected sizes when irradiated with UV rays.</li>
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<h2><b>V08_17 </b></h2><br>
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<b> Purification of the <i>flhDC</i>-promoter constructs </b><br>
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<li>Experiment: <br>
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The restriction products were purified using peqGOLD Gel Extraction Kit (Peqlab) according to the manual.<br></li>
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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/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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Latest revision as of 17:18, 22 September 2012

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#2 Speed Improvement - 17th week

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V08_20


Preparative double digestion of pSB1C3
  • Experiment:
    The vector pSB1C3 in its linearized as well as in its circularized form was 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 bands of the expected sizes when irradiated with UV rays.


V08_21


Purification of the flhDC-promoter constructs
  • Experiment:
    The restricted vector was purified using peqGOLD Gel Extraction Kit (Peqlab) according to the manual.
  • Observations & Results:
    After the purification the DNA concentration was measured with the NanoDrop according to which our samples did not contain any DNA. This is also right for the purified flhDC-promoter constructs. Since NanoDrops are not very accurate, the samples were additionally loaded on a 1% agarose gel, which also did not show any bands expect for those of the marker. Since we already have had problems to receive proper DNA concentration with our purification kit and both preparative gels showed the expected band, we assume that the DNA got lost during purification.


V08_22


Chemical transformation of the eight flhDC-promoter constructs into E. coli BL21
  • Experiment:
    In order to receive fresh colonies for the subsequent preparation of glycerol stocks competent E. coli BL21 cells were transformed with the eight flhDC-promoter constructs. The other strains were neglected for BL21 showed the strongest susceptibility to inserted constructs.
  • Observations & Results:
    The transformation was successful since all plates showed numerous colonies except for the negative control.


V08_23


Preparation of overnight cultures
  • Experiment:
    In order to prepare the glycerol stocks over night cultures of the eight flhDC-promoter constructs in E. coli BL21 were prepared. Additionally, over night cultures of MG1655 were prepared for a subsequent preparation of competent cells.


V08_24


V08_24_1 Preparation of glycerol stocks of the eight flhDC-promoter constructs in E. coliBL21 and the mutation library
  • Experiment:
    The glycerol stocks were prepared according to the following protocol. Furthermore, glycerol stock of the mutation library constructed by group 3 were prepared.

V08_24_2 Chemical transformation of the tar-promoter constructs into E. coli BL21
  • Experiment:
    Since we aim to investigate the constructs using Real-Time Quantitative Reverse Transcription PCR, competent BL21 cells were chemically transformed with the ligation products according to the standard protocol.
  • Observations & Results:
    The transformation was successful since numerous colonies were obtained except for the negative control.

V08_24_3 Miniprep of 18C-flhDC
  • Experiment:
    In order to gain further plasmid material Minipreps were conducted using the PeqGOLD MiniPrep Kit (Peqlab) according to the user manual.

V08_24_4 Preparation of chemically competent cells of MG1655
  • Experiment:
    The preparation of the competent cells was performed according to the following protocol.


V08_26


Preparation of overnight cultures
  • Experiment:
    In order isolate the plasmids for a subsequent retransformation of correct vectors over night cultures of the eight tar-promoter constructs were prepared.


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