Team:Goettingen/week20-1

From 2012.igem.org

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<h2><b>VX_Y </b></h2><br>
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<b>Titel</b><br>
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<h2><b>09_16 </b></h2><br>
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<b>V09_16_1: BL21 <i>tar</i>-Library selection: xx approach, fourth step: plating of the selected clones</b><br>
<ul>
<ul>
-
<li>Experiment: <br>hier text reinschreiben</li>
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<li>Experiment: <br>In the fourth round of the selection of suitable colones from the BL21 <i>tar</i>-library the fastes colonie was determined and the cells plated on LB-agar</li>
 +
<li>Experimental procedure: <br>View <a href="https://2012.igem.org/Team:Goettingen/Project/Methods">methods</a>.</li>
 +
<li>Observations: <br>
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09_17: Growth was observed on every plate. They were placed in the fridge. No clones were picked for sequencing.</li>
</ul>
</ul>
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<br>
 +
 +
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<b>V09_16_2: Testing of the effect of the promotor strength on the swimming behaviour of the strains Δtar and BL21 with pSB1C3_tar_QC with different promotors</b><br>
 +
<ul>
 +
<li>Experiment: <br>In order to qualtify the swimming behaviour and to find ouf whether the promotors have an effect even without ribosomal binding site the strains Δtar and BL21 with pSB1C3_tar_QC with different promotors are used for a standard swimming/cemotaxis assay</li>
 +
<li>Experimental procedure: 100 µl Aspartate solution on the whatmanpaper was used as an attractant and 0.3 tryptone swimming agar was used. <br>View <a href="https://2012.igem.org/Team:Goettingen/Project/Methods">methods</a>.</li>
 +
<li>Observations: <br>
 +
09_17: No swimming could be observed.</li>
 +
</ul>
 +
<br>
 +
 +
<b>V09_16_3: Swimming behaviour of the unmodified strains MG, BL21; XL blue and DH10B on 0.3% tryptone swimming agar </b><br>
 +
<ul>
 +
<li>Experiment: <br>In order to qualtify the swimming behaviour of the unmodified strains they are placed on swimming agar</li>
 +
<li>Experimental procedure: The strains were placed on 0.3% tryptone swimming agar and M9 agar containing either aspartate, the aminoacid mix or the tryptone solution in the whatmanpaper (concentrations: view <a href="https://2012.igem.org/Team:Goettingen/Project/Materials">materials</a>. The standard swimming/chemotaxis assay was applied, view <a href="https://2012.igem.org/Team:Goettingen/Project/Methods">methods</a>.<br>
 +
<li>Observations: <br>
 +
09_17: No swimming could be observed.</li>
 +
</ul>
 +
<br>
 +
 +
<b>09_16_4: Separation assay, continuation of V09_14_2</b><br>
 +
<ul>
 +
<li>Experiment: <br>One of our goals was to invent es protocol that allows the separation of two different fast strains using the antibiotics marker on an inserted plasmid. The plates prepared at the V09_14_2 were used to test the separation assay. On these plates cultures of the strain Δtar with J2006 expressing rfp (amp resistance) and the strain Δtar with pSB1C3-tar-QC-18C were mixed 1:1 (according to the OD600) and dropped on 0.3% tryptone swimming agar plates containing no anibiotic.</li>
 +
<li>Experimental procedure:<br>
 +
- The agar was cut out at the swimming front using a to the first mark shortened yellow Eppendorf tip respectively. Two different mixed cultures and one containing pSB1C3-tar-QC-18C or J2006 expressing rfp only.<br>
 +
- The tips with the agar pieces were inserted into an e-cup filled with 1 ml LB media respectively<br>
 +
- The cultures were incubated for 1 h at 37 °C with approx. 180 rpm<br>
 +
- A 10^-1 to 10^-4 ditutions series was prepared<br>
 +
- 100 µl of the 10^-2 and the 10^-4 dilution was plated on LB agar plates containing either ampicillin or chloramphenicol respecvtively. The cultures containing pSB1C3-tar-QC-18C or J2006 expressing rfp only were only plated on cm or amp plates. <br>
 +
- the plates were incubated in an 33 °C incubator over night.<br>
 +
<li>Observations: <br>
 +
- Mix #1:<br>
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<div style="text-indent:20px;">Cm: 10<sub>-2</sub> 1284</div>
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<div style="text-indent:20px;">Cm: 10<sub>-4</sub> - </div>
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<div style="text-indent:20px;">Amp: 10<sub>-2</sub> 1460</div>
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<div style="text-indent:20px;">Amp: 10<sub>-4</sub> - </div>
 +
- Mix 2#:<br>
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<div style="text-indent:20px;">Cm: 10<sub>-2</sub> 14</div>
 +
<div style="text-indent:20px;">Cm: 10<sub>-4</sub> 1 </div>
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<div style="text-indent:20px;">Amp: 10<sub>-2</sub> -</div>
 +
<div style="text-indent:20px;">Amp: 10<sub>-4</sub> - </div>
 +
- pSB1C3-tar-QC-18C only<br>
 +
<div style="text-indent:20px;">Cm: 10<sub>-4</sub> 76 </div>
 +
- J2006 expressing rfp only <br>
 +
<div style="text-indent:20px;">Cm: 10<sub>-4</sub> 112</div>
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</li>
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</ul>
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<br>
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<br></td></tr>
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Revision as of 18:36, 24 September 2012

Deutsch  / English 

#1 Selection / Swimming - 20th Week

Back to overview

09_16


V09_16_1: BL21 tar-Library selection: xx approach, fourth step: plating of the selected clones
  • Experiment:
    In the fourth round of the selection of suitable colones from the BL21 tar-library the fastes colonie was determined and the cells plated on LB-agar
  • Experimental procedure:
    View methods.
  • Observations:
    09_17: Growth was observed on every plate. They were placed in the fridge. No clones were picked for sequencing.

V09_16_2: Testing of the effect of the promotor strength on the swimming behaviour of the strains Δtar and BL21 with pSB1C3_tar_QC with different promotors
  • Experiment:
    In order to qualtify the swimming behaviour and to find ouf whether the promotors have an effect even without ribosomal binding site the strains Δtar and BL21 with pSB1C3_tar_QC with different promotors are used for a standard swimming/cemotaxis assay
  • Experimental procedure: 100 µl Aspartate solution on the whatmanpaper was used as an attractant and 0.3 tryptone swimming agar was used.
    View methods.
  • Observations:
    09_17: No swimming could be observed.

V09_16_3: Swimming behaviour of the unmodified strains MG, BL21; XL blue and DH10B on 0.3% tryptone swimming agar
  • Experiment:
    In order to qualtify the swimming behaviour of the unmodified strains they are placed on swimming agar
  • Experimental procedure: The strains were placed on 0.3% tryptone swimming agar and M9 agar containing either aspartate, the aminoacid mix or the tryptone solution in the whatmanpaper (concentrations: view materials. The standard swimming/chemotaxis assay was applied, view methods.
  • Observations:
    09_17: No swimming could be observed.

09_16_4: Separation assay, continuation of V09_14_2
  • Experiment:
    One of our goals was to invent es protocol that allows the separation of two different fast strains using the antibiotics marker on an inserted plasmid. The plates prepared at the V09_14_2 were used to test the separation assay. On these plates cultures of the strain Δtar with J2006 expressing rfp (amp resistance) and the strain Δtar with pSB1C3-tar-QC-18C were mixed 1:1 (according to the OD600) and dropped on 0.3% tryptone swimming agar plates containing no anibiotic.
  • Experimental procedure:
    - The agar was cut out at the swimming front using a to the first mark shortened yellow Eppendorf tip respectively. Two different mixed cultures and one containing pSB1C3-tar-QC-18C or J2006 expressing rfp only.
    - The tips with the agar pieces were inserted into an e-cup filled with 1 ml LB media respectively
    - The cultures were incubated for 1 h at 37 °C with approx. 180 rpm
    - A 10^-1 to 10^-4 ditutions series was prepared
    - 100 µl of the 10^-2 and the 10^-4 dilution was plated on LB agar plates containing either ampicillin or chloramphenicol respecvtively. The cultures containing pSB1C3-tar-QC-18C or J2006 expressing rfp only were only plated on cm or amp plates.
    - the plates were incubated in an 33 °C incubator over night.
  • Observations:
    - Mix #1:
    Cm: 10-2 1284
    Cm: 10-4 -
    Amp: 10-2 1460
    Amp: 10-4 -
    - Mix 2#:
    Cm: 10-2 14
    Cm: 10-4 1
    Amp: 10-2 -
    Amp: 10-4 -
    - pSB1C3-tar-QC-18C only
    Cm: 10-4 76
    - J2006 expressing rfp only
    Cm: 10-4 112



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