Team:Goettingen/week14-1

From 2012.igem.org

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<h2><b>VX_Y </b></h2><br>
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<h2><b>V08_02 </b></h2><br>
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<b>Titel</b><br>
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<b>V08_02_1: Performance of motility assay</b><br>
<ul>
<ul>
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<li>Experiment: <br>hier text reinschreiben</li>
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<li>Experiment: <br>Together with group 2 their constructs FliC (DH10B), FliC (Salmonella), yhjH, MotA, MotB and the control with only puc18 in the different strains DH10B,BL21, DH5α and XL1 Blue were tested. Therefore, LB medium + Ampicillin was inoculated with the over night culteres and grown up to an OD600 of 0,4-0,8. After 4 hours of growth, the cells were spinned down and dropped onto fresh prepared M9-minimal agar plates including amp. The central whatmanpaper was filled with 100 µl Tryptone solution. Bacteria were dropped onto dried plates in a distance of 1 cm at four directions around the central attractant.  Plates were put in an incubator at 37°C.</li>
 +
<li>Observations & Results: <br>No swimming was visible. Most cultures displayed only minor growth. Main problem is probably the inocultion of fresh media in the morning. It was observed, that the strain do not grow very well and especially BL21 does not reach the desired OD600 0,4-0,8 in 4 hours. Also additional nutrients might be required in the M9-minimal media.  </li>
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<br>
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<b>V08_02_2: Overnight cultures of chemotaxis strains</b><br>
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<ul><li>Experiment: <br>5ml LB-medium was inoculated with the fast chemotaxis strains MG1655 and RP437, respectively. </li>
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<h2><b>V08_03 </b></h2><br>
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<b>V08_03_1: Preparation of M9-media</b><br>
 +
<ul>
 +
<li>Experiment: <br>The M9-minimal media was prepared as described in the material section. </li>
 +
<li>Observations & Results: <br>M9-media displayed brownish schlieren. Probably CaCl<sub>2</sub> and MgSO<sub>4</sub> should be sterilfiltrated and added to the autoclaved M9-medium afterwards. </li>
 +
<br></ul>
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<b>V08_03_2: Motility assay with MG1655 and RP437</b><br>
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<ul><li>Experiment: <br>The over night cultures were not used to inoculate fresh LB. Instead, the cells were dropped directly onto 0,3% tryptone swimming plates and M9-plates for chemotaxis (as control also 2 LB-plates were used). 5 µl of each culture was dropped in the center of the tryptone plates and in 1 cm distance to the tryptone attractant on M9 plates, the drops were dried for 5min and put in an incubator at 37°C. The experiment was conducted in duplicates.</li>
 +
<li>Observations & Results: <br>Minor growth and swimming was observed after 2 days at 37°C. One colony of MG1655 showed very strong swimming. Maybe lower incubation temperatures should be tested. </li>
 +
</ul>
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Latest revision as of 14:26, 26 September 2012

Deutsch  / English 

#1 Selection / Swimming - 14th Week

Back to overview

V08_02


V08_02_1: Performance of motility assay
  • Experiment:
    Together with group 2 their constructs FliC (DH10B), FliC (Salmonella), yhjH, MotA, MotB and the control with only puc18 in the different strains DH10B,BL21, DH5α and XL1 Blue were tested. Therefore, LB medium + Ampicillin was inoculated with the over night culteres and grown up to an OD600 of 0,4-0,8. After 4 hours of growth, the cells were spinned down and dropped onto fresh prepared M9-minimal agar plates including amp. The central whatmanpaper was filled with 100 µl Tryptone solution. Bacteria were dropped onto dried plates in a distance of 1 cm at four directions around the central attractant. Plates were put in an incubator at 37°C.
  • Observations & Results:
    No swimming was visible. Most cultures displayed only minor growth. Main problem is probably the inocultion of fresh media in the morning. It was observed, that the strain do not grow very well and especially BL21 does not reach the desired OD600 0,4-0,8 in 4 hours. Also additional nutrients might be required in the M9-minimal media.

V08_02_2: Overnight cultures of chemotaxis strains
  • Experiment:
    5ml LB-medium was inoculated with the fast chemotaxis strains MG1655 and RP437, respectively.

V08_03


V08_03_1: Preparation of M9-media
  • Experiment:
    The M9-minimal media was prepared as described in the material section.
  • Observations & Results:
    M9-media displayed brownish schlieren. Probably CaCl2 and MgSO4 should be sterilfiltrated and added to the autoclaved M9-medium afterwards.

V08_03_2: Motility assay with MG1655 and RP437
  • Experiment:
    The over night cultures were not used to inoculate fresh LB. Instead, the cells were dropped directly onto 0,3% tryptone swimming plates and M9-plates for chemotaxis (as control also 2 LB-plates were used). 5 µl of each culture was dropped in the center of the tryptone plates and in 1 cm distance to the tryptone attractant on M9 plates, the drops were dried for 5min and put in an incubator at 37°C. The experiment was conducted in duplicates.
  • Observations & Results:
    Minor growth and swimming was observed after 2 days at 37°C. One colony of MG1655 showed very strong swimming. Maybe lower incubation temperatures should be tested.


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