Team:Goettingen/week19-1
From 2012.igem.org
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- | <h2><b> | + | <h2><b>V09_06 </b></h2><br> |
- | <b> | + | <b>V09_06_1: Observation of the growth of the strains used for EM, BL21 + <i>rfp, +flhDC, +flicC</i>and MG1655</b><br> |
<ul> | <ul> | ||
- | <li>Experiment: <br> | + | <li>Experiment: <br>In order to gain a higher cell density which is necessary of EM, the growth of the cells in different incubation conditions was observed. The cells were grown in 5 mL LB-broth, in 5 mL LB agar topped with 5 mL LB broth, in 10 mL LB-broth and in 10 mL LB agar topped with 10 mL BL-broth.</li> |
+ | <li>Observations: <br> | ||
+ | After 5.5 h an OD600 of over 2 in 10 mL LB agar topped with 10 mL BL-broth can be reached but it is suspected, that the expression of flagella is reduced through the shaking in the erlenmyer flask. Light microscopic test have to follow. | ||
</ul> | </ul> | ||
+ | <br> | ||
+ | |||
+ | <b>V09_06_2: Separation assay</b><br> | ||
+ | <ul> | ||
+ | <li>Experiment: <br>The strains Δ<i>tar</i> J61002_rfp and Δ<i>tar</i> _tar_QC_18C were dropped in the same ratio on 0.3% tryptone swimming agar plates. </a>.</li> | ||
+ | <li>Observations: <br> | ||
+ | Continuation: 09_08_2 | ||
+ | </ul> | ||
+ | <br> | ||
+ | |||
+ | <b>V09_06_3: Selection of clones from the “plating on the chloramphenicol containing LB plates step” for sequencing and observation of the swimming behavior</b><br> | ||
+ | <ul> | ||
+ | <li>Experiment: <br>Clones were selected form the CM plates containing clones form the library. These were again secured on LB agar plates (with CM) and used to inoculate 7 mL LB-broth containing CM..</li> | ||
+ | <li>Observations: <br> | ||
+ | 09_07: all cultures grew, the minipreparation was conducted and the extracted vectors transformed into fresh BL21 cells. | ||
+ | </ul> | ||
+ | <br> | ||
+ | |||
+ | <br></td></tr> | ||
+ | </table> | ||
+ | <br> | ||
+ | <table cellpadding="20 px" border="1" bordercolor="black" valign="top"> | ||
+ | <tr bordercolor="black" valign="top"> | ||
+ | <td width="900" bordercolor="black" valign="top"> | ||
+ | <h2><b>V09_07 </b></h2><br> | ||
+ | <b>V09_07_1: Observation of the swimming behavior of the through the library selection selected clones after they were plates on LB-agar containing chloramphenicol</b><br> | ||
+ | <ul> | ||
+ | <li>Experiment: <br>In order to investigate whether the selected clones containing a library vector still swim with approximately the same speed after they have been plated an LB agar containing chloramphenicol they were dropped again on 0.3 % tryptone swimming agar with the respective attractant and incubated over night at 33 °C</li> | ||
+ | <li>Observations: <br> | ||
+ | 09_08: The clones still swim as fast! | ||
+ | </ul> | ||
+ | <br> | ||
+ | |||
+ | <b>V09_07_2: Retransformation of the extracted plasmids of the selected clones containing the library vectors into fresh BL21 cells</b><br> | ||
+ | <ul> | ||
+ | <li>Experiment: <br>In order to determine whether the observed behavior is dependent on the selected clones or on their vectors, the extracted vectors were transformed into new BL21 cells. View <a href="https://2012.igem.org/Team:Goettingen/Project/Methods">methods</a>.</li> | ||
+ | <li>Observations: <br> | ||
+ | 09_08: growth was observed on each plate! | ||
+ | </ul> | ||
+ | <br> | ||
+ | |||
+ | <b>V09_07_3: Plating of the selected clones of the 3rd approach of the library selection</b><br> | ||
+ | <ul> | ||
+ | <li>Experiment: <br>View <a href="https://2012.igem.org/Team:Goettingen/Project/Methods">methods</a>.</li> | ||
+ | <li>Observations: <br> | ||
+ | 09_08: Growth was observed on every plate, no clones were picked and no vectors were sequenced. | ||
+ | </ul> | ||
+ | <br> | ||
+ | |||
+ | <br></td></tr> | ||
+ | </table> | ||
+ | <br> | ||
+ | |||
+ | <table cellpadding="20 px" border="1" bordercolor="black" valign="top"> | ||
+ | <tr bordercolor="black" valign="top"> | ||
+ | <td width="900" bordercolor="black" valign="top"> | ||
+ | <h2><b>V09_08 </b></h2><br> | ||
+ | <b>V09_08_1: Swimming behavior of the library: selected clones versus retransformed clones</b><br> | ||
+ | <ul> | ||
+ | <li>Experiment: <br>Continuation of 09_07_1. Growth could be observed on every plate, single colonies were picked and used to inoculate 1 ml LB broth. Cultures were shaken for approximately 3 h.</li> | ||
+ | <li>Observations: <br> | ||
+ | 09_09: there is a huge difference between the swimming behavior of the selected clones and the retransformed clones. | ||
+ | </ul> | ||
+ | <br> | ||
+ | |||
+ | <b>V09_08_2: Separation assay</b><br> | ||
+ | <ul> | ||
+ | <li>Experiment: <br>Separation of the strains Δ<i>tar</i> J61002_rfp and Δ<i>tar</i> _tar_QC_18C. The agar was cut out at three different positions and the pieces incubated in 1 mL LB broth for approximately 3 h. The cultures were diluted and the 10^-2 and the 10^-4 dilution was plated on plates containing either ampicillin of chloramphenicol. 3 dopes were treated as described above. </li> | ||
+ | <li>Observations: <br> | ||
+ | 09_09: In one of three cases (one not evaluable) the Δ<i>tar</i> _tar_QC_18C strain was faster. | ||
+ | </ul> | ||
+ | <br> | ||
+ | |||
+ | |||
<br></td></tr> | <br></td></tr> | ||
</table> | </table> | ||
+ | |||
Revision as of 10:40, 26 September 2012
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#1 Selection / Swimming - 19th WeekBack to overview
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