Team:Goettingen/week16-2

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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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                                <!-- Text body -->
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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>
-
In order to perform another test using the methionine containing agar and to determine the effect of <i>tar</i> new over night cultures of <i>tar</i>-18C and RFP-18C in &Delta;<i>tar</i> as well as MG1655 and RP437 without any construct were prepared. <br></li>
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In order to perform another test we used methionine containing agar and determined the effect of new over night cultures of <i>tar</i>-18C and RFP-18C in &Delta;<i>tar</i> as well as MG1655 and RP437 without any construct. <br></li>
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<ul>
<ul>
<li>Experiment: <br>
<li>Experiment: <br>
-
This time 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. Here again the over night cultures were dropped onto the plates directly.  <br></li>
+
This time methionine was added to the prepared M9 agar plates and the Whatman Filter Paper were imbued with an amino acid mixture solution as attractant. Here again the over night cultures were dropped onto the plates directly.  <br></li>
<li>Observations & Results: <br>
<li>Observations & Results: <br>
-
The following day (14th August) already swimming could be observed on all plates. In general MG1655 featured the strongest motility, followed by <i>tar</i>-18C. This pattern intensified over the following days, but chemotactic behavior could not clearly be identified at all.</i></ul>
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The following day (14th August) already swimming could be observed on all plates. In general MG1655 featured the strongest motility, followed by <i>tar</i>-18C. This pattern intensified over the following days, but at all no chemotactic behavior could clearly be identified.</i></ul>
<br>
<br>
<b>V08_13_2 Preparation of over night cultures </b><br>
<b>V08_13_2 Preparation of over night cultures </b><br>
<ul>
<ul>
<li>Experiment:  <br>
<li>Experiment:  <br>
-
For we aimed to test <i>fliC</i> (DH10B), <i>fliC</i> (<i>Salmonella</i>), <i>motA</i>, <i>motB</i> and <i>yhjH</i> again, over night cultures of the constructs in <i>E. coli</i> DH10B, BL21, DH5&alpha; and XL1 Blue were prepared. Furthermore, cultures of pUC18 in the different strains as a reference were made.<br>
+
For testing <i>fliC</i> (DH10B), <i>fliC</i> (<i>Salmonella</i>), <i>motA</i>, <i>motB</i> and <i>yhjH</i> again, over night cultures of the constructs in <i>E. coli</i> DH10B, BL21, DH5&alpha; and XL1 Blue were prepared. Furthermore, cultures of pUC18 in the different strains as a reference were made.<br>
</ul><br>
</ul><br>
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<h2><b>V08_16 </b></h2><br>
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<h2><b>V08_02 </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>
-
<b>V08_02_1  Performance of motility assay </b><br>
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<ul>
<ul>
<li>Experiment: <br>
<li>Experiment: <br>
-
LB medium containing ampicillin was inoculated with the over night culture and shaken until an optical density of 0.4-0.6 was reached. Meanwhile ampicillin containing M9 agar plates were prepared and autoclaved Whatman Filter Papers were imbued with tryptone solution. Then the cultures were dropped on the agar at a distance of 1 cm to the paper. On each plate also colonies hosting the empty vector pUC18 were dropped in order to have a reference. Two drops were rather applied into the agar than onto as done before, because some paper predicted faster motility. Also this time each plate was prepared in duplicates.<br></li>
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The <i>flhDC</i>-promoter constructs were digested with <i>XbaI</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>
-
Unfortunately, even after a week no swimming could be observed only growth. We assume that the medium might not provide adequate swimming conditions and therefore we plan to test more motile strains and alter the agar composition if necessary. </li></ul>
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The gel featured clearly visible bands of the expected sizes when irradiated with UV rays.</li>
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<b>V08_02_2 Preparation of over night cultures </b><br>
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<li>Experiment:  <br>
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Since we recently received new <i>E. coli</i> strains that are more motile than our usually used laboratory stains new over night cultures of MG1655 and RP437 were prepared.<br>
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<h2><b>V08_03 </b></h2><br>
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<h2><b>V08_17 </b></h2><br>
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<b> Performance of motility assay </b><br>
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<b> Purification of the <i>flhDC</i>-promoter constructs </b><br>
<ul>
<ul>
<li>Experiment: <br>
<li>Experiment: <br>
-
This time the over night cultures were directly dropped to the swimming plates. In oder to test whether the medium or indeed our strains are responsible for the immobility of the bacteria the habitual M9 agar plates were prepared. However not antibiotics were used since the new strains are not equipped with any resistance yet. Then the autoclaved Whatman Filter Papers were imbued with tryptone solution were placed in the middle of the plate and the cultures were dropped on the agar at a distance of 1 cm to the paper.  Also this time each plate was prepared in duplicates.<br></li>
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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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The following day (4th of August) no motility could be observed. Since both strains are known to move very quick and are supposed to swarm the whole plate during hours we suggest that the medium composition is indeed not suitable. However, two days after preparation of the swimming assay (5th of August) some evidence for incipient swimming was visible, which grew to small halos the following days. Nevertheless, the slow progress confirmed our resolution to reconsider the composition of the plates. </i>
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Latest revision as of 17:18, 22 September 2012

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#2 Speed Improvement - 16th week

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V08_12


Preparation of over night cultures
  • Experiment:
    In order to perform another test we used methionine containing agar and determined the effect of new over night cultures of tar-18C and RFP-18C in Δtar as well as MG1655 and RP437 without any construct.


V08_13


V08_13_1 Performance of motility assay
  • Experiment:
    This time methionine was added to the prepared M9 agar plates and the Whatman Filter Paper were imbued with an amino acid mixture solution as attractant. Here again the over night cultures were dropped onto the plates directly.
  • Observations & Results:
    The following day (14th August) already swimming could be observed on all plates. In general MG1655 featured the strongest motility, followed by tar-18C. This pattern intensified over the following days, but at all no chemotactic behavior could clearly be identified.

V08_13_2 Preparation of over night cultures
  • Experiment:
    For testing fliC (DH10B), fliC (Salmonella), motA, motB and yhjH again, over night cultures of the constructs in E. coli DH10B, BL21, DH5α and XL1 Blue were prepared. Furthermore, cultures of pUC18 in the different strains as a reference were made.


V08_14


Performance of motility assay
  • Experiment:
    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.
  • Observations & Results:
    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 DH5α are rather poor. The strongest effect can be attributed to fliC (especially the gene originating from DH10B) and motB. However, the comparison to the reference pUC18 is for all strains rather disillusioning. Whereas pUC18 lead to rather small halos in comparison to fliC and motB at some plates, on other plates pUC18 caused the biggest ones. Thus, the results cannot unambiguously be evaluated.

V08_15


Preparation of over night cultures
  • Experiment:
    In order to reproduce the motility assay from the day before new over night cultures of fliC (DH10B), fliC (Salmonella), motA, motB, yhjH and pUC18 in E. coli DH10B, BL21, DH5α and XL1 Blue were prepared.
  • Observations & Results:
    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.


V08_16


Preparative double digestion of 18M-flhDC, 18O-flhDC and 18C-flhDC
  • Experiment:
    The flhDC-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.
  • Observations & Results:
    The gel featured clearly visible bands of the expected sizes when irradiated with UV rays.


V08_17


Purification of the flhDC-promoter constructs
  • Experiment:
    The restriction products were purified using peqGOLD Gel Extraction Kit (Peqlab) according to the manual.


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