Team:Goettingen/week14-2

From 2012.igem.org

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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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<h2><b>V07_09 </b></h2><br>
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<h2><b>V07_30 </b></h2><br>
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<b> Purification of <i>fliC</i> (DH10B), <i>fliC</i> (<i>Salmonella</i>), <i>motA</i>, <i>motB</i> and <i>yhjH</i>  </b><br>
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<b> Preparation of over night cultures
 +
</b><br>
<ul>
<ul>
<li>Experiment: <br>
<li>Experiment: <br>
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The amplicons were separated via application on an 1% agarose gel and subsequently cut out. The DNA was purified out of the gel slices using peqGOLD Gel Extraction Kit (Peqlab) according to the user manual. <br></li>
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In order to perform a further swimming assay new over night cultures of <i>fliC</i> (DH10B), <i>fliC</i> (<i>Salmonella</i>), <i>motA</i>, <i>motB</i> and <i>yhjH</i> in DH10B, BL21, DH5&alpha; and XL1 Blue were prepared. <br></li>
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<h2><b>V08_01 </b></h2><br>
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<h2><b>V07_10 </b></h2><br>
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<b> Preparation of over night cultures </b><br>
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<b>V07_10_1 Preparative double digestion of <i>fliC</i> (DH10B), <i>fliC</i> (<i>Salmonella</i>), <i>motA</i>, <i>motB</i>, <i>yhjH</i> and pUC18 </b><br>
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<ul>
<ul>
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<li>Experiment: <br>
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In order to clone the new genes into pUC18, all components were digested with EcoRI and XbaI according to the protocol. The restricted plasmid pUC18 was subsequently separated on a gel in order to verify the digestions success and purify the desired fragments. The other digestion products were investigated via preparation of an 1% analytical gel and directly purified using the peqGOLD Gel Extraction Kit (Peqlab) according to the manual as well. </li>
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Since some problems with the growing of the cultures appeared new over night cultures of <i>fliC</i> (DH10B), <i>fliC</i> (<i>Salmonella</i>), <i>motA</i>, <i>motB</i> and <i>yhjH</i> in DH10B, BL21, DH5&alpha; and XL1 Blue were prepared. <br></li>
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<li>Observations & Results: <br>
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The digestion was successful. For all PCR products as well as for the vector band of the expected size could be obtained and the fragments can now be used for further cloning. </li>
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        </ul>
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<b>V07_10_2 Insertion of <i>fliC</i> (DH10B), <i>fliC</i> (<i>Salmonella</i>), <i>motA</i>, <i>motB</i> and <i>yhjH</i> into pUC18</b><br>
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                <li>Experiment:  <br>
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The ligation of the digested genes with the plasmid pUC18 was conducted as described in the protocol.
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<h2><b>V07_11 </b></h2><br>
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<b> Chemical transformation of <i>fliC</i> (DH10B), <i>fliC</i> (<i>Salmonella</i>), <i>motA</i>, <i>motB</i> and <i>yhjH</i> into <i>E. coli</i> (DH10B)  </b><br>
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<h2><b>V08_02 </b></h2><br>
 +
<b>V08_02_1  Performance of motility assay </b><br>
<ul>
<ul>
<li>Experiment: <br>
<li>Experiment: <br>
-
For the chemical transformation the standard protocol was followed. <br></li>
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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>
<li>Observations & Results: <br>
<li>Observations & Results: <br>
-
The transformation was successful since all plates showed numeours colonoes except the negative control. </li>
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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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<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>V07_12 </b></h2><br>
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<b> Preparation of over night cultures</b><br>
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Over night cultures of <i>fliC</i> (DH10B), <i>fliC</i> (<i>Salmonella</i>), <i>motA</i>, <i>motB</i> and <i>yhjH</i> in pUC18 were prepared in order to isolate the plasmids. <br></li>
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<h2><b>V08_03 </b></h2><br>
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<h2><b>V07_13 </b></h2><br>
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<b> Performance of motility assay </b><br>
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<b>V07_13_1 Miniprep of <i>fliC</i> (DH10B), <i>fliC</i> (<i>Salmonella</i>), <i>motA</i>, <i>motB</i> and <i>yhjH</i> in pUC18</b><br>
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<li>Experiment: <br>
<li>Experiment: <br>
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Minipreps were conducted with PeqGOLD MiniPrep Kit (Peqlab) according to the user manual.</li></ul>
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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 no 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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<b>V07_13_2 Test digestion of <i>fliC</i> (DH10B), <i>fliC</i> (<i>Salmonella</i>), <i>motA</i>, <i>motB</i> and <i>yhjH</i> in pUC18</b><br>
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<li>Experiment:  <br>
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All vectors were digested with EcoRI and XbaI according to the protocol. The restriction products were subsequently separated on a gel in order to determine successfully ligated plasmids.<br>
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<li>Observations & Results: <br>
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Except for <i>fliC</i> (Salmonella) #1 all picked colonies seem to host plasmids containing the aimed insert. </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 - 14th week

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V07_30


Preparation of over night cultures
  • Experiment:
    In order to perform a further swimming assay new over night cultures of fliC (DH10B), fliC (Salmonella), motA, motB and yhjH in DH10B, BL21, DH5α and XL1 Blue were prepared.


V08_01


Preparation of over night cultures
  • Experiment:
    Since some problems with the growing of the cultures appeared new over night cultures of fliC (DH10B), fliC (Salmonella), motA, motB and yhjH in DH10B, BL21, DH5α and XL1 Blue were prepared.


V08_02


V08_02_1 Performance of motility assay
  • Experiment:
    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.
  • Observations & Results:
    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.

V08_02_2 Preparation of over night cultures
  • Experiment:
    Since we recently received new E. coli strains that are more motile than our usually used laboratory stains new over night cultures of MG1655 and RP437 were prepared.


V08_03


Performance of motility assay
  • Experiment:
    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 no 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.
  • Observations & Results:
    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.

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