Team:Goettingen/week13-2

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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>
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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> on DH10B, BL21, DH5 alpha and XL1 Blue were prepared.<br></li>
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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. </li>
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<h2><b>V07_02 </b></h2><br>
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<b>V07_02_1 Preparative double digestion of 20E-<i>flhDC</i>, 2G-<i>flhDC</i>, 18O-<i>flhDC</i> and pSB1C3</b><br>
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<ul>
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<li>Experiment:  <br>
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In order to clone the <i>flhDC</i>-promoter constructs into pSB1C3, all components were digested with EcoRI and PstI according to the protocol. The restriction products were subsequently separated on a gel in order to verify the digestions success and purify the desired fragments. </li>
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<li>Observations & Results: <br>
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The gel did not feature fragments of the expected size.Obviously, something was wrong with the digestion. </li>
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<br>
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<b>V07_02_2 Preparation of over night cultures</b><br>
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                <li>Experiment:  <br>
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In order to gain more plasmid material of the <i>flhDC</i>-promoter constructs over night cultured were prepared for a subsequent plasmid isolation.<br>
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20E - #2 <br>
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20G - #1 <br>
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2G - #1 <br>
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20I - #2 <br>
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18M - #2 <br>
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18O - #2 <br>
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18C - #2 <br></li>
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<h2><b>V07_25 </b></h2><br>
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<h2><b>V07_03 </b></h2><br>
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<b> Performance of motility assay </b><br>
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<b>V07_03_1 Miniprep of the <i>flhDC</i>-promoter constructs</b><br>
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<ul>
<ul>
<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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LB medium containing ampicillin was inoculated with the over night culture and shaken until an optical density of 0.6-0.8 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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<br>
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<b>V07_03_2 Amplification of the genes <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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The genes <i>fliC</i> (DH10B), <i>fliC</i> (<i>Salmonella</i>), <i>motA</i>, <i>motB</i> and <i>yhjH</i> were amplified by Pfu-Polymerase according to the following protocol. The success of the PCR was subsequently investigated preparing a 1% agarose gel.<br>
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<li>Observations & Results: <br>
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The PCR failed for no bands were visible on the gel whereas the marker was clearly discernible.</li>
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<h2><b>V07_04 </b></h2><br>
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<b> Repetition of the amplification of <i>fliC</i> (DH10B), <i>fliC</i> (<i>Salmonella</i>), <i>motA</i>, <i>motB</i> and <i>yhjH</i> </i></i></b><br>
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<ul>
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<li>Experiment: <br>
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The genes <i>fliC</i> (DH10B), <i>fliC</i> (<i>Salmonella</i>), <i>motA</i>, <i>motB</i> and <i>yhjH</i> were amplified by Pfu-Polymerase according to the following protocol. Additionally, a negative control as well as a positive control were prepared this time. The success of the PCR was subsequently investigated preparing a 1% agarose gel.<br></li>
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<li>Observations & Results: <br>
<li>Observations & Results: <br>
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The did not function again for no bands were visible on the gel; only the marker was clearly discernible. However, since also the positive control did not feature the slightest band, we assume that something might be wrong with our polymerase.</li>
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Here again a certain diversity in the growth rate could be observed. Whereas some cultures had already reached and optical density of 0.8, others were still at merely 0.04. Here again <i>fliC</i> featured a low density, this time in all strains and DH10B and BL21 show a general decreased growth rate. Striking is the fact that the strains that featured the best motility as well as the construct that seems to have the biggest influence show decreased division rate. Whether this phenomenon is a coincidence or not we are not sure about yet.
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At the following day (26th of July) as well as the day after (27th of July) the colonies still looked exactly the same. Neither chemotaxis nor swimming could observed. Almost a weak later (30th of July) still no swimming nor chemotaxis had taken place, solely growth. However, the two colonies that had been applied into the agar not onto featured little swimming. Nevertheless, also these results are lousy considering the long time span. </li>
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<h2><b>V07_05 </b></h2><br>
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<b>V07_05_1 Repetition of the amplification of the genes <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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<li>Experiment: <br>
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To give it a last try the <i>fliC</i> (DH10B), <i>fliC</i> (<i>Salmonella</i>), <i>motA</i>, <i>motB</i> and <i>yhjH</i> were amplified by Pfu-Polymerase according to the following protocol one last time. Here again, a 1% agarose gel was prepared to investigate the PCRs outcome.</li>
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<li>Observations & Results: <br>
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The gel of the PCR delivered the same picture as last time. No band could be observed except for the marker. Even the positive control does not work. Thus, we decided to buy a new polymerase.</li>
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<b>V07_05_2 Repetition of the preparative double digestion of 20G-<i>flhDC</i>, 20I-<i>flhDC</i>, 18K-<i>flhDC</i>, and 18C-<i>flhDC</i></b><br>
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<li>Experiment:  <br>
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The test digestion of the <i>flhDC</i>-promoter constructs EcoRI and PstI  were applied according to the protocol. The restriction products were subsequently separated on a gel in order to verify the digestions success and purify the desired fragments.<br>
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<li>Observations & Results: <br>
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The digestion was successful. Bands of the expected size could be observed and cut out.</li>
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<h2><b>V07_06 </b></h2><br>
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<b> Repetition of the amplification of the <i>fliC</i> (DH10B), <i>fliC</i> (<i>Salmonella</i>), <i>motA</i>, <i>motB</i> and <i>yhjH</i> using the new polymerase </b><br>
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<li>Experiment: <br>
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The genes <i>fliC</i> (DH10B), <i>fliC</i> (<i>Salmonella</i>), <i>motA</i>, <i>motB</i> and <i>yhjH</i> were amplified by Phusion-Polymerase according to the following protocol. We also prepared a negative control as well as a positive control. The success of the PCR was subsequently investigated preparing a 1% analytical agarose gel.<br></li>
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<li>Observations & Results: <br>
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Finally,the PCR was successful! We were able to receive bands of the expected size for each construct. Obviously, the old Pfu-polymerase was not functional anymore and had caused the PCRs failure. </li>
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<b>Important pages</b>:<br>
 
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<a href="https://2012.igem.org/Team:Goettingen/Modeling">Modeling</a>;
 
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<a href="https://2012.igem.org/Team:Goettingen/Notebook">Notebook</a>;
 
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Latest revision as of 17:18, 22 September 2012

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#2 Speed Improvement - 13th week

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V07_23


Preparation of over night cultures
  • Experiment:
    New over night cultures of fliC (DH10B), fliC (Salmonella), motA, motB and yhjH in DH10B, BL21, DH5α and XL1 Blue were prepared.


V07_25


Performance of motility assay
  • Experiment:
    LB medium containing ampicillin was inoculated with the over night culture and shaken until an optical density of 0.6-0.8 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:
    Here again a certain diversity in the growth rate could be observed. Whereas some cultures had already reached and optical density of 0.8, others were still at merely 0.04. Here again fliC featured a low density, this time in all strains and DH10B and BL21 show a general decreased growth rate. Striking is the fact that the strains that featured the best motility as well as the construct that seems to have the biggest influence show decreased division rate. Whether this phenomenon is a coincidence or not we are not sure about yet. At the following day (26th of July) as well as the day after (27th of July) the colonies still looked exactly the same. Neither chemotaxis nor swimming could observed. Almost a weak later (30th of July) still no swimming nor chemotaxis had taken place, solely growth. However, the two colonies that had been applied into the agar not onto featured little swimming. Nevertheless, also these results are lousy considering the long time span.


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