Team:Warsaw/Calendar-Main/1 August 2008

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<h3>Checking if OmpA_omega_A_alpha gives ampicillin resistance </h3>
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<h3> Cloning of truncated fragment of protein A (&Delta;A)</h3>  
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<h4>Piotr</h4>
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<h4>Michał K.</h4>
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<p>Inoculation of OmpA_omega_A_alpha from various IPTG concentrations: 0, 0.1, 0.25, 0.5, 0.75, 1 mM into same IPTG concentrations, but with various ampicillin concentrations (25, 50, 75, 100 mM) in ratio 1:50.</p>
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<table id="result" align="center">
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<thead>
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<b>Bacterial growth (OD) measurement in the evening:</b>
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</thead>
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<tr><th rowspan="2">ampicillin concentration (μg/mL):</th><th colspan="6">IPTG concentration (mmol/mL):</td></tr>
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<tr><th>0</th><th>0.1</th><th>0.25</th><th>0.5</th><th>0.75</th><th>1</th></tr>
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<tr><th>25</th><td class="live">1.054</td><td class="live">1.154</td><td class="live">1.051</td><td class="live">0.99</td><td class="live">1.096</td><td class="live">0.896</td></tr>
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<tr><th>50</th><td class="live">0.94</td><td class="live">0.891</td><td class="live">1.123</td><td class="live">0.847</td><td class="live">0.924</td><td class="live">0,81</td></tr>
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<tr><th>75</th><td class="live">0.5</td><td class="live">0.63</td><td class="live">0.743</td><td class="live">0.782</td><td class="live">0.631</td><td class="live">0.64</td></tr>
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<tr><th>100</th><td class="live">0.02</td><td class="live">0.396</td><td class="live">0.563</td><td class="live">0.678</td><td class="live">0.602</td><td class="live">0.611</td></tr>
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</table>
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<p>Conclusion: we obtained the best induction using 0.5 - 0.25 mM IPTG. Best ampicillin concentration is 50 - 75 μg/ml.</p>
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<h3>
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Checking if degradation of fusion with OmpA is a result of the activity of lon and iompt proteases. (present in <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#top10">top10</a>)<br>
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Piotr</h3>
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<p>Test was conducted in <i>E.coli</i> <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#rosetta">Rosetta</a> strain expressing omp_omega_A_alfa (with and without induction) and omp_A_alfa. </p>
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<p>Transformation of <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#rosetta">Rosettas</a> with omega_A_alpha and A_alpha.</p>
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<h4> Michał K.</h4>
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<p><ol>
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<li> <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#chemotransform">Transformation</a> of <i>E. coli</i> <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#top">TOP10</a> strain with ligation products (pACYC177+OmpA_alpha + deltaA and pACYC177+OmpA_omega + deltaA). </li>
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<li> Transformants plating on LB + kanamycin. </li></ol></p>
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<h3>Checking whether degradation of the fusions with OmpA is caused by Lon and OmpT proteases (present in <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#top10">TOP10</a>)</h3>
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<h4>Piotr</h4>
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<p>
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Test was conducted in <i>E.coli</i> <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#rosetta">Rosetta</a> strain expressing omp_omega_A_alfa (with and without induction) and omp_A_alfa.
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<ol>
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<li>Spinnign</li>
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<li> Colony <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#pcr">PCR</a> on <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-deltaA-alpha>pACYC177+OmpA_alpha + &Delta;A</a> and <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BOmpA-deltaA-omega>pACYC177+OmpA_omega + &Delta;A</a> colonies of tranformants.</li>
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<li>Suspending</li>
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<li> Gel electrophoresis of PCR products (<a href="https://2008.igem.org/Team:Warsaw/Calendar-Main/1_August_2008#fig1">Fig. 1.</a>).  </li>
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<li>Adding of lysis buffer</li>
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<li>Boiling</li>
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<li>Putting into poliacrylamide gel</li>
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<li>Transfer onto nitrocellulose</li>
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<li>Blocking</li>
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<li>Anti-A antibody binding</li>
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<li>Washing</li>
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<li>Anti-rabbit antibody binding</li>
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<li>Developing with BCIP and NBT</li>
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</ol>
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</p>
 
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[photo of the gel is to be placed here]
 
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<p>We didn't observe differences in espression and degradation in <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#rosetta">Rosettas</a> nor in <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#top10">TOP10</a>. Therefore we suppose that degradation of the fusions is caused by other factor than Lon and OmpT proteases.</p>
 
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<a name="fig1"><img src="https://static.igem.org/mediawiki/2008/7/7c/Cos.jpg" width=300/></a> <var><b>Fig. 1.</b> Colony PCR to find proper clones of pACYC177_OmpA_alpha+&Delta;A and pACYC177_OmpA_omega+&Delta;A<br>
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Upper gel:<br>
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1. Marker<br>
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2-13. colony PCR products to find proper clone of pACYC177_OmpA_alpha+&Delta;A <br>
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Lower gel:<br>
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1. Marker<br>
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2-11. colony PCR products to find proper clone of pACYC177_OmpA_omega+&Delta;A<br>
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12. positive control - pACYC_OmpA_A_alpha<br>
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13. negative control - pACYC_OmpA_Z_alpha<br></var>

Latest revision as of 18:42, 26 October 2008

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Cloning of truncated fragment of protein A (ΔA)

Michał K.

  1. Colony PCR on pACYC177+OmpA_alpha + ΔA and pACYC177+OmpA_omega + ΔA colonies of tranformants.
  2. Gel electrophoresis of PCR products (Fig. 1.).

Fig. 1. Colony PCR to find proper clones of pACYC177_OmpA_alpha+ΔA and pACYC177_OmpA_omega+ΔA
Upper gel:
1. Marker
2-13. colony PCR products to find proper clone of pACYC177_OmpA_alpha+ΔA
Lower gel:
1. Marker
2-11. colony PCR products to find proper clone of pACYC177_OmpA_omega+ΔA
12. positive control - pACYC_OmpA_A_alpha
13. negative control - pACYC_OmpA_Z_alpha



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