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| - | <center><h4>Checking if degradation of fusion with OmpA is a result of Top10 proteases' activity (lon, iompt)</center>Piotr:</h4>
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| - | <br>
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| - | Inoculation of OmpA_omega_A_alpha and OmpA_A_alpha with and without inductor (0,5 mm/mL IPTG) in <i>E. coli</i> Rosetta strain.
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| - | <p>1. Isolation of plasmids from cultures inocluated on previous day.<br>
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| - | 2. Control digest of isolated plasmids with BamHI and SacI (we confirmed 4 colonies but A in our constructs turned out to be trunctated and contain only one from two highly similar domains).
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| - | </p>
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| - | <h3>Checking if OmpA_omega_A_alpha gives ampicillin resistance<br> | + | <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> <h4>Piotr:</h4> |
| - | Piotr</h3>
| + | <p><ol><li> |
| - | | + | Inoculation of <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-omega-A-alpha>OmpA_omega_ΔA_alpha</a> and <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-A-alpha>OmpA_A_alpha</a> with and without inductor (0,5 mm/mL IPTG) in <i>E. coli</i> Rosetta strain. </li> |
| - | <p>Inoculation OmpA_omega_A_alpha from various IPTG concentrations (in mmol/mL) (0, 0.1, 0.25, 0.5, 0.75, 1) into same IPTG concentrations, but with various ampicillin concentrations (in mmol/mL)(25, 50, 75, 100) in ratio: 1:50.</p>
| + | <li> |
| - | | + | 5 hours culture</li> |
| - | <center><h4 style="text-align: center">Measurement of bacterial culture growth (OD) in the evening:</h4> </center>
| + | <li>Bacteria centrifuged and frozen. |
| - | | + | </li></ol> |
| - | | + | |
| - | <table id="result" align="center">
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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">1.558</td><td class="live">1.469</td><td class="live">1.587</td><td class="live">1.49</td><td class="live">1.566</td><td class="live">1.311</td></tr>
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| - | <tr><th>50</th><td class="live">1.425</td><td class="live">1.435</td><td class="live">1.524</td><td class="live">1.055</td><td class="live">0.920</td><td class="live">0.935</td></tr> | + | |
| - | <tr><th>75</th><td class="live">1.09</td><td class="live">0.989</td><td class="live">1.447</td><td class="live">0.971</td><td class="live">0.951</td><td class="live">0.992</td></tr> | + | |
| - | <tr><th>100</th><td class="live">0.09</td><td class="live">0.685</td><td class="live">1.378</td><td class="live">1.078</td><td class="live">0.977</td><td class="live">0.992</td></tr>
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| - | | + | |
| - | </table> | + | |
| - | <p>
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| - | Inoculation of OmpA_omega_A_alpha into various IPTG concentrations: 0, 0.1, 0.25, 0.5, 0.75 mmol/mL (replication is necessary)</p>
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| - | | + | |
| - | <h3>Checking OmpA_omega_A_alpha and OmpA_A_alpha expression<br>
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| - | Piotr</h3>
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| - | <ol>
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| - | <li>Spinning</li>
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| - | <li>Suspending</li> | + | |
| - | <li>Adding of lysis buffer</li>
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| - | <li>Boiling</li> | + | |
| - | <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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| - | | + | |
| - | [a photo of the gel is top be put here]
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| - | <h4>Michał L., Ewa, Marcin</h4>
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| - | <p>
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| - | <ol>
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| - | <li>Separate transformant colonies (transformation from previous day) inoculated to liquid LB with kanamycin. </li>
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| - | <li>Inoculation of pACYC177+OmpA_alpha and pACYC177+OmpA_omega - liquid LB with kanamycin.</li></ol>
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| | </p> | | </p> |
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| - | <h3>Checking for presence of A on the cell membrane</h3>
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| - | <h4>Piotr</h4>
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| - | <p>Inoculation of omp_A_alfa, omp_Z_alfa and omp_omega_A_alfa (with and without induction).</p>
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