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Team:Warsaw/Calendar-Main/30 July 2008
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| - | + | <li>Isolation of plasmids from cultures inocluated on previous day.</li> | |
| + | <li>Control <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#digest">digest</a> of isolated plasmids (pACYC177+omega_deltaA) with HindIII and SacI (7 from 8 colonies confirmed). </li></ol></p> | ||
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| - | + | <h3>Checking if OmpA_omega_A_alpha gives ampicillin resistance <br> | |
| + | Piotr</h3> | ||
| - | Bacterial growth (OD) measurement in the evening: | + | <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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| + | <center><h4>Bacterial growth (OD) measurement in the evening: </h4></center> | ||
<table id="result" align="center"> | <table id="result" align="center"> | ||
<tr><th rowspan="2">ampicillin concentration (μg/mL):</th><th colspan="6">IPTG concentration (mmol/mL):</td></tr> | <tr><th rowspan="2">ampicillin concentration (μg/mL):</th><th colspan="6">IPTG concentration (mmol/mL):</td></tr> | ||
| - | <tr><th>0</th><th>0 | + | <tr><th>0</th><th>0.1</th><th>0.25</th><th>0.5</th><th>0.75</th><th>1</th></tr> |
| - | <tr><th>25</th><td class="live">1 | + | <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> |
| - | <tr><th>50</th><td class="live">0 | + | <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> |
| - | <tr><th>75</th><td class="live">0.5</td><td class="live">0 | + | <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> |
| - | <tr><th>100</th><td class="live">0.02</td><td class="live">0 | + | <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> |
</table> | </table> | ||
| - | + | <p>Conclusion: we obtained the best induction using 0.5 - 0.25 mM IPTG. Best ampicillin concentration is 50 - 75 μg/ml.</p> | |
| + | <h3> | ||
| + | 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> | ||
| + | Piotr</h3> | ||
| + | <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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| - | Transformation omega_A_alpha and A_alpha | + | <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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Checking if OmpA_omega_A_alpha gives ampicillin resistance
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| ampicillin concentration (μg/mL): | IPTG concentration (mmol/mL): | |||||
|---|---|---|---|---|---|---|
| 0 | 0.1 | 0.25 | 0.5 | 0.75 | 1 | |
| 25 | 1.054 | 1.154 | 1.051 | 0.99 | 1.096 | 0.896 |
| 50 | 0.94 | 0.891 | 1.123 | 0.847 | 0.924 | 0,81 |
| 75 | 0.5 | 0.63 | 0.743 | 0.782 | 0.631 | 0.64 |
| 100 | 0.02 | 0.396 | 0.563 | 0.678 | 0.602 | 0.611 |
Conclusion: we obtained the best induction using 0.5 - 0.25 mM IPTG. Best ampicillin concentration is 50 - 75 μg/ml.
Checking if degradation of fusion with OmpA is a result of the activity of lon and iompt proteases. (present in top10)
Piotr
Test was conducted in E.coli Rosetta strain expressing omp_omega_A_alfa (with and without induction) and omp_A_alfa.
Transformation of Rosettas with omega_A_alpha and A_alpha.
