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Team:Warsaw/Calendar-Main/6 August 2008
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| - | < | + | <h3>Checking if <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-omega-A-alpha>OmpA_omega_ΔA_alpha</a> gives ampicillin resistance </h3> |
| + | <h4>Piotr, Weronika</h4> | ||
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| + | <p>Repetition of the test from previous day. Inoculation of <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-omega-A-alpha>OmpA_omega_ΔA_alpha</a> 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"> | ||
| + | <thead> | ||
| + | <b>Bacterial growth (OD) measurement in the evening:</b> | ||
| + | </thead> | ||
| + | <tr><th rowspan="2">ampicillin concentration (μg/mL):</th><th colspan="6">IPTG concentration (mmol/mL):</td></tr> | ||
| + | <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">1.054</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">0.94</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">0.63</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">0.396</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> | ||
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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>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>Emilia</h4> | ||
| + | <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 <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-omega-A-alpha>ompA_omega_ΔA_alpha</a> (with and without induction) and <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-A-alpha>ompA_A_alpha</a>. </p> | ||
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| + | <p>Transformation of <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#rosetta">Rosettas</a> with <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>.</p> | ||
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| + | <h3>Preparing <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-omega-deltaA>pACYC177+OmpA_omega_ΔA</a> construct</h3><h4>Michał K.</h4> | ||
| + | <p> Inoculation of cultures from colonies grown on plate with <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-omega-deltaA>pACYC177+OmpA_omega_ΔA</a> transformation. </p> | ||
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Checking if OmpA_omega_ΔA_alpha gives ampicillin resistancePiotr, WeronikaRepetition of the test from previous day. Inoculation of OmpA_omega_ΔA_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.
Conclusion: we obtained the best induction using 0.5 - 0.25 mM IPTG. Best ampicillin concentration is 50 - 75 μg/ml. Checking whether degradation of the fusions with OmpA is caused by Lon and OmpT proteases (present in TOP10)EmiliaTest was conducted in E.coli Rosetta strain expressing ompA_omega_ΔA_alpha (with and without induction) and ompA_A_alpha. Transformation of Rosettas with OmpA_omega_ΔA_alpha and OmpA_A_alpha. Preparing pACYC177+OmpA_omega_ΔA constructMichał K.Inoculation of cultures from colonies grown on plate with pACYC177+OmpA_omega_ΔA transformation.
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