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_&Delta;A_alpha</a> gives ampicillin resistance </h3>
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<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_&Delta;A_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">
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<b>Bacterial growth (OD) measurement in the evening:</b>
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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>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>Emilia</h4>
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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 <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-omega-A-alpha>ompA_omega_&Delta;A_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_&Delta;A_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_&Delta;A</a> construct</h3><h4>Michał K.</h4>
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<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_&Delta;A</a> transformation. </p>
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</html>

Latest revision as of 16:47, 28 October 2008

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Checking if OmpA_omega_ΔA_alpha gives ampicillin resistance

Piotr, Weronika

Repetition 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.

Bacterial growth (OD) measurement in the evening:
ampicillin concentration (μg/mL):IPTG concentration (mmol/mL):
00.10.250.50.751
251.0541.1541.0510.991.0960.896
500.940.8911.1230.8470.9240,81
750.50.630.7430.7820.6310.64
1000.020.3960.5630.6780.6020.611

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)

Emilia

Test 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 construct

Michał K.

Inoculation of cultures from colonies grown on plate with pACYC177+OmpA_omega_ΔA transformation.


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