Team:Warsaw/Calendar-Main/6 August 2008

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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>
<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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<h3>Checking if degradation of fusion with OmpA is a result of the activity of 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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Checking if degradation of fusion with OmpA is a result of the activity of 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>
<h4>Piotr</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 omp_omega_A_alfa (with and without induction) and omp_A_alfa. </p>
<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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<h3>Preparing pACYC177+OmpA_omega_deltaA construct<h4>Michał K.</h4>
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<h3>Preparing pACYC177+OmpA_omega_deltaA construct</h3><h4>Michał K.</h4>
<p> Inoculation of cultures from colonies grown on plate with pACYC177+OmpA_omega_deltaA transformation. </p>  
<p> Inoculation of cultures from colonies grown on plate with pACYC177+OmpA_omega_deltaA transformation. </p>  

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Checking if OmpA_omega_A_alpha gives ampicillin resistance

Piotr

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.

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 if degradation of fusion with OmpA is a result of the activity of lon and ompt 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.

Preparing pACYC177+OmpA_omega_deltaA construct

Michał K.

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