Team:Warsaw/Calendar-Main/8 August 2008

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<h3>Checking if degradation of fusion with OmpA is a result of Top10 proteases' activity (lon, ompt) </h3> <h4>Piotr:</h4>
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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>Piotr:</h4>
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<p><ol><li>
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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. </p>
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Inoculation of <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> with and without inductor (0,5 mm/mL IPTG) in <i>E. coli</i> Rosetta strain. </li>
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5 hours culture</li>
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<li>Bacteria centrifuged and frozen.
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</li></ol>
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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 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>
 

Latest revision as of 20:26, 26 October 2008

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Checking whether degradation of the fusions with OmpA is caused by Lon and OmpT proteases (present in TOP10)

Piotr:

  1. Inoculation of OmpA_omega_ΔA_alpha and OmpA_A_alpha with and without inductor (0,5 mm/mL IPTG) in E. coli Rosetta strain.
  2. 5 hours culture
  3. Bacteria centrifuged and frozen.




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