Team:Warsaw/Calendar-Main/12 August 2008

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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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<h3>Checking for presence of protein A on the cell membrane</h3>
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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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<h4>Emilia</h4>
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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>Inoculation of <A href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-A-alpha>pACYC177+OmpA_A_alpha</a>, <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-Z-alpha>pACYC177+OmpA_Z_alpha</a> and <a href=https://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-omega-A-alpha>pACYC177+OmpA_omega_&Delta;A_alpha</a> (with and without induction).</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>
 
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<h3>Purification of proteins: Z-alpha and Z-omega</h3><h4>Piotr</h4>
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<p><ol><li>Bacteria centrifuged and sonicated (according to <a href=https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#Z_a_Z_o>protocol</a>).</li>
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<li>Sonicate centrifuged. </li>
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<li>Supernatant discarded, pellet resuspended in ddH20, centrifuged and again resuspended in ddH20.</li>
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</ol></p>
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Latest revision as of 19:52, 25 October 2008

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Checking for presence of protein A on the cell membrane

Emilia

Inoculation of pACYC177+OmpA_A_alpha, pACYC177+OmpA_Z_alpha and pACYC177+OmpA_omega_ΔA_alpha (with and without induction).

Purification of proteins: Z-alpha and Z-omega

Piotr

  1. Bacteria centrifuged and sonicated (according to protocol).
  2. Sonicate centrifuged.
  3. Supernatant discarded, pellet resuspended in ddH20, centrifuged and again resuspended in ddH20.

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