Team:Warsaw/Calendar-Main/1 August 2008

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<h4> Michał K.</h4>
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<li> <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#chemotransform">Transformation</a> of <i>E. coli</i> <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#top">TOP10</a> strain with ligations (pACYC177+OmpA_alpha + deltaA and pACYC177+OmpA_omega + deltaA). </li>
<li> <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#chemotransform">Transformation</a> of <i>E. coli</i> <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#top">TOP10</a> strain with ligations (pACYC177+OmpA_alpha + deltaA and pACYC177+OmpA_omega + deltaA). </li>
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<h3>Checking whether degradation of the fusions with OmpA is caused by lon iompt proteases (present in <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#top10">TOP10</a>)<br>
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Piotr</h3>
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Sprawdzenie czy degradacja fuzji z Ompa jest wynikiem działania proteaz lon iompt (obecne w top10)
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<p>
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PIOTR
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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.
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<li>Spinnign</li>
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<li>Suspending</li>
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<li>Adding of lysis buffer</li>
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<li>Boiling</li>
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<li>Putting into poliacrylamide gel</li>
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<li>Transfer onto nitrocellulose</li>
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<li>Blocking</li>
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<li>Anti-A antibody binding</li>
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<li>Washing</li>
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<li>Anti-rabbit antibody binding</li>
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<li>Developing with BCIP and NBT</li>
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Zwirowanie, zawieszenie, dodanie buforu lizującego (skład), zagotowanie, nałożenie na żel poliakryl, transfer na nitrocelulozę, blokowanie, przeciwciała anty-A, płukanie, przeciwciała anty-królik, wywołanie BCIP i NBT (omp_omega_A_alfa, z i bez indukcji, omp_A_alfa)
 
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(Zdjęcie żelu)
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[photo of the gel is to be placed here]
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<p>We didn't observe differences in espression and degradation in <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#rosetta">Rosettas</a> nor in <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#top10">TOP10</a>. Therefore we suppose that degradation of the fusions is caused by other factor than lon iompt proteases.</p>
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Brak rużnic ekspresji i degradacji w top10 i rosettach => degradacja nie wynika z działania proteaz- z czego?
 

Revision as of 22:30, 8 October 2008

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Michał K.

  1. Transformation of E. coli TOP10 strain with ligations (pACYC177+OmpA_alpha + deltaA and pACYC177+OmpA_omega + deltaA).
  2. Transformants plating on LB + kanamycin.

Checking whether degradation of the fusions with OmpA is caused by lon iompt 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.

  1. Spinnign
  2. Suspending
  3. Adding of lysis buffer
  4. Boiling
  5. Putting into poliacrylamide gel
  6. Transfer onto nitrocellulose
  7. Blocking
  8. Anti-A antibody binding
  9. Washing
  10. Anti-rabbit antibody binding
  11. Developing with BCIP and NBT

[photo of the gel is to be placed here]

We didn't observe differences in espression and degradation in Rosettas nor in TOP10. Therefore we suppose that degradation of the fusions is caused by other factor than lon iompt proteases.

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