From 2008.igem.org
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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> | + | <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>)<br> |
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Revision as of 07:02, 9 October 2008
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Michał K.
- Transformation of E. coli TOP10 strain with ligation products (pACYC177+OmpA_alpha + deltaA and pACYC177+OmpA_omega + deltaA).
- Transformants plating on LB + kanamycin.
Checking whether degradation of the fusions with OmpA is caused by 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.
- Spinnign
- Suspending
- Adding of lysis buffer
- Boiling
- Putting into poliacrylamide gel
- Transfer onto nitrocellulose
- Blocking
- Anti-A antibody binding
- Washing
- Anti-rabbit antibody binding
- 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 and OmpT proteases.
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