Elongation time: 30s

- Optimization of annealing temperature (gradient from 55°C to 75°C)
- Optimization of number of cycles(15, 20, 25, 30, 35)

2. PCR to obtain truncated A protein DNA fragment

Primers: AL+SacI AP+NotI

Elongation time: 30s

Annealing temperature: 60°C

20 cycles

3. Gel electrophoresis and isolation of 250 bp band.
4. Digest of isolated PCR product, pACYC177+OmpA_alpha and pACYC177+OmpA_omega with NotI and SacI.
5. Clean-up of digestion reaction.
6. Gel electrophoresis for estimation of DNA concentration.
7. Overnight ligation: pACYC177+OmpA_alpha + deltaA and pACYC177+OmpA_omega + deltaA.

Michał K.

1. <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#chemotransform">Transformation</a> of E. coli <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#top">TOP10</a> strain with ligation products (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 and OmpT proteases (present in <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#top10">TOP10</a>)

Piotr

Test was conducted in E.coli <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.

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 <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 and OmpT proteases.

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