Checking if OmpA_omega_A_alpha gives ampicillin resistance

Piotr

Inoculation of OmpA_omega_A_alpha from various IPTG concentrations: 0, 0.1, 0.25, 0.5, 0.75, 1 mM into same IPTG concentrations, but with various ampicillin concentrations (25, 50, 75, 100 mM) in ratio 1:50.

Conclusion: we obtained the best induction using 0.5 - 0.25 mM IPTG. Best ampicillin concentration is 50 - 75 μg/ml.

Checking if degradation of fusion with OmpA is a result of the activity of lon and 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.

Transformation of Rosettas with omega_A_alpha and A_alpha.

Michał K.

1. Transformation of E. coli TOP10 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 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

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.