Cloning of truncated fragment of protein A (ΔA)

Emilia

1. Isolation of plasmids from cultures inoculated on previous day.
2. Control digest with SacI and BamHI (BamHI buffer).
3. Gel electrophoresis - proper clones found for both products of ligation.

Checking if OmpA_omega_ΔA_alpha gives ampicillin resistance

Piotr

Inoculation OmpA_omega_ΔA_alpha from various IPTG concentrations (in mmol/mL) (0, 0.1, 0.25, 0.5, 0.75, 1) into same IPTG concentrations, but with various ampicillin concentrations (in mmol/mL)(25, 50, 75, 100) in ratio: 1:50.

Inoculation of OmpA_omega_ΔA_alpha into various IPTG concentrations: 0, 0.1, 0.25, 0.5, 0.75 mmol/mL (repetition is necessary)

Checking OmpA_omega_ΔA_alpha and OmpA_A_alpha expression

Piotr

1. Spinning.
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 (Fig. 1.).

Preparing pACYC177+OmpA_omega_ΔA construct

Michał K.

1. Transformation of E. coli TOP10 strain with ligation.
2. Transformants plating on LB + kanamycin.