MutD₅ testing

Piotr

Inoculation of MutD₅ carrying: pACYC177+OmpA_Z_alpha, pACYC177+OmpA_Z_omega, pACYC177+Omp_A_omega and pACYC177+OmpA_omega_ΔA + induction using 0.25mM IPTG.

'Hunter and prey' system tests: Competition tests

Weronika

1. Plasmid isolation from 19 September cultures.
2. Digest with SacI and BamHI.
3. Electrophoresis.

Conclusion: cells with interracting protein survive competition!

Mutagenesis of protein A

Paweł

Mutagenesis of protein A was performed using 2 pairs of primers: ADelL+KpnI and ADelP (deletion of aminoacids involved in interaction with protein Z) and AMutL+KpnI and AMutP (changing of few aminoacids involved in interaction with protein Z).

Mutagenesis were performed on 3 vectors: pACYClac+ompA-ΔA-omega, pACYClac+ompa-ΔA-alpha and pACYClac+ ompa-omega-ΔA.

Each mutagenesis was performed using each primer at final concentration 0.1 μM, dNTPs at final concentration 0.25 μM and Walk (Pwo) polymerase (shipped by A&A Biotechnology), with 100 ng of DNA template.

PCR program (15 cycles):

94°C 5 min

94°C 30 s
55°C 30 s
72°C 10 min

72°C 8 min

4°C hold

Preparation of ΔA (BBa_K103003)

Michał K.

Inoculation of only one colony which grown from transformation (19 September) - pSB1A3+ΔAplasmid into liquid LB + ampicillin.