Piotr

Inoculation of mutD5 into 100 ml of LB and rendering them chemocompetent. Electroporation with various OmpA variants.

Optimisation of primers for preparation of parts

Michał K.

1. Repetition of PCR on pACYC177+OmpA_omega_deltaA_alpha plasmid using LinLSXNE and AlfaPSpe primers (elongation length 60s) to obtain link_alpha fragment. Reaction was carried out with 25 cycles and in temperature gradient from 55 to 75 °C (four reactions).
2. Gel electrophoresis of PCR products.

Preparation of BioBricks

Michał K.

1. PCR on pACYC177+OmpA_omega_deltaA_alpha plasmid using AL_BNXNE and APSacSpe primers (annealing temperature 58 °C ; elongation length 45s) to obtain deltaA fragment.
2. PCR on pACYC177+OmpA_omega_deltaA_alpha plasmid using LinLSXNE and AlfaPSpe primers (annealing temperature 65 °C ; elongation length 60s) to obtain link_alpha fragment.
3. PCR on pACYC177 + OmpA_omega plasmid using LinLSXNE and OmegPSpe primers (annealing temperature 55 °C ;elongation length 60s) to obtain link_omega fragment.
4. PCR on pACYC177+OmpA_omega_deltaA_alpha plasmid using OmpLNXNE and LinP_BS primers (annealing temperature 58 °C ;elongation length 90s) to obtain Omp_omega fragment.
5. Gel electrophoresis of PCR products and gel-out of proper bands (deltaA - 250 bp, linker_alpha - 600 bp and linker_omega - 350 bp, Omp_omega - 900 bp).
6. Digest of purified PCR products and RFP(??????) plasmid with EcoRI and BcuI (BamHI buffer) and pET15b+OmpA_alfa plasmid with NdeI and SacI (BamHI buffer).
7. Gel electrophoresis of digested plasmids and gel-out of proper bands (Omp_link - 500 bp and RFP (??????) - 2200 bp).