MutD₅ testing

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, linker_omega - 350 bp and 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. Clean-up of digested PCR products.
8. Gel electrophoresis of digested plasmids and gel-out of proper bands (Omp_link - 500 bp and RFP (??????) - 2200 bp).