MutD₅ testing

Piotr

Sequencing results: there weren't any mutations in plasmids isolated from MutD₅ - maybe this strain is too weak as a mutator.

Preparation of BioBricks

Michał K.

1. Digest of pGeneArt-Z and pSB1A3 carrying ΔA (BBa_K103003) plasmids with NdeI and BcuI (BamHI buffer).
2. Digest of pSB1A3 carrying ΔA (BBa_K103003) plasmid with NdeI and SacI (BamHI buffer).
3. Dephosphorylation (CIAP) of plasmids.
4. Gel electrophoresis of digested plasmids and gel-out of proper bands (Z - 200 bp and both pSB1A3's - 2200 bp).
5. Gel electrophoresis to estimate concentration of purified DNA from previous day.
6. Overnight ligation of isolated DNA fragments: pSB1A3 + Z (BBa_K103004) and pSB1A3 + OmpA_linker (BBa_K103006) (from 18 September).
7. PCR on pACYC177+OmpA_omega_ΔA_alpha plasmid using OmLNXNEB and LinPBSNP primers (annealing temperature 58 °C; elongation length 90s) to obtain OmpA_omega fragment.
8. PCR on pACYC177+OmpA_omega_ΔA_alpha plasmid using PlacL_XNE and LinP_BS primers (annealing temperature 58 °C; elongation length 90s) to obtain pLac_OmpA_omega fragment.
9. PCR on pACYC177+OmpA_omega_ΔA_alpha plasmid using AL_BNXNE and APSacSpe primers (annealing temperature 58 °C; elongation length 45s) to obtain ΔA fragment.
10. PCR on pACYC177 + OmpA_omega plasmid using OmegLNde and LinP_BS primers (annealing temperature 55 °C; elongation length 60s) to obtain omega_link fragment.
11. Gel electrophoresis of PCR products and gel-out of proper bands (ΔA - 250 bp, pLac_OmpA_omega_ - 1200 bp, omega_linker - 350 bp and OmpA_omega_ - 900 bp).

Piotr

Inoculation of bacteria received from iGEM HQs, carrying pSB2K3 and BBa_I739204 (pACYC177 converted into BioBrick vector) plasmids.