Mutagenesis of protein A

Paweł

Results of sequencing: unfortunately all sequences were wild-type.

Preparation of vectors for Biobricks

Michał K., Piotr

1. Isolation of pSB2K3 and BBa_I739204 (pACYC177 converted into BioBrick vector) plasmids (Fig. 1.).
2. Digest of isolated plasmids with EcoRI and BcuI (BamHI buffer). Dephosphorylation (CIAP) of plasmids.
3. Inoculation of bacteria received from iGEM HQs, carrying pSB2K3 and BBa_I739204 (pACYC177 converted into BioBrick vector) plasmids.
4. Gel elctrophoresis and gel-out of proper bands: 4500 bp - pSB2K3 and 3000 bp - BBa_I739204 (pACYC177 converted into BioBrick vector).

Preparation of OmpA-linker-omega-linker (BBa_K103016)

Michał K.

1. Digest of OmpA-linker-omega-linker (BBa_K103016) PCR product with BglII and PstI (Orange buffer).
2. Clean-up of above digest reaction.
3. Digest of pACYC177+OmpA_omega_deltaA_alpha with BamHI and PstI (BamHI buffer) (Fig. 2.). Dephosphorylation (CIAP) of plasmid.
4. Gel elctrophoresis and gel-out of proper band - 3800 bp.

Preparation of OmpA-linker (BBa_K103006)

Piotr

1. E. coli TOP10 transformation with overnight ligation pSB1A3+OmpA-linker (BBa_K103006).
2. Plating on LB + ampicillin.

Preparation of omega_linker under PT7 (BBa_K103020)

Michał K.

1. Digest of omega_linker fragment (PCR from previous day) with EcoRI and BcuI (BamHI buffer).
2. Clean-up of above digest reaction.

Preparation of Z(BBa_K103004)

Piotr

1. E. coli TOP10 transformation with overnight ligations pSB1A3 + Z(BBa_K103004).
2. Plating on LB + ampicillin.

Preparation of OmpA_linker_omega_linker under Plac (BBa_K103018)

Michał K.

Overnight digest of purified pLac_OmpA_omega fragment (from previous day) with EcoRI (EcoRI buffer). DNA ends blunting with Klenow fragment.