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.
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 pACYC177 + 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. 1.). Dephosphorylation (CIAP) of plasmid.
4. Gel elctrophoresis and gel-out of proper band - 3800 bp.

Preparation of pSB1A3+Omp_

Piotr

1. E. coli TOP10 transformation with overnight ligation pSB1A3 + OmpA_link.
2. Plating on LB + ampicillin.

Preparation of BioBricks

Michał K.

1. Digest of omega_link fragment (PCR from previous day) with EcoRI and BcuI (BamHI buffer)Fig. 2
2. Clean-up of above digest reactions.
3. Overnight digest of purified pLac_OmpA_omega fragment (from previous day) with EcoRI (EcoRI buffer). DNA ends blunting with Klenow fragment.

Piotr

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