Mutagenesis of protein A

Paweł

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

Preparation of BioBricks

Michał K.

1. Isolation of HQs(??????) plasmids.
2. Digest of isolated plasmids with EcoRI and BcuI (BamHI buffer).
3. Digest of pACYC177+OmpA_omega_deltaA_alpha with BamHI and PstI (BamHI buffer).
4. Dephosphorylation (CIAP) of plasmids.
5. Gel elctrophoresis and gel-out of proper band: ??????.
6. Digest of omega_link fragment (PCR from previous day) with EcoRI and BcuI (BamHI buffer)
7. Digest of OmpA_omega with BglII and PstI (Orange buffer).
8. Clean-up of above digest reactions.
9. 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 (RFP(??????) + Z and RFP(????) + OmpA_link).
2. Plating on LB + ampicillin.
3. Inoculation of bacterias received from iGEM HQs ( ).