Preparation of constructs with OmpA protein fusions

1. Isolation of plasmids from cultures inocluated on previous day.
2. Control digest of isolated plasmids with BamHI and NotI (we confirmed pCACYC177 + OmpA_omega). We didn't obtain pCACYC177 + OmpA_alpha probably because of a mistake in plating.
3. Ligation of pACYC177 and OmpA_alpha (1 hr)
4. Transformation of E. coli TOP10 strain with ligation products: pACYC177 and OmpA_alpha.
5. Transformants plating on LB + kanamycin.

Cloning of protein Z DNA to OmpA constructs

1. Digest of Geneart_Z and pACYC177+OmpA_omega with SacI and NotI (pACYC177 was also dephosphorylated with CIAP)(3 hr).
2. Gel electrophoresis and gel-out of proper bands 220 bp (for Geneart_Z lane)and 4050 bp (pACYC177+OmpA_omega lane).
3. Ligation of pACYC177+OmpA_omega and Z (1 hr).
4. Transformation of E. coli TOP10 strain with ligation.
5. Transformants plating on LB + kanamycin.

Preparation of construct pKS with A protein
Michał L., Marcin:

1. Inactivation of digestion enzymes and CIAP.
2. Ligation of digested PCR product and pKS 2h at room temperature.
3. Transformation of E. coli TOP10 strain with 7 µl of ligation mix.
4. Transformants plating on LB + ampicillin + X-gal + IPTG.

Preparation of construct pKS with A protein

1. <a href="https://2008.igem.org/wiki/index.php?title=Wiki/Team:Warsaw/protocols#plasmid_DNA_isolation">Isolation of plasmid DNA</a> from cultures inocluated on previous day.
2. Control <a href="https://2008.igem.org/Wiki/Team:Warsaw/igem_notebook.htm#digest">digest</a> of isolated plasmid with SacI and NotI; 2 h
3. Gel electrophoresis of digested DNA
4. We are glad to find the proper clone ;-) and inoculate liquid LB with ampicillin to freeze bacteria carrying pKS-A construct

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