Preparation of constructs with OmpA protein fusions

1. Isolation of plasmids from cultures inocluated on previous day.
2. Control digestion 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 pACYC177 and OmpA_alpha.
5. Transformants plating on LB + kanamycin.

Cloning of protein Z DNA to OmpA constructs

1. Digestion 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

1. Isolation of plasmid DNA from cultures inocluated on previous day.
2. Control digestion 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