Preparation of constructs with OmpA protein fusions
1. Isolation of plasmids from cultures inocluated on previous day.
2. Control digestation of isolated plasmids with BamHI and NotI (we confirmed pCACYC177 + OmpA_omega). We didn't obtain pCACYC177 + OmpA_alpha probably because 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.
Ligation of protein Z DNA to OmpA constructs
1. Digestation 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.