Engineering Multicolor Cyanobacterial Pigment Expression in E. coli

The pACYCDuet-1 Vector was digested with restriction endonucleases (Thermo Fisher Scientific) BamHI and SalI, and then the genes pcBA and apcAB were respectively connected to the linear vector with T4 ligase (TransGen Biotech) to obtain plasmids pACYCDuet-pcBA and pACYCDuet-apcAB. The plasmid pACYCDuet-pcBA was then digested with restriction endonucleases NdeI and XhoI, and the gene apcAB was connected to it to obtain the plasmid pACYCDuet-pcBA-apcAB. The above three plasmids were respectively transformed into E. coli BL21 (DE3) to obtain a strain expressing phycocyanin (E. coli PC), a strain expressing allophycocyanin (E. coli APC), and a strain expressing both phycocyanin and allophycocyanin (E. coli PC-APC).
The genes ho (digested by BamHI and SacI), pcyA (digested by SacI and PstI) were inserted into the pETDuet-1 Vector to obtain the plasmid pETDuet-ho-pcyA, and transformed into E. coli BL21 (DE3) to obtain the strain E. coli HP.
Then the chromophore lyase genes cpcU (digested by PstI and SalI), cpcS (digested by SalI and HindIII), cpcT (digested by HindIII and NotI), cpcE (digested by NdeI and AatII), cpcF (digested by AatII and KpnI) were inserted into the plasmid pETDuet-ho-pcyA to obtain the plasmid pETDuet-ho-pcyA-cpcU-cpcS-cpcT-cpcE-cpcF.
The three strains E. coli PC, E. coli APC, and E. coli PC-APC were transformed with the plasmid pETDuet-ho-pcyA-cpcU-cpcS-cpcT-cpcE-cpcF respectively to obtain three kinds of double plasmid expression strains E. coli PC-APC-HPUSTEF, E. coli PC-HPUSTEF, and E. coli APC-HPUSTEF.