Genomic DNA from C. trachomatis serovar D was used as the source for all genes cloned in ![image]()
this study. Using Platinum pfx DNA amplification enzymes (Life Technologies), the following PCR products were generated (note: subscript denotes amino AZD-3463 number): CopB1–100 with a 5′ BamHI restriction site and 3′ EcoRI restriction, CopD1–100 with a 5′ EcoRI restriction site and a 3′ SalI restriction site, full length CT584 with a 5′ SalI restriction site and 3′ HindIII restriction site. PCR products were digested with their respective endonuclease (New England Biolabs) (Fig. 2). The multiple cloning site (MCS) 1 of pET-DUET was restriction digested with BamHI and HindIII (New England Biolabs). Restriction digested CopB1–100, CopD1–100, CT584, and pET-DUET were ligated in a population 3:3:3:1 ratio using T4 DNA Ligase (Invitrogen) and transformed into NEB Turbo Cells (New England Biolabs). Prior to protein expression, all constructs were verified by Sanger sequencing at the MOBIX laboratory (McMaster University).
this study. Using Platinum pfx DNA amplification enzymes (Life Technologies), the following PCR products were generated (note: subscript denotes amino AZD-3463 number): CopB1–100 with a 5′ BamHI restriction site and 3′ EcoRI restriction, CopD1–100 with a 5′ EcoRI restriction site and a 3′ SalI restriction site, full length CT584 with a 5′ SalI restriction site and 3′ HindIII restriction site. PCR products were digested with their respective endonuclease (New England Biolabs) (Fig. 2). The multiple cloning site (MCS) 1 of pET-DUET was restriction digested with BamHI and HindIII (New England Biolabs). Restriction digested CopB1–100, CopD1–100, CT584, and pET-DUET were ligated in a population 3:3:3:1 ratio using T4 DNA Ligase (Invitrogen) and transformed into NEB Turbo Cells (New England Biolabs). Prior to protein expression, all constructs were verified by Sanger sequencing at the MOBIX laboratory (McMaster University).