T4 dna ligase hc

The coding sequence (CDS) of the bgc33 and bgc86 NRPS biosynthetic genes were synthesized with BsaI flanking sites by GeneArt (Thermo Fisher Scientific) with E. coli codon optimization using the GeneOptimizer algorithm. The synthesized gene was cloned by Golden Gate assembly into E. coli expression vector under T7 promoter control. In brief, PCR fragment of expression vector pET28a (Novagen) was generated with Q5 High-Fidelity DNA Polymerase (New England Biolabs) following manufacturer protocol with primers pET28a-BsaI-F and pET28a-BsaI-R (Table S3). PCR fragment of pET28a was ligated to synthesized bgc33 or bgc86 construct in a 5 μl one-pot digestion/ligation reaction mix consisting of 10 fmol of bgc33 construct, 10 fmol of pET28a PCR product, 2.5 u of BsaI (New England Biolabs) and 2.5 u of T4 DNA ligase HC (Promega). Reaction conditions: 1 cycle of 37 °C for 5 hr; 10 cycles of 37 °C for 2 min, 16 °C for 5 min; 1 cycle of 50 °C for 15 min; 1 cycle of 80 °C for 15 min; 4 °C hold. The re sulting construct bgc33-pET28a was verified by complete plasmid sequencing service (Massachusetts General Hospital DNA core facility). bgc33-pET28a and bgc86-pET28a were transformed into E. coli BAP1 containing T7 DNA polymerase and sfp phosphopantetheinyl transferase.