Construction of Pectinase Gene Cluster Plasmid

A DNA fragment containing 1.08 kb of the 5’ flanking region and 1.0 kb of the 3’ flanking region of the pectinase gene cluster (Cbes1853-1856) was generated by overlap extension polymerase chain reaction (OE-PCR) using primers JF021, JF20.3, JF15.2, and JF014 with KpnI sites added to the 5’ end and an ApaLI site at the 3’ end. Wild-type C. bescii gDNA was used as a template. A 4.3-kb DNA fragment, containing an apramycin resistance gene cassette, the pyrF cassette [18 (link)], and sequences related to the E. coli pSC101 replication origin were amplified from pDCW88 [21 (link)] using primers DC081 (w/KpnI) and DC262 (w/ApaLI). The two linear DNA fragments were digested with KpnI and ApaLI and ligated to generate pJFW54 using a Fast-link DNA Ligase kit (Epicentre Biotechnologies, Madison, WI) according to the manufacturer’s instructions. The primers used in this construction are shown in Additional file 1: Table S2, and a detailed diagram of the vector is shown in Additional file 1: Figure S1. E. coli strain DH5α was transformed by electroporation in a 2-mm-gap cuvette at 2.5 V, and transformants were selected for apramycin resistance. The DNA sequence of the final vector was determined to confirm its structure (Macrogen, Cambridge, MA). All plasmids are available upon request.