Fast link dna ligase kit

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.

Extraction of fosmids containing metagenomic DNA: 5 ml of bacterial culture was grown overnight with 12.5 μg/ml Cm. One millilitre of culture was used to inoculate 4 ml of fresh LB broth. To this, 5 μl of 1000× Copy Control Induction solution (Epicentre Biotechnologies) and 12.5 μg/ml Cm were added. The mixture was incubated at 37°C for 5 h with vigorous shaking (200–250 rpm) to ensure maximum aeration. Cells were harvested from the whole 5 ml of induced culture by centrifuging at 2100 × g for 12 min. Qiagen QIAprep Spin mini-prep kit was used to extract fosmids as per manufacturer's instructions. PCR products were purified with a Qiagen PCR purification kit and digested with XbaI and PstI (Roche Applied Science) for mfsT and with SalI and XbaI for sdtR, followed by ligation using the FastLink DNA ligase kit (Epicentre Biotechnologies) to similarly digested plasmid pCI372. Electrocompetent E. coli MKH13 and E. coli EPI300 were transformed with the ligation mixture and plated on LB agar plates containing 20 μg/ml Cm for selection. Colony PCR was performed on all resistant transformants using primers across the multiple cloning site (MCS) of pCI372 to confirm the presence and size of the insert.

Induction of fosmids from LOW to high copy number was performed as per the manufacturer’s instructions. The Qiagen QIAprep Spin mini-prep kit was used to extract fosmids using the protocol outlined by manufacturer. The brpA_L, brpA_S and brpAatfA genes were amplified using ReddyMix PCR mastermix (Thermo Scientific). PCR products were purified with a Qiagen PCR purification kit and digested with restriction enzymes XbaI and PstI (Roche Applied Science), followed by ligation using the Fast-Link DNA ligase kit (Epicentre Biotechnologies) to similarly digested plasmid pCI372. Electro-competent E. coli MKH13 were transformed with the ligation mixture and plated on LB agar plates containing 20 µg/ml Cm for selection.
The pBAD TOPO TA expression kit (Invitrogen, Carlsbad CA, USA) was used to clone the PCR products into the pBAD expression vector according to the manufacturer’s instructions. The brpA_L, brpA_S and brpAatfA genes were amplified as outlined above. The resulting plasmids, containing the genes of interest were electroporated into freshly competent E. coli EPI300 and plated on LB agar containing 100 µg/ml of ampicillin.
Colony PCR was performed on resistant transformants using a gene and plasmid (pCI372 or pBAD) specific primer combination to confirm the presence and size of the insert. Inserts were sequenced to confirm the correct nucleotide sequence (GATC Biotech, Germany).