Homologous recombination with linear DNA for deletion of the orig

Homologous recombination with linear DNA for deletion of the original Selleckchem Regorafenib target gene was performed according to the procedure previously reported with modifications (Datsenko & Wanner, 2000). In brief, PCR products containing the kan gene from pKD4 or pKD13 were electroporated into the E. coli strain harboring pKD46r and grown in LB agar containing KM (either 5 or 25 μg mL−1) and/or 3-β-indoleacrylic acid (IAA, inhibitor of TrpR) (25 μg mL−1). Deletion of the target gene was examined by PCR. Colonies grown in LB agar containing KM (5 μg mL−1) and IAA (25 μg mL−1) were suspended with sterile saline. Suspensions were diluted 10-fold serially with sterile

saline. Then, one hundred microliters of samples was spread onto LB agar without any supplement, LB agar containing IAA (25 μg mL−1), LB agar containing tryptophan (Trp, 1 mg mL−1), or LB agar containing Trp (1 mg mL−1) plus IPTG (10 mM) and then cultured at 37 °C for > 24 h. Finally, the number of colonies grown on the plates was enumerated. Colony-forming capacity was determined selleck chemicals by the appearance of visible colonies within

48 h of cultivation, and as a positive control, approximately 1000 colony-forming units (CFU) per plate of bacteria were spread on a plate. Colonies grown on LB agar containing KM (5 μg mL−1) and IAA (25 μg mL−1) were suspended with sterile saline and adjusted to OD600 nm = 0.08–0.10. These solutions were diluted 10 000-fold in LB broth and incubated in a shaking incubator at 120 r.p.m. at 37 °C for 2 h. After

incubation, IAA (25 μg mL−1) or IPTG (10 mM) plus Trp (1 mg mL−1) was added to each tube. Aliquots from each tube were removed at −1, 0, 1, 3, and 6 h, and then 10-fold serial dilutions were spread onto LB agar plates containing KM (5 μg mL−1) and IAA (25 μg mL−1). Viable colonies were enumerated after 24–48 h incubation at 37 °C with the limit of detection for the time-kill studies being 10 CFU mL−1. When no viable colony was detected in the undiluted culture, the sample was defined as 10 CFU mL−1. The wild-type lacI promoter is Megestrol Acetate very weak (Calos, 1978). For efficient lacI gene expression, the lacI promoter of E. coli K-12 MG1655 was replaced with lacI-35-10 promoter (Glascock & Weickert, 1998) by homologous recombination, and then the promoter of the clpA gene was replaced with lacUV5 promoter (Lanzer & Bujard, 1988), and the ORF of HA tag was fused in-frame to 3′-end of the clpA gene ORF. Next, the ORF region of the sdaB (Shao & Newman, 1993), a homologue of sdaA that is not essential for bacterial survival, was replaced with CP25e promoter, a constitutive promoter with modification for optimal sequences for E. coli (Jensen & Hammer, 1998), and the ubp1 ORF fused in-frame with FLAG tag ORF at 3′-end. No apparent phenotypic change by deletion of sdaB was observed. The protein expression of ClpA-HA (IPTG supplemented condition) and UBP1-FLAG in this strain was confirmed by Western blotting using anti-HA and anti-FLAG antibody respectively (data not shown).

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