Plasmid pET30a was used as expression vector in E. coli BL21 (DE3). Escherichia coli–Bacillus shuttle vector pKSV7 (Smith & Youngman, 1992), which has a Bacillus temperature-sensitive
(ts) origin of replication, was used for gene replacement via homologous recombination at a nonpermissive temperature (30 °C) Chromosomal DNA of B. thuringiensis was isolated as described by Sambrook et al. (1989). PCR was performed with Pfu DNA polymerase (TaKaRa BioInc.) using the chromosomal DNA of B. thuringiensis as a template. The primers were designed according to the conserved region of the related proteins to clone the calY gene and its flanking sequences (Fig. 1a). The calY gene fragment was analyzed by 1% agarose gel LY2835219 supplier electrophoresis, purified, and cloned into pET30a check details vector according to the manufacturer’s instructions. The resultant plasmid was sequenced completely (Invitrogen, Shanghai, China) and designated pETCA. Escherichia coli transformation was carried out according to the method of Sambrook et al. (1989). Bacillus thuringiensis transformation was performed by electroporation in a Bio-Rad Gene Pulser Apparatus (Bio-Rad Ltd, Richmond, CA) according to the methods of Hu et al. (2009) and Xia et al. (2009). The plasmid pETCA was transformed
into E. coli BL21 (DE3). The overnight culture was diluted 100 times with fresh LB medium supplemented with 100 μg mL−1 kanamycin and incubated at 37 °C with shaking until the OD600 nm reached 0.6. Camelysin expression was then induced by adding isopropyl β-d-1-thiogalactopyranoside to a final concentration of 1 mmol L−1 and incubation for a further 4 h. The induced camelysin protein was purified by affinity
chromatography according to the protocol of HisTrap FF crude 1-mL column (GE Healthcare, Milwaukee, WI) and then used for antiserum production in rabbits as described previously (Chen et al., 2002). The E. coli–B. subtilis shuttle vector (pKSV7) which contained a temperature-sensitive B. subtilis Urease origin of replication (Smith & Youngman, 1992) was used to construct a calY replacement mutant. The general method is outlined in Fig. 1b. A 780-bp upstream fragment of gene calY was amplified with primer pair P7/P8 (Table 2). Its PCR fragment was digested with HindIII/SalI and cloned into the corresponding site of pUE containing an erythromycin-resistant cassette (erm) to generate pUES. An 800-bp downstream fragment was amplified with primer pair P9/P10 (Table 2). Its PCR fragment was digested with BamHI/EcoRI and cloned into the pUES to generate pUESX. A 2.8-kb HindIII/EcoRI fragment containing upstream and downstream fragments, erm was ligated into the corresponding site of pKSV7 to generate pKESX. The properties of pKESX that allow it to be used as a B. thuringiensis integration vector are as follows: (1) pKESX replicates in E. coli and B.