Bioinformatics analysis of B pseudomallei SDO The B pseudomalle

Bioinformatics analysis of B. pseudomallei SDO The B. pseudomallei SDO amino-acid sequence was subjected to basic local Ilomastat alignment search (BLAST) [15]; further alignment was then performed using ClustalW [16]. The sequence with maximum identity, Bacillus megaterium glucose 1-dehydrogenase, was used as a template for homology modeling using SWISS-MODEL [17]. The constructed model was validated

Talazoparib price by PROCHECK [18]. Construction of B. pseudomallei SDO deletion mutant and complemented strain Deletion mutagenesis of the SDO gene was performed by homologous recombination (Additional file 1), as previously described by Lopez et al. [19]. The B. pseudomallei K96243 SDO gene sequence was obtained from GenBank (accession number NC_ 006351 and locus_tag = “BPSS2242” [14]). Primers used in this study were designed using Primer-BLAST (http://​www.​ncbi.​nlm.​nih.​gov/​tools/​primer-blast). The primer sequences are shown in Table 3. Molecular cloning was carried out on 5′ 298 bp upstream and 3′ 288 bp downstream fragments of the B. pseudomallei SDO gene. The 5′ upstream and 3′ downstream fragments of the SDO gene were ligated www.selleckchem.com/products/pnd-1186-vs-4718.html by PCR using BPSS2242-F1 and BPSS2242-R2; this was facilitated by a tail on the 3′ forward primer to give a new PCR product with

a deletion in the region (631 bp) between BPSS2242-R1 and BPSS2242-F2. Table 3 Oligonucleotide primers used for PCR Primer names Oligo sequences (from 5′–3′) Purpose Reference BPSS2242-F1 ACCGCGCGACCGATATGAACG Forward primer for upstream fragment of SDO gene This study BPSS2242-F2 GGACTCCTTGCCGAACGGGC Reverse primer for upstream fragment of SDO gene This study BPSS2242-R1 GCCCGTTCGGCAAGGAGTCC AACGTCGAGGCGAAGCTGCC Forward primer for downstream fragment of SDO gene

This study BPSS2242-R2 TCCCTTCGCGCTCGTGCAAC Chlormezanone Reverse primer for downstream fragment of SDO gene This study OriT-F CAGCCTCGCAGAGCAGGATTC Forward primer for oriT [50] OriT-R TCCGCTGCATAACCCTGCTTC Reverse primer for oriT [50] This constructed fragment was cloned into pGEM®-T Easy Vector and transformed into Escherichia coli strain DH5α. White colonies were selected using β-galactosidase indicator medium, using 50 μg/ml 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-Gal) (Promega) plates containing 100 μg/ml ampicillin. Colonies harboring the desired plasmid were analyzed by PCR using primers flanking the mutant allele (BPSS2242-F1 and BPSS2242-R2). Products were checked for correct size by agarose gel electrophoresis and verified by DNA sequencing. The unmarked knockout cassette assembled by PCR containing the deletion of the SDO gene was cloned into the non-replicative plasmid, pEXKm5 [19]. The pEXKm5-mutant allele construct was then transformed into E. coli strain DH5α. Plasmids were extracted and checked by PCR, with primers BPSS2242-F1 and BPSS2242-R2, for correct product sizes of the target gene. The pEXKm5-mutant plasmid was transformed into E.

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