Computational protocol: Probiotic Potential of Lactobacillus Strains with Antimicrobial Activity against Some Human Pathogenic Strains

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Protocol publication

[…] Overnight culture (1.5 mL) of each of the nine LAB isolates in MRS broth was centrifuged at 5000 ×g for 10 min at room temperature. The cell pellet was used for extraction of total genomic DNA using the DNeasy Blood and Tissue Kit (Qiagen, Germany). For amplification of the 16S rRNA gene, universal primers F27 (5′-AGAGTTTGAT CMTGGCTCAG-3′) and R1492 (5′-TACGGYTACCTTGTTACGACTT-3′) were used [, ] with expected PCR product size of 1.5 kb. The PCR amplification was performed in 25 μL reaction mixtures using a MyCycler Thermal Cycler (BioRad, USA). The PCR reaction mixture contained 2.5 μL of PCR buffer (10X PCR amplification buffer containing 500 mM KCl, 100 mM Tris-HCl (pH 9.0), and 1% Triton X-100), 0.5 μL of deoxynucleotide triphosphate (dNTP, i-DNA Biotechnology, Singapore) in concentration of 10 mM, 0.5 μL of each primer in concentration of 10 μM, 20 μL of deionised water, 0.5 μL of Taq DNA polymerase (Viogene, Taiwan, 2 U/μL), and 0.5 μL of template DNA (corresponding to approximately 50 to 100 ng of DNA). The PCR conditions were as follows: initial denaturation at 94°C for 4 min, 30 cycles of denaturation at 94°C for 1 min each, primer annealing at 55°C for 30 s and primer extension at 72°C for 2 min, and a final step of primer extension at 72°C for 5 min. The PCR product with expected size of 1.5 kb was excised from the gel and purified using MEGAquick-spin PCR and Agarose Gel DNA Extraction System (iNtRON Biotechnology, Korea). Each purified PCR product was ligated into PCR 2.1 TOPO vector using a TOPO TA cloning kit (Invitrogen, USA) and cloned into E. coli TOP 10 according to the manufacturer's instructions. The DNA sequence analysis was carried out for plasmid with the unique insert using an ABI 373XL automated sequencer (Applied Biosystems, USA) at both directions to obtain the full sequence of the amplicons. DNA sequence data sets were assembled using the Bioedit sequence alignment editor software, version 7.0.9.0 []. Sequence similarity values were determined using the basic local alignment search tool (BLAST) of the National Centre of Biotechnology Information (NCBI) []. Sequences with ≥97% similarity to the previously published sequences were used as the criteria to indicate species identity. Sequence alignment was carried out using CLUSTAL W program of the Bioedit software version 7. A phylogenetic tree was constructed based on the 16S rRNA gene sequence analysis where the analysis involved 51 nucleotide sequences comprising nine sequences of LAB obtained in this study, 41 sequences belonging to Lactobacillus species that were obtained from the GenBank and the sequence of Lactococcus lactis (AB100803.1) which was used as an outgroup. Evolutionary analyses were conducted using molecular evolutionary genetic analyses (MEGA) version 5. The evolutionary history was inferred using the neighbor-joining method. Bootstrapping was performed for 1000 replicates and the evolutionary distances were computed using the Tamura 3-parameter method []. Potential anomalous sequences of the 16S rRNA gene were examined by the Mallard [] and the Bellerophon [] programs. Nucleotide sequences of nine LAB isolates determined in this study were deposited in the GenBank (NCBI) database. […]

Pipeline specifications

Software tools BioEdit, BLASTN, Clustal W
Application Phylogenetics
Organisms Lactobacillus casei, Homo sapiens
Chemicals Bile Acids and Salts, Lactic Acid