Computational protocol: Antitumor activity of bacterial exopolysaccharides from the endophyte Bacillus amyloliquefaciens sp. isolated from Ophiopogon japonicus

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Protocol publication

[…] The isolated endophytic bacteria were resuspended in 200 μl TE buffer with 50 μl lysozyme and then incubated in a water bath at 37°C for 60 min with gentle agitation. A total of 10 μl proteinase K was added to the suspension, followed by incubation at 37°C for 30 min with gentle agitation. The suspension was then supplemented with 40 μl 10% SDS and incubated at 37°C for 30 min with gentle agitation. Subsequent to being mixed with 100 μl 5 M NaCl and 200 μl 3 M sodium acetate solutions, an equal volume of chloroform:isoamylol was added and then the suspension was gently shaken. Following being left to stand at room temperature for 5 min, the mixture was centrifuged at 2,609 × g for 10 min and the supernatant was transferred into precooled isopropanol and maintained for 5 min. Following centrifugation at 3,914 × g for 10 min, the deposit was washed with 1 ml 70% ethanol and dried by evaporation at room temperature. The final deposit was dissolved with 30 μl high-salt TE buffer and collected as extracted DNA for the following analysis.The extracted DNA was electrophoresed in 1% (w/v) agarose gel, stained with ethidium bromide and UV-visualized. To determine the 16S rDNA gene sequence, PCR was conducted using the universal primers (forward, 5′-AGAGTTTGA TCCTGGCTCAG-3′; reverse, 5′-AAGGAGGTGATCCAG CCGCA-3′). PCR amplification consisted of an initial denaturation at 94°C for 5 min followed by 30 cycles (denaturation at 94°C for 1 min, annealing at 55°C for 1 min and extension at 72°C for 2 min) and a final extension at 72°C for 8 min. Amplified DNA was purified using a Takara agarose gel DNA purification kit (Takara Biotechnology Co., Ltd., Dalian, China) and sequenced by Takara Biotechnology Co., Ltd. The 16S rDNA sequence was subjected to BLAST analysis with the NCBI database and aligned by using the multiple sequence alignment program CLUSTAL W (). A phylogenetic analysis was performed using CLUSTAL X () and MEGA 4.0 () software, based on the neighbor-joining (), maximum-likelihood () and maximum-parsimony methods (). […]

Pipeline specifications

Software tools Clustal W, MEGA
Application Phylogenetics
Organisms Bacillus amyloliquefaciens, Ostichthys japonicus
Diseases Congenital Abnormalities, Stomach Neoplasms