Computational protocol: Effect of quorum sensing signals produced by seaweed-associated bacteria on carpospore liberation from Gracilaria dura

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Protocol publication

[…] The genomic DNA of different bacteria was extracted using the cetyltrimethylammonium bromide buffer [CTAB 2%, NaCl 1.4 mM, EDTA 50 mM, Tris 100 mM, PVP 20%] method (Chen and Kuo, ). Purification of genomic DNA was confirmed with 0.8% agarose gel electrophoresis. The universal 16S rRNA primers 27F and 1492R were used for PCR amplification and sequencing (Lane, ). The reaction mixture and PCR conditions were the same as previously described (Singh et al., ). In brief, the PCR reaction mixture contained 2.5 μl 10 × PCR buffer with MgCl2, 25 mM of each deoxynucleoside triphosphate (dATP, dCTP, dGTP, dTTP), 100 ng of each of the forward and reverse primers, 1 unit of Taq DNA polymerase and 10 ng of template DNA. The PCR protocol included a 5-min initial denaturation at 95°C, followed by 30 cycles at 94°C for 40 s, 55°C for 40 s, and 72°C for 2 min, with a final cycle of 10 min at 72°C. The amplified products were analyzed on 1.2% (w/v) agarose gels stained with ethidium bromide and the bands were visualized under UV light. The PCR products were purified using a QIAquick PCR purification kit (QIAGEN, no. 28104). The sequences were manually trimmed and their sequence homology was checked against other sequences available at the NCBI GenBank. The sequence alignment of 16S rRNA was carried out by ClustalW2 software (http://www.ebi.ac.uk/Tools/msa/clustalw2/) and the aligned sequences were clustered into operational taxonomic units (OTUs) at 0.03 cut off values using sequence homology. Finally, the aligned 16S rRNA bacterial sequences were used to construct the phylogenetic trees with the neighbor joining method using the MEGA-5 software (Tamura et al., ). The bootstrap test was performed with 1000 replicates in the phylogenetic trees. The sequences were taxonomically classified using the Ribosome Database Project (RDP) using Naive Bayesian rRNA classifier version 2.4 with an 80% confidence threshold (Wang et al., ). […]

Pipeline specifications

Software tools Clustal W, MEGA
Application Phylogenetics
Organisms Ulva fasciata
Chemicals Sodium Dodecyl Sulfate