Computational protocol: Evidence of the Generation of Isosaccharinic Acids and Their Subsequent Degradation by Local Microbial Consortia within Hyper-Alkaline Contaminated Soils, with Relevance to Intermediate Level Radioactive Waste Disposal

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Protocol publication

[…] Total genomic DNA was extracted from the soil microcosm using a Powersoil DNA extraction kit (Mo-BIO, Carlsbad, US). A ∼1500 bp fragment of the eubacterial 16S rRNA gene was amplified using broad specificity primers pA and pH as previously described [] and a ∼750 bp fragment of the archaeal 16S rRNA gene was amplified using primers Ar and Af []. PCR reaction mixture contained 5 μL of extracted DNA, 1.5 μL of each primer (10pmol μL−1 concentration), and 25 μL of BIOMIX red master mix (BIOLINE, UK) made up to 50 μL volume with PCR grade water. The reaction mixture was then incubated at 94°C for 5 mins, and then cycled 35 times through three steps: denaturing (94°C, 1 min), annealing (60°C, 1min), primer extension (72°C, 1min 30s). This was followed by a final extension step of 72°C for 5 minutes. PCR was verified by electrophoresis of 5 μL samples of product in a 1.0% agarose TAE gel with ethidium bromide staining. The remaining 45 μL of product was purified using a Qiaquick PCR purification kit (Qiagen, UK). PCR products were ligated into the standard cloning vector PGEM-T easy (Promega, US) and transformed into E.coli JM109 competent cells (Promega, US). Transformed cells were grown on Luria Bertani (LB) agar containing 100μg mL−1 ampicillin overlaid with 40 μL of 100 mM IPTG and 40 μL of 40mg mL−1 X-GAL (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside) in N’N dimethylformamide for blue-white colour screening for 16 hours at 37°C. Insert containing colonies were sub-cultured on to LB plates containing ampicillin/IPTG/X-GAL as described previously and incubated for 24 hours at 37°C. Colonies were then transferred to 96 well plates containing LB agar with 150 mg mL−1 ampicillin and sequenced using Sanger sequencing technology (GATC Biotech, Germany). Inserts were amplified using a universal M13 forward primer as a sequencing start point. The resulting 16S rRNA gene sequences were grouped into Eubacterial and Archaeal divisions. The sequences were then aligned using the MUSCLE-Multiple Sequence Alignment package (http://www.ebi.ac.uk/Tools/services/web/toolform.ebi?tool=muscle). Aligned sequences were then chimera checked using Mothur [] and sequences analysed against the NCBI database using Basic Local Alignment Search Tool (MegaBLAST) utilising the 16S ribosomal RNA sequences for Bacteria and Archaea. Sequences were then placed into phylogenetic families based on the closest sequence from the MegaBLAST output. […]

Pipeline specifications

Software tools mothur, BLASTN
Application Phylogenetics
Organisms Citrus aurantiifolia
Chemicals Carbon, Methane