Computational protocol: Molecular cloning, expression, and functional characterization of the β-agarase AgaB-4 from Paenibacillus agarexedens

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Protocol publication

[…] The genomic DNA of P. agarexedens was isolated and quantified using a Quant-iT dsDNA BR assay (Thermo Fisher Scientific). The quality of the extracted genomic DNA was verified on a 0.6% agarose gel. DNA libraries were constructed using an Illumina TruSeq DNA LT Sample Prep Kit. Mate-pair libraries were constructed using an Illumina Mate Pair Library Prep Kit v2. For the de novo assembly of the complete genome, we used Velvet (Zerbino and Birney ) to assemble paired-end and mate-pair reads to scaffolds. Genes on these assembled scaffolds were predicted using GeneMark.hmm (Besemer and Borodovsky ) and were annotated using a BLAST (blastp) search against the NCBI nr protein database, with an e-value cutoff of 0.00001. For the functional annotation of predicted genes, the accession numbers from BLAST hits were mapped to GO terms by querying the GO database (Ashburner et al. ). Enzyme code annotations were retrieved by mapping to GO terms, and enzyme codes were retrieved by querying the GO database. [...] The kinetic parameters of purified rAgaB-4 (1.54 μg) were determined in 50 mM phosphate buffer (pH 6) containing LMP agarose and HMP agarose (molecular mass, 120 kDa), ranging in concentration from 2 to 30 mg/mL. The reaction mixture was incubated at 55 °C for 10 min. Km and Vmax for LMP agarose and HMP agarose were determined from Lineweaver–Burk plots using SigmaPlot 12 software (Systat Software, San Jose, USA). Subsequently, the Kcat (turnover number) and Kcat/Km (catalytic efficiency) values were calculated based on the Vmax, Km, and [E] (concentration of rAgaB-4) values. […]

Pipeline specifications

Software tools Velvet, GeneMark.hmm, BLASTP, SigmaPlot
Applications Miscellaneous, Genome annotation, WGS analysis
Organisms Escherichia coli BL21(DE3), Escherichia coli
Chemicals Amino Acids