PMC:3395577 / 22404-23262 JSONTXT

{"project":"MicrobeTaxon","denotations":[{"id":"T51","span":{"begin":67,"end":99},"obj":"1517"},{"id":"T52","span":{"begin":650,"end":657},"obj":"562"}],"namespaces":[{"prefix":"_base","uri":"http://purl.bioontology.org/ontology/NCBITAXON/"}],"text":"Plamid constructions\nThe β-glucosidase gene bgl was amplified from T. thermosaccharolyticum DSM 571 genomic DNA by PCR using primers bgl-1 and bgl-2 (Table 3), the PCR products were digested with Nde I and Xho I and inserted into pET-20b at Nde I and Xho I sites, yielding the plasmid pET-20-BGL.\nTable 3 Nucleotide sequences of used primers The boldface italic nucleotides represented mutations for optimizing codons. In order to improve the expression level of recombinant BGL, the internal region from 1st to 19th amino acids in open reading frame of bgl was mutated in situ by inverse-PCR to replace the rare codons with the optimal codons of E. coli; the primers for the inverse-PCR were designated as bgl-3 and bgl-4 (Table 3). Inverse-PCR with primers was carried out using Pyrobest with pET-20-BGL as template, generating the plasmid pET-20-BGLII."}