Secretion assay of putative enzymes.
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posted on 2019-01-02, 18:29 authored by Mackenzie Thornbury, Jacob Sicheri, Patrick Slaine, Landon J. Getz, Emma Finlayson-Trick, Jamie Cook, Caroline Guinard, Nicholas Boudreau, David Jakeman, John Rohde, Craig McCormickLog-phase BL21(DE3) E. coli containing a putative enzyme or empty vector control were treated with 0.1 mM IPTG to induce T7-promoter-dependent transcription. After 3 hours, A) cells were lysed, B) cells were treated with cold osmotic shock to extract periplasmic protein fraction, or C) the supernatant was filtered, and protein was precipitated by addition of TCA. All protein fractions were prepared for SDS-PAGE and analyzed by western blot with an anti-His-antibody.
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β- glucosidase fusion protein6- chloro -4-methylumbelliferyl xylobioside substratenovel lignocellulose-degrading enzymesNPGα- L-arabinofuranosidase enzymesplasmid expression vectorsinducible protein expressionmetagenomic sequencing approachporcupine microbiomegeneporcupine microbiome encodingpHmetagenomic library screening1.16 x 10Nitrophenyl β- D-glucopyranosideencode lignocellulose-degrading enzymeshemicellulose-degrading β- xylosidaseNorth American porcupinenovel hemicellulose-degrading enzymemetagenomics Plant cell walls
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