The characterization of point mutants of β-glucosidase B from P. polymxa ("Bagel") is an ongoing project in the Siegel group. This repository contains all the laboratory protocols for the production and assay of mutants of this enzyme.
- We use an automated Kunkel mutagenesis procedure that runs on Transcriptic's workcell. Single-stranded DNA for this protocol is available for free from our lab, please email us. If you would like to perform this procedure by hand, you can follow the Siegel group protocol for a manual Kunkel mutagenesis procedure.
The following protocols are used in our lab:
production_productionfor production of protein beginning from purified plasmidgel_and_yieldsfor determining protein purity by SDS-PAGE and protein yield by A280kinetic_assayfor determining Michaelis-Menten parameters from a plate assay (also see data analysis tools)thermal_stability_assayfor an assay to determine an enzyme's melting temperature
The entire pipeline:
- deep prep from
recipes.md ssDNA_prep.mdfor preparation of single-stranded DNA template- automated Kunkel mutagenesis on Transcriptic
protein_production.mdgel_and_yields.md(and, if necessary, production again)kinetic_assay.mdandthermal_stability_assay.md- data analysis using the fitter
The outputs for each protein are the soluble expression (yes/no), quantitative protein yield by A280 (in units of mg/mL), and the Michaelis-Menten constants kcat and KM.
Data for the native BglB sequence:
| Protein yield (mg/mL) | Expresses | kcat (1/min) | Percent error, kcat | KM (mM) | Percent error, KM | Tm (C) |
|---|---|---|---|---|---|---|
| 1.13 | Yes | 895.6 | 2.1 | 4.79 | 9.8 | 39.6 |