Constructing a ToolboxOur goal was to create a toolbox for developing customized site-specific methylases. Like any good toolbox, it includes three components: the tool, the assay, and the automation.
The Tool: a fusion between a DNA binding domain and a methylase enzyme
The Assay: a digestion based assay, called MaGellin, to measure both methylase activity and sequence specificity
The Automation: a unique software package that predicts experimental outcomes and analyzes gel electrophoresis images to measure methylase functionality
Toolbox with the three essential components.
Our toolbox satisfies 6 requirements:
Robust – includes functional fusion proteins for comparison
Standardized – all inclusive in one-plasmid with simple multiple cloning sites, standard bisulfite sequencing primers, and a digestion based methylation assay built in
Easy to Use – we programmed a software package to predict experimental outcomes and automate gel electrophoresis analysis
Inexpensive – methylase activity and specificity can be screened by simply digesting and running a gel
Noiseless – the bacterial chassis has no background CpG methylation
Open Source – we BioBrick’ed the full plasmid and the methylases
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