Toolbox

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.

1. The Tool: a fusion between a DNA binding domain and a methylase enzyme
2. The Assay: a digestion based assay, called MaGellin, to measure both methylase activity and sequence specificity
3. The Automation: a unique software package that predicts experimental outcomes and analyzes gel electrophoresis images to measure methylase functionality

Our toolbox satisfies 6 requirements:

1. Robust – includes functional fusion proteins for comparison
2. Standardized – all inclusive in one-plasmid with simple multiple cloning sites, standard bisulfite sequencing primers, and a digestion based methylation assay built in
3. Easy to Use – we programmed a software package to predict experimental outcomes and automate gel electrophoresis analysis
4. Inexpensive – methylase activity and specificity can be screened by simply digesting and running a gel
5. Noiseless – the bacterial chassis has no background CpG methylation
6. Open Source – we BioBrick’ed the full plasmid and the methylases