Assay Validation

For a detailed, graphical explanation of the MaGellin work flow, please download the MaGellin Workflow Specifications Sheet, which includes all of the steps in the MaGellin workflow.

Standardized Bisulfite Sequencing Primers.

Bisulfite sequencing is a good next step to further characterize functional site-specific methylases but it is inherently very difficult to design good primers. People will use advanced algorithms for primer design, and are still not guaranteed to be successful for some sequences. We went through 8 sets of primers, most of which did not show the proper bias to amplify only bisulfite converted DNA. Primer Set 2 was successful and is included with our MaGellin plasmid, much like VF and VR are included as standardized biobrick sequencing primers (Figure 3).

In Vitro.

First, we tested MaGellin with a purified methyltransferase in vitro. The results made it clear that MaGellin can detect methylation, at both the “target” and “off-target” site (Figure 4). MaGellin is also sensitive to various degrees of methylation (Figure 5). These experiments helped us optimize the ideal amount of plasmid and restriction enzyme to use in any study moving forward.

In vivo.

Then, we expressed M.SssI in vivo and compared it with purified M.SssI used on the plasmid in vitro. In both cases, we saw similar full methylation of the plasmid, confirming that MaGellin can express methylases and report their activity in vivo (Figure 6).

Summary

We have created MaGellin, a new technology that facilitates screening novel DNA binding domain – methyltransferase fusion proteins
1. Our assay is less expensive and faster than existing methods
2. We have eliminated noise associated with previous studies
3. We have a system with clear input/output
4. Our assay lends itself to high throughput screening of many different proteins
5. We are releasing it alongside an open source data analysis software package which streamlines the entire screening process