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).
Figure 3: Validating standardized bisulfite sequencing primers. Primer Set 2 successfully amplify converted DNA but not unconverted DNA, as desired.
Figure 4: Plasmid DNA treated in vitro with purified M.SssI. The first three lanes were not treated and show zero methylation detection by our assay. The last three lanes were methylated and show 100% methylation. This figure validates that MaGellin is capable of clear input/output.
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.
Figure 5: Plasmid DNA treated in vitro with purified M.SssI. Each lane was treated for a different amount of time, this figure shows MaGellin's sensitivity.
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).
Figure 6: M.SssI expressed in vivo compared with in vitro methylation.
Summary
We have created MaGellin, a new technology that facilitates screening novel DNA binding domain – methyltransferase fusion proteins
Our assay is less expensive and faster than existing methods
We have eliminated noise associated with previous studies
We have a system with clear input/output
Our assay lends itself to high throughput screening of many different proteins
We are releasing it alongside an open source data analysis software package which streamlines the entire screening process
"
