Team:Penn/MethylaseCharacterization
From 2013.igem.org
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We developed the MaGellin assay to optimize the development process for site-specific methylases. Having validated the assay, we determined to design and test three site-specific methylases, two of which had never been constructed before.</br> </br> | We developed the MaGellin assay to optimize the development process for site-specific methylases. Having validated the assay, we determined to design and test three site-specific methylases, two of which had never been constructed before.</br> </br> | ||
- | <h7>The process further validated our MaGellin assay: | + | <h7>The process further validated our MaGellin assay:</br> |
- | </br>1. We recapitulated published results with a zinc finger-methylase and shed light on the significant magnitude of its off target effects. MaGellin is an excellent assay for this purpose, because of the noiseless chassis and because it's simpler to detect off target effects on a plasmid than a genome. | + | </br>1. We recapitulated published results with a zinc finger-methylase and shed light on the significant magnitude of its off target effects. MaGellin is an excellent assay for this purpose, because of the noiseless chassis and because it's simpler to detect off target effects on a plasmid than a genome.</br> |
- | </br>2. We further characterized our promising novel TALE-methylase and were able to de-noise this noisy, complex system. MaGellin was in agreement with COBRA that the TALE exhibited on and off target methylation. This could have implications for the multitude of TALE-effector systems that have recently been developed. The MaGellin workflow is well suited to solve this optimization problem. | + | </br>2. We further characterized our promising novel TALE-methylase and were able to de-noise this noisy, complex system. MaGellin was in agreement with COBRA that the TALE exhibited on and off target methylation. This could have implications for the multitude of TALE-effector systems that have recently been developed. The MaGellin workflow is well suited to solve this optimization problem.</br> |
</br>3. We demonstrated the ease-of-use of the MaGellin workflow by assaying 20 conditions in less than one week.</h7> | </br>3. We demonstrated the ease-of-use of the MaGellin workflow by assaying 20 conditions in less than one week.</h7> | ||
Revision as of 02:42, 29 October 2013
Methylase Characterization
Zinc Finger-M.SssI Fusion
The zinc finger is a small DNA binding domain, with limited sequence specificity. Previous studies showed it was prone to off-target methylation, which we verified.TALE-M.SssI Fusion
TALEs have a greater sequence specificity than zinc fingers, and are easier to customize and less expensive to construct. They have already been validated for use in genome engineering and are replacing zinc fingers for some applications. We followed the MaGellin protocol to clone a TALE-M.SssI fusion and induced its expression. We repeated this experiment numerous times with varying induction conditions and found the TALE-M.SssI was methylating at both sites, as reported by the MaGellin software.TALE-M.SssI actively methylates DNA as reported by our MaGellin Assay
MaGellin reported our TALE-M.SssI was detectably methylating DNA at both the target site and off-target site. We expected a certain degree of off target methylation simply because the TALEs could occupy all the binding sites on our low copy plasmid; the molar ratio is one of the problems in developing site-specific methylases that the inducible MaGellin system is designed to address. MaGellin is designed to screen multiple fusion protein constructs in a high-throughput manner, and a user would normally select only constructs that methylate in a highly site-specific manner. However, we were interested in using MaGellin to study the TALE-M.SssI further before going back to the drawing board to redesign the linker length, binding site, and other variables.
Validated COBRA is in agreement with our new MaGellin Assay
Varied Induction Conditions Suggests TALE-M.SssI is Prone to Off Target Activity
Summary
MaGellin was designed to optimize the development of robust tools for site-specific methylation. To those ends, we successfully cloned and expressed three fusion methylases, two of which are novel constructs with advantages over the previously published zinc finger. Our constructs have shown methylase activity and DNA binding activity, which we could measure with our new assay. They are ready to be further optimized, using our MaGellin workflow.We picked up on the noisiness of our TALE-M.SssI using MaGellin, which could have implications for the noisiness of other TAL-Effector systems being used in mammalian systems. To do so, we used MaGellin to its full extent: swapping out DNA binding domains and binding sites, varying induction conditions, applying COBRA, and depending on our original algorithm to properly predict methylation-sensitive digestion patterns.