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 | + | We developed the MaGellin assay to optimize the development process for site-specific methylases. Having validated the assay, we determined to design and test two site-specific methylases, one of which had never been constructed before.</br> </br> |
<h7>The process further validated our MaGellin assay:</h7> | <h7>The process further validated our MaGellin assay:</h7> | ||
</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. | ||
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<h1>Zinc Finger-M.SssI Fusion</h1> | <h1>Zinc Finger-M.SssI Fusion</h1> | ||
- | 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. This was also validation that the MaGellin assay accurately reports the site-specificity of methylation, effectively demonstrating our assay | + | 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. This was also validation that the MaGellin assay accurately reports the site-specificity of methylation, effectively demonstrating our assay can detect site specific methylation in vivo. |
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<font color="red">SHOW ZINC FINGER DATA</font> | <font color="red">SHOW ZINC FINGER DATA</font> | ||
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- | </br><i>Figure 1: The | + | </br><i>Figure 1: The zinc finger-M.SssI was cloned into MaGellin with and without its binding site present. We ran the standard MaGellin assay on both plasmids, using methylation sensitive restriction enzymes to report the methylase activity.</i></br></br> |
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<h1>TALE-M.SssI Fusion</h1> | <h1>TALE-M.SssI Fusion</h1> | ||
- | </br>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 and found the TALE-M.SssI was methylating at both sites, as reported by the MaGellin software. | + | </br>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. |
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<center><h4>Varied Induction Conditions Suggests TALE-M.SssI is Prone to Off Target Activity</center></h4> | <center><h4>Varied Induction Conditions Suggests TALE-M.SssI is Prone to Off Target Activity</center></h4> | ||
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- | Given the quick turnaround and cost-effectiveness of the MaGellin assay, it was feasible to test our TALE-M.SssI construct at 20 different conditions to get a better idea of its <i>in vivo</i>. We hoped to find the optimal induction point for reducing off target methylation. This study would have cost us approximately $7,000 to do by bisulfite sequencing, based on the prices at our university core facility. MaGellin only required restriction enzymes and gel electrophoresis. | + | Given the quick turnaround and cost-effectiveness of the MaGellin assay, it was feasible to test our TALE-M.SssI construct at 20 different conditions to get a better idea of its fuction <i>in vivo</i>. We hoped to find the optimal induction point for reducing off target methylation. This study would have cost us approximately $7,000 to do by bisulfite sequencing, based on the prices at our university core facility. MaGellin only required restriction enzymes and gel electrophoresis (less than 5% the cost of bisulfite sequencing). |
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<div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/thumb/9/9e/New-3d-Plot-Converted.png/800px-New-3d-Plot-Converted.png" alt="Workflow" width="700px"><figcaption><i>Figure 4: The TALE-M.SssI with and without the TALE binding site present was induced with 0, .1, 1, and 2 mM of IPTG for 0, 2, 6, and 24 hours to find optimal expression conditions. Representative bands’ intensities were quantified and normalized by background intensity. The plot is missing samples marked * because we ran out of miniprepped DNA. The dotted white circle marks the conditions of our initial experiments.</i></figcaption></figure></div> | <div style="margin-left:auto;margin-right:auto;text-align:center"><figure><img border="0" src="https://static.igem.org/mediawiki/2013/thumb/9/9e/New-3d-Plot-Converted.png/800px-New-3d-Plot-Converted.png" alt="Workflow" width="700px"><figcaption><i>Figure 4: The TALE-M.SssI with and without the TALE binding site present was induced with 0, .1, 1, and 2 mM of IPTG for 0, 2, 6, and 24 hours to find optimal expression conditions. Representative bands’ intensities were quantified and normalized by background intensity. The plot is missing samples marked * because we ran out of miniprepped DNA. The dotted white circle marks the conditions of our initial experiments.</i></figcaption></figure></div> |
Revision as of 02:19, 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. This was also validation that the MaGellin assay accurately reports the site-specificity of methylation, effectively demonstrating our assay can detect site specific methylation in vivo. SHOW ZINC FINGER DATA Figure 1: The zinc finger-M.SssI was cloned into MaGellin with and without its binding site present. We ran the standard MaGellin assay on both plasmids, using methylation sensitive restriction enzymes to report the methylase activity. To be sure of the targeting specificity, we cloned the MaGellin plasmid with and without the zinc finger’s binding site present at the target cut site. This demonstrated how the presence of a zinc finger binding site shifts the methylation pattern (Figure 1).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.