Team:Penn/AssayOverview
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Revision as of 02:04, 28 October 2013
Assay Overview
![COBRA Methylation Assay](http://upload.wikimedia.org/wikipedia/commons/thumb/f/fe/Cobra_workflow.svg/776px-Cobra_workflow.svg.png)
![](http://upload.wikimedia.org/wikipedia/commons/thumb/7/7f/Cobra_quantification.svg/776px-Cobra_quantification.svg.png)
The MaGellin Methylation Assay
![MaGellin](https://static.igem.org/mediawiki/2013/9/9d/Plasmid-Schematic-Updated.png)
- CpG Methyltransferase (M.SssI) with a generic linker sequence in the cloning site. For a working fusion protein and assay, only a DNA binding domain must be cloned into the plasmid. This inherently standardizes MaGellin and lessens the time a user of the assay must spend cloning.
- Multiple cloning site downstream of T7 promoter for orthogonal expression of fusion protein in T7 Express competent E. coli.
- Cloning site for a smaller DNA sequence, specific to the fusion protein being screened – named the “target site”, where the protein will bind. This can be the binding site for a CRISPR-Cas, TALE, Zinc Finger, or transcription factor
- AvaI restriction site 4 bases downstream of the target site – the AvaI restriction enzyme is blocked by methylated CpG sites, thus screening for site specific methylation becomes equivalent to screening for AvaI digestion
- AvaI restriction site sufficiently further downstream of the target site – named the off-target site. This site screens for non-specific DNA methylation as it is spatially removed from where the fusion protein binds to the plasmid.
- XbaI site for linearization of the plasmid. Linearizing the plasmid simplifies analysis of the AvaI digestion by gel electrophoresis.
- Validated bisulfite conversion primer binding sites, so users do not need to go through the time-consuming primer design process if they choose to fortify MaGellin’s results with bisulfite sequencing results
- sgRNA cloning site for users who want to target a CRISPR-Cas binding domain. The sgRNA is constitutively expressed and can be swapped by restriction digest.
- Validated bisulfite conversion primers for users who choose to advance to bisulfite sequencing, for even higher resolution in detecting methylation, after proving their enzyme’s efficacy with our MaGellin assay.
- Kanamycin resistance as a selection marker
- T7 RNA Polymerase in the lac operon allows us to turn on expression of fusion protein after induction with IPTG
- In the T7 Express cell line, genes for several restriction enzymes known to target methylated DNA are knocked out (McrA-, McrBC-, EcoBr-m-, Mrr-). This ensures that our assay plasmid is not cleaved in vivo. Results are difficult, if not impossible, to interpret in the commonly used BL21 cell line.
Assemble the MaGellin backbone together with a DNA-binding protein and target sequence of your choosing.
- Forward: CAGGAGGAATTC[ATG] (add start codon only if not included in gene).
- Reverse: CTCTAGAAGCGGC (make sure to remove the stop codon).
- Transform the completed MaGellin plasmid into T7 Express.
- Induce culture with 1 mM IPTG.
- Incubate in a shaker at 37C for 5 hours.
- Miniprep to isolate the plasmid.
- Digest 600 ng of miniprep DNA in a 15 uL reaction with 10 U of both XbaI and AvaI.
- Incubate reaction for 1 hour at 37C.
- Look for 3 distinct band patterns that correspond to specific and interpretable methylation outcomes.
- The presence of large one band corresponds to non-site-specific DNA methylation (AvaI was blocked at both the target and off target sites, and thus only XbaI cut the plasmid)
- The presence of two bands corresponds to site-specific DNA methylation (AvaI was only blocked at the target site, thus AvaI cut in the off target site and XbaI cut the plasmid)
- The presence of three bands corresponds to no DNA methylation – or an inactive fusion protein (AvaI was not blocked at either the target or off target sites and XbaI cut the plasmid)
![Workflow](https://static.igem.org/mediawiki/2013/1/11/Workflow_Schematics_copy.jpg)