Team:Penn/Parts

Parts Submitted

CpG Methylase M.SssI (BBa_K1128000)

This protein de novo methylates CpG sites. It is derived from Spiroplasma, and functions by covalently bonding a methyl group (-CH₃) to the cytosine residue within the site. CpG methylation, which normally ocurs in eukaryotes, is completely orthoganol to methylation by the DNA Adenine Methyltransferase enzyme found in E.Coli, which methylates GATC sites, and can be used for in vivo methylation studies in E. coli. It should be noted that CpG methylated DNA in E. coli is subject to Mcr and Mrr restriction, and should be used only in Mcr- and Mrr- E. coli strains.

CpG Methylase M.SssI with Linker (BBa_K1128002)
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For full characterization see BBa_K1128000. This is an M.SssI methyltransferase protein with a linker on the N terminus. The part is ready for fusion to transcription factors. The linker is composed of a string of glycine residues.

MaGellin Plasmid Backbone (BBa_K1128001)

This part allows a user to quickly and easily screen site specific methylating proteins for activity and specificity. It uses a modified version of Novagen's pET-26(b)+ vector with additional sites cloned in, as well as the standard BioBrick prefix and suffix for cloning in standard parts from the registry. The plasmid backbone contains two AvaI restriction sites, which are composed of CYCGRG and are cut by the AvaI restriction enzyme. These sites can be CpG methylated, which blocks the activity of the AvaI enzyme. The backbone also contains a 9 base pair region that can be removed and replaced. This site is upstream of one of the AvaI sites and can act as a transcription factor "target site", used to determine where certain DNA transcription factors bind. It has the BBa_J04450 insert, which is an RFP expression cassette with a pLac promoter. This backbone has a lac repressor gene, and will NOT express the RFP in vivo. The backbone has a gene for Kanamycin resistance.