Difference between revisions of "Part:BBa K323058"

Revision as of 08:44, 27 October 2010

Gli1 link nCFP under T7 promoter

[http://2010.igem.org/Team:Slovenia Team Slovenia 2010] further characterized Gli-1 DNA-binding protein BBa_K165007. Gli-1 belongs to zinc finger family of proteins. Zinc fingers are small protein structural motifs that can coordinate one or more zinc ions to help stabilize their folds. They can be classified into several different structural families and typically function as DNA binding proteins. Zinc fingers coordinate zinc ions with a combination of cysteine and histidine residues and can be classified by the type and order of these zinc coordinating residues. Gli-1 is composed of five tandem fingers that bind to DNA sequence: GAC CAC CCA AGA CGA. For isolation, purification and other experiments we added HIS tag to N-terminal side of Gli1 sequence and N-terminal split CFP on C-terminal part of Gli1 after an extension linker. We deposited a sequence for a modified variant of Gli1 as Gli1_link_nCFP BBa_K323016 and additionally placed it under T7 promoter for expression in production E.coli BL21(De3)pLysS strain and further characterized it with several methods, including SDS-page, Western Blot and circular dichroism spectroscopy. In vitro binding of Gli-1 to target DNA sequence was determined with surface plasmon resonance (SPR) and in vivo with betagalactosidase assay as a reporter. See results under [Experience.

Sequence and Features