Part:BBa_K165007:Experience

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Gli1 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. Gli1 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 modified variant of Gli1 as Gli1_link_nCFP (BBa_K323016) and additionally characterized it with several methods, including SDS-page, Western Blot and circular dichroism spectroscopy. In vitro binding of Gli1 to target DNA sequence was determined with surface plasmon resonance (SPR) and in vivo with betagalactosidase assay as a reporter.

SDS-page and Western blot

Gli1 was produced in E.coli BL21(De3)pLysS and further purified. Above figure represents SDS-page and Western blot of Gli1_link_nCFP after purification. Arrows on both figures indicate Gli1_link_nCFP (42.303 kDa). Molecular weight was determined in silico, using sequence and ProtParam online tool. [http://2010.igem.org/Team:Slovenia/METHODS_and_PARTS Check out the protocol on Team Slovenia iGEM 2010 web site]

Circular dichroism spectroscopy:

Circular dichroism is a method that refers to the differential absorption of left and right circularly polarized light, a phenomenon exhibited in the absorption bands of optically active chiral molecules. Circular dichroism spectroscopy is usually used after protein purification to determine its secondary structure. Using CD spectroscopy we showed (figure below) that Gli1_link_nCFP refolds mostly in α-helical structure.

[http://2010.igem.org/Team:Slovenia/METHODS_and_PARTS Check out the protocol on Team Slovenia iGEM 2010 web site]

Surface plasmon resonance:

Surface plasmon resonance is a method used for qualitative and quantitive analysis of molecular interactions, in our case binding of zinc fingers to DNA. We used surface plasmon resonance to demonstrate if Gli1_link_nCFP binds specifically to its target DNA sequence. Figure below implays specific binding of Gli1 to target DNA.

[http://2010.igem.org/Team:Slovenia/METHODS_and_PARTS Check out the protocol on Team Slovenia iGEM 2010 web site]

Betagalactosidase activity:

Since all above experiments are done in in vitro system, we further characterise if Gli1 binds to its target binding site in vivo. For that purpose betagalactosidase assay was used where instead of lac operator Gli1 binding sequence was inserted. Below figure represents binding of Gli1 to its binding site, resulting in transcription inhibition, lower betagalactosidase production and as a result lower betagalactosidase activity.

Reference:

Kasper M., Regl G., Frischauf A., Aberger F. 2006. GLI transcription factors: Mediators of oncogenic Hedgehog signalling. EUROPEAN JOURNAL OF CANCER 42 (2006) 437–445

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