Part:BBa_K165007:Experience

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Applications of BBa_K165007

[http://2010.igem.org/Team:Slovenia Team Slovenia 2010] further characterized Gli-1 DNA-binding protein. 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 sequence of a modified variant of Gli1 as Gli1_link_nCFP BBa_K323016 and additionally placed it under T7 promoter BBa_K323058 to ensure production in E.coli(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.

Production and isolation of Gli-1 confirmed with SDS-page and Western blot

Gli1_link_nCFP was cloned behind T7 promoter to ensure its strong production in E.coli BL21(De3)pLysS strain and further purified. [http://2010.igem.org/Team:Slovenia Team Slovenia 2010] also deposited this part. Left 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 right) that Gli1_link_nCFP refolds mostly in α-helical structure, what is known for zinc finger proteins.

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

In vitro study of Gli-1 binding characteristics with 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 target DNA. We used surface plasmon resonance to demonstrate if Gli1_link_nCFP binds specifically to its target DNA sequence. Figure left implays specific binding of Gli-1 to target DNA. [http://2010.igem.org/Team:Slovenia/METHODS_and_PARTS Check out the protocol on Team Slovenia iGEM 2010 web site]

In vivo study of Gli-1 binding characteristics with our device for testing binding of DNA binding proteins to their target sequences in vivo

Since all above experiments were done in in vitro system, we further characterise if Gli-1 binds to its target binding site in vivo. For that purpose we designed a device composed of several parts:

1. a synthetic promoter pSYN in which a DNA binding sequence, particular for each zinc finger to be tested, was inserted between -35 and -10 sites using BbsI restriction site (DTER_pSYN_BbsI, BBa_K323088).

2. lacZ reporter gene, which expression is controlled by pSYN

3. DNA binding protein (cut with XbaI/NotI) to be tested under arabinose inducible (pBAD) promoter in lacZ_DTER_pBAD_BsaI_DTER cut with BsaI BBa_K323089.

The successful binding of DNA binding protein to the synthetic promoter would prevent transcription of lacZ resulting in lower beta-galactosidase activity. Figure on the right represents binding of Gli-1 to its binding site.

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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