Part:BBa_K165006:Experience

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Applications of BBa_K165006

[http://2010.igem.org/Team:Slovenia Team Slovenia 2010] further characterised HivC. HivC 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. HivC is composed of three fingers that bind to DNA sequence: GAT GCT GCA and is derived from zinc finger Zif268. HivC binding characteristics were analysed with several methods, including mobility shift analysis, surface plasmon resonance (SPR) and in vivo test of binding using beta-galactosidase as a reporter.

Production and isolation of HivC confirmed with SDS-page and Western blot

For isolation, purification and experimental purposes, we deposited a modified variant of HivC termed cCFP_link_HivC BBa_K323071. cCFP_link_HivC 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 presents SDS-page and Western blot for cCFP_link_HivC. Arrows on both figures indicate cCFP_link_HivC (22.164 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 cCFP_link_HivC refolds mostly in α-helical structure and some random coil.

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

In vitro study of HivC binding characteristics with surface plasmon resonance and electrophoretic mobility shift assay (EMSA)

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 cCFP_link_HivC binds specifically to its target DNA sequence. Figure left implays specific binding of HivC to target DNA.

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

Electrophoretic mobility shift assay (EMSA):

Principle of EMSA assay is a shift in DNA mobility as a result of protein binding. This shift can be observed under UV light after running samples of DNA with protein in agarose gel stained with ethidium bromide. EMSA proved that cCFP_link_HivC binds to its binding site. Test was repeated using three different quantities of target DNA. The control lane (DNA without proteins) contains a single band of target DNA, whereas a sample containing target DNA and HivC contains a band shifted upwards, because target DNA with bound HivC travels slower in agarose gel.

In vivo study of HivC 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 HivC 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 HivC to its binding site.

Reference:

Reynolds L., Ullman C., Moore M., Isalan M., West M.J., Clapham P., Klug A., Choo Y. 2003. Repression of the HIV-1 5LTR promoter and inhibition of HIV-1 replication by using engineered zinc-finger transcription factors. PNAS 2003 (100) 4:1615–1620

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