Part:BBa_K165006:Experience

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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 betagalactosidase as a reporter.

SDS-page and Western blot:

HivC was produced in E.coli BL21(De3)pLysS and further purified. For isolation, purification and experimental purposes, we deposited a modified variant of HivC termed cCFP_link_HivC (BBa_K323071). Above 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 below) 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]

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 cCFP_link_HivC binds specifically to its target DNA sequence. Figure below 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.

Betagalactosidase activity:

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

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