Team:Slovenia/METHODS and PARTS/protocols/ba

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protocols - binding assays


Contents


β-Galactosidase enzyme assay

β-galactosidase has been commonly used as a reporter for measuring transcription strength of various promoters. We used β-galactosidase assay to measure activity of synthetic promoter comprised of lac operator in which a binding site for the DNA binding protein tested was inserted. Bacterial cultures carrying selected BioBricks were incubated in 10 ml LB broth supplemented with 5µl IPTG (1M) (isopropyl-β-D-thio-galactoside) and increasing concentrations of (0%, 0,0025%, 0,1% and 1%) L-arabinose at 37°C on a rotary shaker at 180 rpm for 18 hours. Bacterial density was determined by optical density (OD600). The measurements of β-galactosidase activity were done in an ELISA-reader, preheated on 28°C. 5 μl of each culture was transferred to a 96-well microtiter clear-bottom plate to which 100 μl of Z-buffer with chloroform (Z-buffer: 0,06 M Na2HPO4 x 7H20, 0,04 M NaH2PO4 x H20, 0,1M KCl, 0,001 M MgSO4x7H2O, pH 7; Z-buffer with chloroform: Z-buffer, 1% β-mercaptoethanol, 10% chloroform) was added. Bacterial cells were lysed by addition of 50 μl of Z-buffer with SDS (Z-buffer, 1,6 % SDS) followed by incubation for 10 min at 28°C. 50 μl of 0.4% ortho-Nitrophenyl-β-galactoside (ONPG) solution in Z-buffer was added to each well and changes in OD405 were measured for a period of 20 min at 30 sec intervals.

SPR - Surface plasmon resonance (Binding of Zn fingers to program DNA)

SPR is a method used for qualitative and quantitave analysis of molecular interactions, in our case binding of Zn fingers to DNA. SPR analysis was performed on Biacore T100 (GE Healthcare). ImmunoPure├▓ Avidin from hen egg white (Pierce) was immobilised on the surface of two flow cells of Series S sensor chip CM5 through the amine coupling following the protocol recommended by the producer. Briefly, the carboxymethylated surface was activated by 7 min pulse of 0.05 M N-hydroxysuccinimide and 0.2 M N-ethyl-N'-(3-diethylaminopropyl) carbodiimide and ethanolamine (pH 8.5) (GE Healthcare). The avidin in Na-acetate pH 5.5 was then injected across both flow cells to around 3000 response units (RU). Unreacted sites on the sensor surface were blocked with a 7 min injection of 1 M ethanolamine (pH 8.5). Single stranded biotinylated DNA was immobilized on the second flow cell to reach approximately 300 RU. The first flow cell was left unreacted and served as a control for the nonspecific binding of analytes to the dextran matrix of a sensor chip. Hybridization of program DNA was performed by injection of 0,5-2 μM of DNA in running buffer (10 mM HEPES, 150 mM NaCl, 0,1mM EDTA, 0.005 % P20, 2mM DTT in 10 mM ZnCl2, pH 7.4) for 300 s to get the final response of approx. 300 RU. Finally, purified ZnF were injected across such a surface for 1 min at concentration of around 1 µM at flow rate 30 µl/min and the dissociation was followed for at least 5 min. The regeneration of the surface was achieved with two 30 s injections of 50 mM NaOH and one 24 s injection of 0,5 % SDS. After that the new DNA was injected to obtain fresh binding surface.

EMSA – Electrophoretic mobility shift assay

Specific DNA binding of synthetic zinc finger domains to the program nucleic acid was tested by electrophoretic mobility shift assay. 1 µg of purified fusion proteins were incubated with various amounts of program nucleic acid sequence – 500ng, 750ng, 1000ng – for 3 hours. Samples diluted with high grade laboratory water to 20 µl were loaded on a 2,0 % agarose gel prestained with ethidium bromide and run at 70 V for 40 minutes. Nucleic acid – protein – complexes were detected under UV light.