Team:Slovenia/METHODS and PARTS/protocols/ba

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Revision as of 17:15, 27 October 2010

Fun fact:



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). ImmunoPureò 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.