Team:Slovenia/METHODS and PARTS/protocols

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Cloning & Proteins Binding Assays Biosynthesis Methodology Split/FRET Methodology protocols - Cloning & Proteins

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Cloning of BioBricks

We used synthetic and PCR amplified genes, or constructs obtained from the Registry. All DNA sequences were constructed and assembled to conform BioBrick standard. The DH5α E.coli strain was used as a host for cloning of all parts, with the exception of plasmids containing the ccdB domain, in which case we used the DB3.1 E.coli strain. The sequence of final constructs was verified by sequencing.

Protein expression and isolation protocol

Plasmids encoding fusion proteins were transformed with chemical transformation into competent E. coli BL21 (DE3) pLysS cells. Selected bacterial colonies grown on LB plates with an appropriate antibiotic (ampicillin) were inoculated into 10 mL of LB broth supplemented with antibiotic. After several hours of growth at 37°C 10-100 µL of the culture were inoculated into 100 mL of growth media and left overnight shaking at 37°C. The overnight culture was diluted 20-50-times reaching the OD₆₀₀ of diluted culture between 0.1 and 0.2. Culture flasks with 500 ml of diluted culture were put on the shaker and bacteria were grown at 37°C until OD₆₀₀ reached 0.6-0.8, when protein expression was induced by addition of inducer IPTG (1 mM). Four hours after induction culture broth was centrifuged and bacterial cells were resuspended in lysis buffer (10 mM Tris pH 8.0, 1M NaCl, 0.1% deoxycholate, 0,1mM zinc sulphate, DTT and supplemented with protease inhibitor cocktail) and frozen at -80°C for at least overnight. Thawed cell suspension was lysed with sonication and then centrifuged. Precipitate (cell membranes, inclusion bodies) and supernatant were checked for expression of our constructs by SDS-PAGE and Western blot, using anti-His-tag antibodies as primary antibodies when necessary. Designed fusion proteins were mainly present in insoluble part (inclusion bodies), which was composed of >80% of the chosen protein. Inclusion bodies where dissolved in 8 M urea in 50 mM Tris pH 8.0. Purification under denaturing conditions was performed according to manufacturer’s instructions. Protein refolding was carried out by dialysis against buffer containing 50 mM Tris pH 8.0, 500 mM NaF, 500 µM ZnSO₄, 5 mM DTT, and 10 % glycerol. Dialysis was carried out at for 12h at 4 °C with no mixing. Upon completion of the process the dialysate was carefully removed from the dialysis tubes and placed into centrifuge tubes. Protein that failed to refold precipitated and was separated from the refolded protein in the soluble phase via centrifugation at 10,000 rpm. Protein concentration was determined using spectrophotometer and Bradford protein assay and specific protein size determined by SDS-PAGE/commasie stain and Western blot analysis using anti-His antibodies.

SDS-PAGE and westeren blot

SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) is a widely used technique which separates proteins according to their molecular weight. Western blot is an analitical method used for detection of specific proteins. Proteins are transfered from SDS-PAGE gel to nitrocelulose membrane and are probed using antibodies specific to target protein. Samples to be analyzed were dissolved in sample buffer and denatured by boiling. SDS-PAGE was performed and separated proteins were transferred to a nitrocellulose membrane using electroblotting. The efficiency of the transfer was determined either by staining the membrane with Ponceau dye or by using the pre-stained molecular weight markers, which are visible on the membrane after the transfer. After blotting, the free binding sites on the membrane were blocked with i-Block solution in order to prevent unspecific binding of antibodies to the membrane. Then the nitrocellulose membrane was incubated with primary antibody for 1 h at room temperature or overnight at 4°C with gentle shaking. After rinsing the membrane to remove unbound primary antibody, the membrane was exposed to secondary horseradish peroxidase-labelled antibody for 1 h at room temperature with gentle shaking. The signal was finally detected using chemiluminiscence imaging system.

Circular dichroism

Circular dichroism (CD) is a type of spectroscopy based on the differential absorption of left- and right-handed circularly polarized light. The far-UV CD spectrum of proteins can reveal important characteristics of their secondary structure. We used this method to confirm that intact protein with its proper secondary structure was isolated. CD spectra were taken in the far-UV region between 200 nm and 280 nm on a Chirascan CD spectrometer (Applied Photophysics, UK). The path length of the cell was 1 mm, concentration of proteins was between 0.1 and 0.5 mg/ml in 10mM Tris buffer.