Team:Slovenia/Methods.html

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Methods

CLONING, PROTEIN EXPRESSION AND PURIFICATION
The genes we used were of two different origins, synthetic or obtained by PCR. Coding sequences were cut according to our improved BioBrick standard and inserted in our functionalized vector (BBa_K245005). This enabled us to combine different parts in a desired way. The four new inner restriction sites added in multiple-cloning site (NgoMIV and AgeI in prefix; XmaI and BspEI in suffix) enabled an extension of part cloned into BBa_K245005 with SG on N-terminal or TG on C-terminal; extension of linker and creating a friendly scar during parts fusion thus allowing flexibility. The functionalized vector BBa_K245005 was designed in such way, that genes cloned into this vector gained His tag on N-terminal. DNA sequences of synthetic origin had to be optimized for protein expression in desired hosts (Escherichia coli). They were designed from amino acid sequences with tool program Gene Designer from DNA2.0 Inc., version 1.0.0.1 (DNA2.0 Headquarter 1430 O'Brien Drive, Suite E, Menlo Park, CA 94025, USA). Undesired restriction sites in coding sequences of proteins were also eliminated using this program. Genes were then ordered from MR.Gene GmbH (Im Gewerbepark B32, D-93059 Regensburg) and cut out from their vector with restriction endonucleases. DNA constructs were prepared using methods of molecular biology that are basically described in molecular biology handbook (Sambrook et al., 1989). After DNA sequence verification E. coli BL21(DE3) pLysS were transformed with individual construct for the overexpression of protein. Overnight cultures were diluted and grown in LB media (Luria-Bertani) supplemented with ampicillin at 37 °C with shaking at 180 rpm. To induce protein expression 1 mM IPTG (isopropyl β-D-1-thiogalactopyranoside) was added when OD600 was 0.7-1. Bacterial cells were further cultivated for four hours and then harvested by centrifugation (5000g, 10 min at 4 °C). Bacteria were resuspended either in lysis buffer (0.1% sodium deoxycholate, 10 mM Tris/HCl, pH 8.0) or dissolved in a buffer with a denaturant. Protease inhibitors (CPI, Sigma) were added to the lysate and homogenized by sonication. After centrifugation, supernatant was separated from the pellet. Our proteins were either in the soluble fraction or in inclusion bodies in the pellet. Isolation of soluble proteins was performed on a Ni-NTA column under native conditions.I Inclusion bodies were washed several times and if necessary dissolved in denaturant and purified on Ni-NTA column under denaturing conditions. Purity and integrity of proteins was assessed by SDS-PAGE and Western blot.

SDS-PAGE and WESTERN 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 they 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 gel 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 using non-fat dry milk 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 4oC with gentle shaking. After rinsing the membrane to remove unbound primary antibody, the membrane is exposed to secondary horseradish peroxidase-labelled antibody for 1 h at room temperature with gentle shaking. The signal was finally detected on the specialized photographic film using a chemiluminescent substrate.

UV/VIS SPECTROSCOPY
UV/Vis spectroscopy is a method that uses light in visible and UV ranges to determine the quantity of a given compound. We measured spectra of protein solutions which reveal the maximum absorption at wavelength of 280 nm.

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 190 nm and 250 nm on a Chirascan CD spectrometer (Applied Photophysics, UK). The path length of cell used was 1 mm, sample concentration was 0.1 - 0.5 mg/ml in MQ water or in proper buffer.

GEL FILTRATION
Gel filtration is a method in which molecules are separated based on their size. An aqueous solution is used to transport the sample through the column and due to different abilities of various sample molecules to enter pores of column they are separated according to their size. We used this method to detect monomers or multimers of our protein present in a sample. Gel filtration was performed on HPLC (high-performance liquid chromatography) system (Agilent 1100) using column of BioSep-SEC S4000 (Phenomenex).

AFM
Atomic force microscope (AFM) is a high resolution type of scanning probe microscope. Piezoelectric elements move the probe over the sample in a raster pattern and force between the tip and the surface is measured. This is later used to form an image of the relief of the specimen. Because the method shows the surface the sample has to be fixed on a very even medium (for example mica). A drop of protein sample was put on freshly cut mica (grade V1, Ted Pella) and left to adsorb for 10 min. After that the sample was washed several times with MQ water and dried under nitrogen stream. Protein structures deposited on mica were observed by Agilent Technologies 5500 Scanning Probe Microscope operating in acoustic alternative current AFM mode using silicon cantilever PPP-NCH with force constant 42 N/m and tip radius less than 7 nm (Nanosensors).

TEM
Transmission electron microscopy (TEM) is a microscopy technique where a beam of electrons is used to generate an image of the sample. Electrons pass trough a thin sample, which has been previously fixed on a special grid and stained with uranyl acetate, interact with the sample and are detected via fluorescent screen, photographic film or CCD camera. Holey formvar carbon-coated copper grids (SPI supplies) were placed on the drop of protein sample for 3 minutes. The samples on grids were negatively stained with 1% (w/v) aqueous uranyl acetate and observed under Philips CM 100 electron microscope operating at 20 keV. Some experiments were performed on JEM-200FX microscope.

SEM
Scanning electron microscopy (SEM) is a microscopy technique where an image is generated by scanning the sample surface with a high energy beam of electrons. We used this technique to determine the surface of the protein membranes. Samples were put on sample holder and dried. Samples were observed by SUPRA 35 VP electron microscope.

DLS
Dynamic light scattering (DLS) is a technique which can be used to determine the size distribution profile of polymers in solution. When the light hits particles the light scatters in all directions and one observes a time-dependent fluctuation in the scattering intensity. The dynamic information of the particles is derived from an autocorrelation of the intensity trace recorded during the experiment. After complex data analysis the hydrodynamic radius (RH) is obtained. We used this method to verify the RH of our protein assemblies. We measured our samples on instrument 3D-DLS (LS Instruments, CH).

MASS SPECTROMETRY
Mass spectrometry (MS) is an analytical technique also used for elucidating the chemical structures of molecules, such as peptides and proteins. The MS principle consists of ionizing chemical compounds to generate charged molecules or molecule fragments and measurement of their mass-to-charge ratios. From these data molecular mass can be calculated. We used this method to verify the right molecular mass of produced proteins.

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