Ljubljana/methods
From 2007.igem.org
Parts Design
Since almost all of the parts used by our team had to be designed de novo, a huge portion of our time in the lab was spent on cloning. For each basic part, we designed primers containing standard BioBrick restriction sites. We had to introduce point mutations into some genes, because they contained one or more standard restriction sites inside the coding region. These mutations were introduced by PCR-driven overlap extension method (REFERENCA).
The genes, amplified by PCR, were therefore flanked by XbaI site upstream and SpeI, NotI and PstI downstream of the gene. After restriction, fragments were cloned into BBa_J52017, a vector already containing a eukaryotic terminator sequence downstream of the cloning region. This vector has previously been constructed by the Slovenian iGEM 2006 team.
In addition to basic parts, we also designed dozens of composite parts, mainly constructs expressing chimeric proteins. Composite parts were assembled by three-point ligations (REFERENCA?), leaving 6 nucleotides long SpeI-XbaI mixed restriction site inbetween the two domains. Some of our composite parts contain as many as 6 basic parts. Such composite parts were constructed by repeating three point ligations.
Cell cultures
In our experiments two different cell lines were used: human embrional kidney cell line HEK293T and human cervical cancer cell line HeLa. Cells were grown in DMEM medium containing 10% FBS and antibiotics streptomycin and penicillin, except during transfection when antibiotics were absent from the medium. For luciferase assays, cells were tipically seeded in 96-well plates, for microscopy in slide chambers, whereas for Western blotting and cytometry, where larger numbers of cells were needed, they were grown in 6-well plates, 35 mm dishes or even 10 cm2 dishes. Depending on the type of experiment Lipofectamine 2000 or GeneJuice were used for transfection according to manufacturer´s protocol. Cells were transfected with the desired plasmids, stimulated if necessary and left grown before detection.
Bioluminescence
Bioluminescence is emission of light, produced by enzymatic reaction. In molecular biology, genes coding for enzymes, which catalize reactions where light is emitted, are used as reporter genes. The most widely used reporter enzymes are luciferases.
In our experiments we used firefly luciferase placed under a specific inducible promoter of interest. The amount of sythesized luciferase was therefore dependent on the activation rate of cells and was semiquantified by measuring emitted light after substrate cleavage. As the number of cells and transfection efficiency vary, the results were normalized by a second reporter luciferase (the Renilla luciferase) placed under a constitutive promoter (expression of which is therefore cell activation independent) and with specificity for a different substrate. The measurements were conducted directly on 96-well plates in a luminometer.
Luciferase assay proved to be our most valuable tool due to the extremely low detection limits. We used this assay to test both our split protein and HIV-protease systems. Cells were transfected with CD4 and CCR5 receptors fused with components of split ubiquitin or split TEV protease systems and reporter constructs. T7 promoter-luciferase reporter construct was used to measure the cellular response to viral attack. HIV surface protein g120 induces cleavage of of T7 RNA polymerase fused with a transmembrane segment and its translocation to nucleus, where it starts to transcribe the reporter gene.
Western blotting
Western blotting is a method of protein transfer from SDS-PAGE (SDS-polyacrylamide gel electrophoresis) gels to nitrocelulose membrane, which enables detection of proteins of interest in cell lysates using specific primary antibodies (pAb) and enzyme-conjugated secondary antibodies (sAb), which bind to pAb. In the first step, transfected cells grown in cell cultures were lysed and the insoluble membrane fraction was removed by centrifugation. Loading buffer containing SDS and a reducing agent was then added to the cleared lysate and the samples were denatured by boiling. Next, samples were applied to PAGE, where proteins were separated according to their molecular weight. Separated proteins were then transferred to a nitrocellulose membrane by electrical current. 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 by 5% non-fat dry milk in order to prevent unspecific binding of antibodies to the membrane. Then, the nitrocellulose membrane was incubated with pAb for 1 h at room temperature or overnight at 4oC with gentle shaking. The membrane was then washed to remove the unbound pAb and incubated with horseradish peroxidase-labelled sAb for 1 h at room temperature with gentle shaking. The signal was finally detected on the film by using a chemiluminescent substrate.
Western blotting proved to be a very useful technique for determining whether proteolytic cleavage has occured. In the split ubiquitin assay, CMV-CD4-CUb-GFP and CMV-CCR5-NUb constructs were used. By comparing to molecular weight standards, we could detect whether the GFP was cut off from the transmembrane segment or remained attached to it when using anti-GFP antibodies. This method was also used in the HIV-protease project; the construct CMV-CD4-cleavage site-GFP was used and again GFPs of different sizes were detected when cotransfection with plasmid expressing HIV protease was performed.
Confocal microscopy
The basis of fluorescence as a technique is excitation of a fluorophore, which then emits light of a specific wavelenght. The main feature of this technique is its very high sensitivity. Confocal microscopy enables three dimensional detection and allocation of flourescent molecules inside the cells. This technique was used to determine the location of CD4 fused to mCerulean or mCherry inside the cells. Additionally, microscopy of cells after transfection with CD4 and CCR5 receptor constructs fused with split protein systems or HIV-protease cleavage site was used to show that after cell activation (with gp120, pseudoviruses or HIV protease) cleavage of mCerulean or mCherry from the receptor occured. Thus, we can expect that the same occurs when T7 polimerase is fused instead of mCerulean or mCherry.
In our experiments, cell membranes were stained with SynaptoRed C2 (5 mM) for 5 minutes at 25 μM concentration, endoplasmic reticulums with ER-Tracker Red (1 mM) for 10 minutes at 2,5 μM and lysosomes with LysoTracker Green (1 mM) for 5 minutes at 1 μM. Cells were viewed using a confocal microscope with an oil-immersion objective. The 458, 488, 514 (all multi-line Argon laser) and 543 (He-Ne laser) wavelengths were used to excite mCerulean, LysoTracker Green, SynaptoRed C2 and mCherry (or ER-Tracker Red), respectively.
Flow cytometry
Flow cytometry is a method which allows monitoring of cells in a flow stream on the basis of light scattering and fluorescence on single cell level. This method allows measuring of more parameters in a time and determination of a share of cells with a specific characteristic. There is a possibility of wide range of applications, however, we used two: annexin V and 7-AAD stainings for detection of apoptotic cells and antibody staining for detection of surface expression of transmembrane proteins.
Apoptotic cells are characterized by certain morphological changes, one of them being translocation of phosphatidylserine (PS) from inner to the outer layer of plasma membrane bilayer. This change occurs early in the process. Annexin V is a protein, which binds specifically to PS. As this translocation of PS occurs early in the process, Annexin V is a sensitive probe to detect early apoptotic cells. Annexin V can be conjugated to a fluorochrome and thus detected by means of flow cytometry.
In addition to staining with Annexin V-PE (emission 575 nm), cells were also stained with 7-Amino actomycin (7-AAD, emission 670 nm), a dye that binds to necrotic cells. Staining of cells with both markers allows differentiation between early apoptotic cells, as they stain positive only for annexin, and necrotic cells, which stain positive for 7-AAD or both. This system was also used to present the idea as a whole - by using an effector protein which is an activator of apoptosis (such as caspase-3) as a reporter, binding of the virus to CD4 and CCR5 receptors expressed on cells would lead to their death even before the virus itself is able to replicate. This would then be reflected in positive staining of cells as detected by flow cytometry.
Antibody staining was used for detection of transmembrane receptors CD4, CCR5 and CXCR4 on cell surfaces. For this purpose composite parts with receptors and Myc or HA tags, which flutter on the cell surface were designed. After transfection with plasmids expressing these receptors, cells were incubated with primary anti-tag antibodies, and then with secondary antibodies, which bind to constant, species-specific region of primary antibodies. Secondary antibodies were conjugated with a fluorophores (PE or DyeMer), which emit light at 575 nm and 610 nm, respectively. While using specific anti-Myc or anti-HA tag antibodies only cells transfected with our receptor constructs and not endogenous receptors were detected.
ELISA
ELISA (enzyme-linked immunosorbent assay) is an immunological technique which enables semiquantitative determination of the amount of molecules of interest in the cell lysate using specific antibodies. Antibodies are bound to the surface of a microtiter plate and molecules of interest present in the cell lysate bind to them. The amount of bound molecules is detected using another primary antibody conjugated with biotin. Streptavidin conjugated with horse radish peroxidase specifically interacts with biotin. Peroxidase enzymatically cleaves a chromogenic substrate and the absorbance is measured. ELISA was used to determine the amount of caspase-3 protein after the activation of the engineered system with HIV protease, gp120 or pseudoviruses.
