Constucts used in individual detection system and short description of detection systems:

Flow cytometry

Used composite parts: NFκB-MyD-Luc ([http://partsregistry.org/Part:BBa_J52014 BBa_J52014]).

Figure 1: Ota working on the flow cytometer.

Flow cytometry is a sophisticated method, which gives very precise and highly valuable results. The main advantage of this method is that measurements are done on individual cells, so the results are not the average value of the population. In our project we could detect cellular activation based on the phosphorylation cascade activation and anti-phosphoprotein antibodies. In our experiments we used primary antibodies (Ab) from mouse, which specifically detect only phosphorylated form of ERK kinase (pERK). The secondary antibodies used were anti-mouse Abs labelled with a fluorescence marker phycoerithrin (PE), which we can detect on flow cytometer. Cells were transfected with TLR5 or TLR4/MD-2 and with or without dnMyD88, which is under NF-κB promoter. On the second day the cells were stimulated with flagellin or LPS (depends on the selected TLR receptor). According to the literature phosphorylation of ERK can already be detected after 15 min. The amount of pERK kinase increases for additional 20 min and then it starts to decline. After 40 min no pERK is detected.

We used pulsed stimulation of cells. First we stimulated cells with 5ng/ml of flagellin to activate the expression of dnMyD88. We repeated the stimulation with the second, stronger stimulus (50ng/ml of flagellin) at different time intervals (2, 4, 6 and 8 hours after the first stimulation. 30 min after the second stimulus cells were fixed in 2% paraformaldehid, permeabilized with ice cold methanol and stained for pERK and secondary Ab labelled with PE.

Expected results: The activation of the signaling pathway also activates the expression of dominant negative protein MyD88, which should block the signaling, so we expect to detect decreased amount of pERK kinase after second stimulation in comparison to cells without of inducible dnMyD88.

Luminometry

Used composite parts: NFκB-MyD-luc ([http://partsregistry.org/Part:BBa_J52014 BBa_J52014]), NFκB-MyD-luc-PEST([http://partsregistry.org/Part:BBa_J52024 BBa_J52024]), NFκB-luc-PEST([http://partsregistry.org/Part:BBa_J52023 BBa_J52023]), CMV-luc([http://partsregistry.org/Part:BBa_J52038 BBa_J52038]) and CMV-luc-PEST([http://partsregistry.org/Part:BBa_J52039 BBa_J52039]).

Figure 2: Mithras luminometer.

Luminometry is a very used method to measure activation of signaling pathways. In many cases it replaced ELISA because it is much more sensitive. The basis of the method is the measurment of light, which is emited after substrate cleavage. We used two different enzymes (firefly luciferase - fLuc and renilla luciferase - rLuc), which recognize and cleave different substrates, so we can measure the activity of both in one sample.

Different variants of the method were used:

1. To be able to measure the activation of dnMyD88 expression after stimulation, we prepared dnMyD88 construct under NF-κB inducible promoter in the fusion with rLuc (construct NF-κB-MyD-luc). We transfected cells with TLR receptor plasmid, biobrick and fLuc under NF-κB promoter plasmid as a positive control. Cells were stimulated and the expression of luciferases was measured in time intervals. We expect that amount of rLuc will increase in time only if cells are stimulated with a ligand.

2. CMV-luc-PEST biobrick was constructed to measure the kinetics of degradation of proteins in fusion with PEST. Cells were transfected with the construct, which is under constitutive promoter. We found out that it takes four hours after the transfection to detect luciferase activity. To calculate half-life of rLuc and rLuc-PEST we transfected cells with those two constructs, incubated to allow proteins to sythesize. New protein synthesis was inhibited by adding cycloheximide and in time intervals the activity of rluc was measured. Because the addition of cycloheximide stops the sythesis of new proteins after time there should be a decrease in rLuc activity due to protein degradation. A construct with PEST should decrease more rapidly as rLuc alone.

3. The main experiment was....

ELISA

Figure 3: Schematic representation of the detection of free NFκB, which can bind to the promoter sequence.

Used composite parts: NF-κB–dnMyD88 ([http://partsregistry.org/Part:BBa_J52036 BBa_J52036]) and NFκB–TRAF6–GFP ([http://partsregistry.org/Part:BBa_J52022 BBa_J52022]).

With ELISA detection system we can detect the free NF-κB transcription factor, which is one of the most direct measurements of the pathway activation. Active form of this protein is present after the activation of signaling pathway when the protein is released from the complex with IκB inhibitor. Free NF-κB migrates into the nucleus where it acts as a transcription activator. We are using high bind 96-well plates coated with [http://en.wikipedia.org/wiki/Streptavidin streptavidin](we prepare it ourselves). The probe for free NF-κB is biotinylated double stranded NF-κB binding sequence. The NF-κB protein from whole cell lysate is captured by a probe, whose biotin group binds to the streptavidin coated on the plate. We detect NF-κB with primary mouse antibodies and secondary peroxidase-conjugated rabbit anti-mouse antibodies. Peroxidase cleaves the substrate which emits light, which we measure on the luminometer.

Confocal microscopy

File:Celica2.gif

Microscope allows localization of signaling molecules within each individual cell, such as translocation of transcription factors, trafficking of proteins fused with reporter fluorescent proteins etc. Therefore this technique should be very usefull particularly for monitoring the expression of dnMyD88 inhibitory domain fused with fluorescent protein and the timecourse of its degradation. Theerfore we designed Parts and several Devices, which included fusion with fluorescent protein:
<tr><td>[http://partsregistry.org/Part:BBa_J52021 BBa_J52021]</td></tr> <tr><td>[http://partsregistry.org/Part:BBa_J52021 BBa_J52022]</td> <tr><td>[http://partsregistry.org/Part:BBa_J52021 BBa_J52026]</td> <tr><td>[http://partsregistry.org/Part:BBa_J52021 BBa_J52027]</td> <tr><td>[http://partsregistry.org/Part:BBa_J52021 BBa_J52040]</td> <tr><td>[http://partsregistry.org/Part:BBa_J52021 BBa_J52048]</td>.

Problems and Troubleshooting

Construction of Parts using bad oligos
We can only suggest to the other teams in the future to order oligos from a reliable and well established company (not necessarily the lowest price). When we repeatedly failed to produce PCR products it could have been the beginner's bad luck but when we determined a huge number of point mutations in the primer region even the mentors decided that we should better avoid this company in the future (although they offered us the replacement of oligos for free). There'll be no next time for this company (from one of our neighboring countries).

Theory versus practise
Parts Data are not necessarily true. We spent a lot of time to find an answer to the following question: "Why didn't our transformants grow on the LBTc plates?". First we doubted to our tetracycline, than to the propper concentration, but in the end we found out that one of the previous year's parts was false registered. Now we know the importance of checking everything twice...

Folding question
Does the linker allow efficient & undependent folding of both fusion parts?

Expression levels
Are low signals due to low expression levels of recombinant proteins in human cells? Is low expression a result of low efficient cell transfection?

Luminometry
We have attempted to monitor "real time" cell activation using luminescence. We could not measure luminescence in vivo since coelentrazine decays too quickly, while the luciferin could not penetrate the cells.

Optimization of activation intervals
Much od lab work (more than initially planned) was devoted to the optimization of cell activation. We had to find out the optimal concentration of inducers, timespan between the first and second activation, lenght of experiment in which difference between genetically engineered and wild-type cells would be sufficiently prominent etc. Methods from the literature could not simply be adopted into our experimental procedure.