Team:UGent Belgium/Proof
Proof of concept
Protein Attachement
Setup
To show we were able to attach a protein to our biotinylated PLA we made mGFPuv2 containing fusion proteins. The attachment of the fusion proteins was tested in 2 different cases. On one hand the biotin impregnated filament (Filament page, Method 2) was used to apply the protein to and on the other hand glass slides coated with PLA and biotine as described on the Filament page, Method 3. After applying the lysates which contain the protein, the PLA is washed with physiologic solution.
Two biological treatments were used here:
- mSA2-mGFPuv2
- mGFPuv2 (control without the biotin binding streptavidin)
Results
After applying the biological treatments and a subsequent wash step, the filaments and the glass slides are put under a UV light (365 nm).
In picture (A), the filaments labeled with ‘10’ are coated with the mSA2-mGFUuv2 protein and filaments labeled “11” with mGFPuv2. There was no green color visible for either protein coatings. In picture B, the same lysates are applied to PLA + biotin coated glass slides. Here we clearly see under UV light that the mSA2-mGFPuv2 (left slide) sticks to the surface while the mGFPuv2 does not (right slide).
We prove in picture B that our fusion proteins can indeed stick to the biotinylated PLA but for the case of our filaments, this method seems not sensitive enough.
Therefor we conducted a second experiment with our filaments with a much more sensitive method described on the measurement page.
Setup
Same as previous, lysates are prepared and applied to the filament. Here we also used normal PLA without biotin as an extra control.
The biological treatments were:
- mGFPuv2 (control without the biotin binding streptavidin)
- mGFPuv2-Strept (fusion protein which should stick to the bitinylated filament)
- mGFPuv2-mSA2 (fusion protein which should stick to the bitinylated filament)
Our customized ELISA protocol (measurement page) was used to detect protein on the PLA.
Results
The results are displayed in the following picture. As expected we don’t see any/little coloring on the normal PLA filaments since no protein should attach without the biotin present. As for the biotinylated PLA, the two fusion proteins are indeed colored as expected. Unfortunately the biotinylated PLA with the mGFPuv2 applied also resulted in a colored filament. On our measurement page, a possible cause and solution is already given.
Water Collection
Before testing the biological treatments on the Dewpal water collectors, these were first put to the test in a more controlled setup using microscope slides. For this experiment, we used a mixture of PLA and biotin dissolved in dichloromethane. We dipped four microscope slides in the PLA-biotin solution and subsequently applied all biological treatments to each of the slides. Afterwards, all slides were rinsed thoroughly with physiological solution. The coated slides are left to dry overnight in a laminar flow cabinet, allowing the solvent to evaporate.
We considered four biological treatments:
- INP + INP_NC-mSA2: whole cells expressing both full length INP and membrane-bound monomeric streptavidin (mSA2)
- RFP + INP_NC-mSA2 (control): cells expressing membrane-bound mSA2 and a red fluorescent protein
- INP_RC-mSA2: protein extract: INP nucleating domain - mSA2 fusion protein
- mGFPuv2-mSA2 (control): mGFPuv2 - mSA2 fusion protein
In each experiment, the measurements were performed in a controlled humidified chamber. Both the temperature and the humidity were constantly measured and the latter was actively controlled using bang-bang control.
Setup
The slides were placed straight up in a falcon tube, allowing moist air to reach the treated surface. All four slides in a falcon were weighted on the analytical scale. The falcons were placed randomly in the humidified chamber for six hours. Their places were changed periodically to decrease the chance of confounding errors. Afterwards, the accumulation of water was determined for each experimental unit.
Results
The amount of water collected of the four slides, by treatment, is shown in the bar chart below.
The difference between the treatments is relatively small and the treatments with INP do not seem to collect more water. As for the previous experiment, this experiment could not prove that PLA bonded with INP could efficiently extract moisture from the air.
