Proof of concept

Protein Attachement

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:

1. mSA2-mGFPuv2
2. mGFPuv2 (control without the biotin binding streptavidin)

Setup

Results

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:

1. INP + INP_NC-mSA2: whole cells expressing both full length INP and membrane-bound monomeric streptavidin (mSA2)
2. RFP + INP_NC-mSA2 (control): cells expressing membrane-bound mSA2 and a red fluorescent protein
3. INP_RC-mSA2: protein extract: INP nucleating domain - mSA2 fusion protein
4. 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.