Team:UGent Belgium/Measurement
Measurements
Characterization of a new part: Truncated Ice Nucleation Protein as part of a fusion protein
Two aspects of a truncated INP fusion protein can be analyzed for normal functionality.
- Protein foldingw: to visualize correct folding of the protein we also fused, instead of the mSA2 protein, the mGFPuv2 protein to INP(RC).
- Ice Nucleating capability:
- Lysates were prepared as described on the SOP page
- Deionized water was super-cooled to approximately -10°C in a cooled 50% glycerol water bath.
- The capability of ice nucleation was tested in two ways:
- In the same tube, first 50 µL of lysate containing mGFPuv2 was added and subsequently 50 µL of one of the lysates with a INP(NC)-fusion protein.
- To prove the ice nucleation reaction is not just caused by increasing the volume from 50 to 100 µL with any lysate, 50 µL of the lysate containing mGFPuv2 was also added multiple times to the same tube.
The fusion with mGFPuv2 exhibits fluorescence and both the lysates with INP(RC)-mSA2 and mGFPuv2 were active as a nucleus for ice formation.
Methods to demonstrate the attachment of a protein to PLA filament
Fluorescent protein visualization
In this project a protein was attached to a PLA surface by taking advantage of the strong biotin – streptavidin binding. To prove the proteins do indeed stick to the filament, one option is to make fusion proteins containing both streptavidin and a fluorescent protein.
This was tested in 2 different setups. On one hand the biotin impregnated filament was used (Filament page, Method 2) and on the other hand glass slides coated with PLA and biotine as described on the Filament page, Method 3. For both cases, lysates containing mSA2-mGFPuv2 or mGFPuv2 were prepared as described on the SOP page. The lysates were applied to the PLA and afterwards washed with physiologic solution.
In the following picture (A), the filaments labeled with ‘10’ are coated with the mSA2-mGFUuv2 protein and filaments labeled “11” with mGFPuv2. Under UV light, there was no green color visible for either protein coatings. In the 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).
This tells us that making fusion proteins which include a fluorescent protein can work in some cases to prove the presence of a protein of interest, but clearly the method was not sensitive enough in case of our filament and creating fusion proteins to prove the binding of a protein can be very labor intensive.
Elisa
Because of the results with the fluorescent protein, a second, more sensitive method was though of which makes use of antibodies and an enzymatic reaction. Here we do not rely on the color/fluorescence of a protein itself for detection but on a color coming from an enzymatic reaction. Because many substrate molecules can be converted into a color by one enzyme, this intensifies the signal enormously.
The method is a customized ELISA protocol that can be adapted to detect any protein on any (3D printed) PLA structure without the requirement of an extra protein being fused to the protein of interest.
