Team:UBonn HBRS/Description/Binding Assays
Binding Assays
To determine the impact of our final product we were planning to perform binding assays to analyse biofilm formation of Streptococcus mutans on a pellicle protein coated surface.Obtaining pellicle proteins
To obtain the pellicle proteins, saliva from a single donor was collected. The donor was not allowed to eat 4 hours before the donation and only drunk water in this period. The saliva was filtered over a sterile 0.22 µm PES low protein-binding filter unit. The saliva was diluted 1:1 (v/v) in adsorption buffer.
During the procedure, the solutions were constantly kept on ice to prevent protein degradation. One batch of pellicle proteins was always freshly prepared for each experiment.
Binding Assay
We used the study from Lemos et. al (2011) as an inspiration of our assay.
For the binding assay a sterile 96-well plate was coated with sterile saliva at 37°C overnight. BHI-Medium was inoculated with our strain of S. Mutans to serve as a preculture for the further experiment.
The saliva solution was blotted out, washed 2 times with ddH₂O and a few wells were tested for protein content perorming a Bradford assay. 90 µL of BHI medium + 10 µL pre-culture (OD600nm adjusted to 0.5) were transferred into the empty wells and grown at 37°C for 24h.
The medium was removed and the wells were washed 2 times with ddH₂O. 50 µL of 0.1% crystalviolet solution were added to the wells and incubated 15 minutes. Afterwards the wells were washed twice with ddH₂O again. The remaining crystal violet is therefore caught in the cells still adsorbed to the plate. For eluting the dye, the wells were treated with 200 µL of 33% acetic acid and incubated for 10 minutes. Afterwards the solution was transferred to a new plate and the OD575nm was read. This value was characterised by previous studies (Lemos et. al. 2011).
Uncoated wells with bacteria and coated wells with/without bacteria were included as controls.
Due to problems in the cloning part of our project, we did not manage to run the assay in quantitative numbers.
Preliminary Results are suggesting that the assay works, however further experiments need to be done to confirm this and show the significance of the results.
For the future, we are planning on including controls of other Biofilm inhibiting substances and compare it with our end product.
Media
Adsorption Buffer (AB)
pH adjusted to 6.5. Stored at room temperature.
| Substance | Concentration |
|---|---|
| KCL | 50 mM |
| K₂PO₄ | 0.35 mM |
| KH₂PO₄ | 0.65 mM |
| CaCl₂ | 1 mM |
| MgCl₂ | 0.1 mM |
References
- 1. Lemos et. al 2011 “Protocols to Study the Physiology of Oral Biofilms” https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3130507
