The anaerobic bacterium Porphyromonas gingivalis is one of the main pathogens in the development of periodontitis. This oral inflammatory disease can lead to severe bone-loss of the teeth supporting tissues, leading to tooth loss. Recently, research from our department showed that P. gingivalis is associated to delayed epithelial wound healing as well. The goal of this project is to create a knock out bank of P. gingivalis and identify the genes which are associated with delayed epithelial wound healing.
This project includes two aspects:
1. Create a random gene knockout bank of P. gingivalis.
Via transposon mutagenesis using E. coli carrying a suicide plasmid and P. gingivalis, random knockouts will be created in P. gingivalis. PCR using semi-random priming will be performed in order to determine the chromosomal location of the transposon insertion.
2. P. gingivalis knockout screening with a high throughput in vitro wound healing assay.
Currently the in vitro wound healing assay uses a 24-well plate to identify influencing factors. A high throughput wound healing model using 96-well plates will be introduced and optimized in this project. This new model will be applied for P. gingivalis knockout screening.
Techniques that will be used are:
- transposon mutagenese
- anaëroob kweken;
- microscopisch onderzoek;
- cel migratie assay