Proof of concept
Proof of concept
In order to verify perspectives of our idea, the proof of concept is essential step in our work. The realisation of our project’s feasibility was achieved by:
- Showing that the PAL enhances cell’s activity to reduce concentration of L-Phe in extracellular environment.
- Validating that synthesis of Polyphe protein in cell increases its consumption of L-Phe.
Experiments and results
One of the most essential ideas in our project is to create live system, which could easily reproduce and live in guts and be beneficial for PKU patients. To fulfil this idea, our final system was tested in E. coli chasis organism. Firstly, we proved that our recombinant bacteria, which express PAL, has better characteristics to reduce concentration of phenylalanine. The whole experiment was conducted by growing cells in a special synthetic medium. After that cells were collected and submerged in a solution, which acted as a simulation of a gastrointestinal tract environment, by mimicking the pH 7.4, the amount of daily phenylalanine intake (1.1 g) and temperature (37ºC). By measuring trans-cinnamic acid with spectrophotometer at 280 nm we collected data, which proved that 5 g of E. coli with PAL enzyme overcomes control cells activity by reducing almost one-fifth of daily phenylalanine intake. In addition, the improvement of whole system with the membrane permease PheP, which increases the transfer of L-Phe into cell, even more validates prove of our concept.
Phenylalanine reduction activity of E. coli cells improved with PAL and PAL+PheP over 20 minute period in human’s gastrointestinal conditions.
Our demonstrated new approach for reduction of phenylalanine by creating a synthetic proteins derived from Gp45 and Csm4, which are enriched in phenylalanine also proved their ability to minimize target amino acid in reaction mixture. The recombinant cells were grown in synthetic medium and after 12 hours they were induced in order to begin translation of synthetic proteins. After 30 minutes of incubation those cells in 37ºC, pH 7.4, with a daily amount of phenylalanine 1.1 g, the results verified that 2.5 grams of E. coli cells with Gp45 (37) protein managed do decrease nearly 20 % of daily phenylalanine intakes. Csm4 (11) and Csm4 (42) showed slightly lower activity, but all those result are significantly better when compared with control.
Cell activity in reduction of phenylalanine in growth medium while expressing PolyPhe over 30-minute period in human gastrointestinal conditions.
The realisation of projects feasibility was proved by demonstrating activity in reduction of L-phenylalanine by two different approaches: one with PAL enzyme and other by expression of novel synthetic proteins highly enriched in phenylalanine amino acid.