Design

Our project has three main approaches which enable more efficient and liable performance of the designed probiotic.

PAL

The first approach is described by the expression of phenylalanine metabolizing enzyme – phenylalanine ammonia lyase (PAL). Originally, human body uses another enzyme called phenylalanine hydroxylase (PAH) to break down this amino acid; however, this enzyme requires oxygen as a co-factor and acts in the bloodstream. Normally, oxygen is not abundant in the intestinal tract, so PAL happens to be more applicable. When breaking down phenylalanine, cinammic acid and ammonia are produced, both of which are considered to be harmless to human body in the occuring concentrations.

For this approach, PAL sequence from already existing biobrick in iGEM parts registry was used K1692004. We have slightly changed the sequence by adding His-Tags to C-terminal, N-terminal and to both C- and N-termini instead of the Flag-Tags which originally existed in the biobrick. His-Tags were added with the help of oligonucleotide primers.

Polyphe proteins

This approach can be viewed as a distinctive creation of synthetic biology. We have designed a unique tool that minimizes the concentrations of phenylalanine in the medium. This tool is comprised of a synthetic gene, which is rich in phenylalanine codons. The translated protein consists of [kiekis procentais] of phenylalanine. Therefore, the expressed gene will use the majority of phenylalanine in the surroundings during the process of translation.

In order to find the most stable folds having multiple phenylalanine residues, we have developed a script based on R programming language. This script uses FoldX protein modeling program as a tool for mutagenesis and protein stability calculation. The script and its manual can be accessed at GitHub .

We have chosen five aromatic and hydrophobic amino acids for mutation (Tyr, Trp, Leu, Ile, Met), as they will most likely be hidden in hydrophobic pockets of the protein and not affect the tertiary structure. For the mutant candidates, we chose the proteins that were reported to have a high inducible expression rates in laboratory conditions: T4 virus clamp protein gp45, Streptococcus thermophilus Csm4. Also, we found that the transmembrane protein lactose permease (LacY) also has a good percentage of phenylalanine and amino acid candidates mentioned above. For this protein, only the transmembrane segments (TMS), excluding TMS1, were selected for mutagenesis.

Next, we loaded our developed script to acquire the most stable desired mutants (see Fig.X). As an additional step of refinement, we loaded the sequences of these mutants on Phyre2 or I-TASSER servers for tertiary structure prediction. The simulated structures were aligned using TM-Align and the mutants with the least identical sequence (lowest sequence identity) and the most similar structure (highest TM score) were chosen as candidates for our experiments.

Additional mechanisms

To increase the effectiveness of our system, we came up with two advances which will enable better intake of phenylalanine. These include the overexpression of phenylalanine tRNA and phenylalanine transporters. The plasmids containing tRNAphe and pheP were introduced into E. coli cells.

Generally speaking, the probiotic will have two major advances which will let it to efficiently take up and breakdown phenylalanine. These include the expression of phenylalanine ammonia lyase to breakdown phenylalanine, and Polyphe protein to collect excess phenylalanine. These elements are accompanied by the riboswitch and the enhanced tRNA-Phe and pheP expression. The proof that the designed system performs at the expected capacity is presented in the following sections.