Our whole cell biosensor design is sensing the phenylalanine (Phe) or tyrosine (Tyr) by relation between the TyrR and PtyrP regulated by the input (Phe), resulting in turning on the reporter gene (lacZ), or a relation between Parof and PtyrP regulated by Tyr resulting in turning off LacZ gene
TyrR, a DNA-binding protein, controls the biosynthesis of Phe and Tyr in E. coli. TyrR substantially stimulates the expression of lacZ alpha by activating the TyrP promoter in the presence of Phe and ATP. And the ParoF promoter regulates the expression of L7Ae. When Tyr levels are high, a TyrR dimer self-assembles to create a hexamer that inhibits ParoF and limits the production of L7Ae.
The LacZ-alpha fragment is encoded by this region and is derived from the pUC19 cloning vector. Because it is smaller than the LacZ-omega region, the LacZ-alpha fragment can be easily incorporated into a plasmid when the two non-functional LacZ gene fragments (alpha and omega) are co-expressed. X-gal (5-bromo-4-chloro-3-indoyl-d-galactopyranoside), a soluble colorless molecule that is a substrate of ß-galactosidase and generates a blue product when cleaved, is used to detect the alpha-complementation. It's linked to KP-SP ISA secreting peptide signal at the N-terminus of the reporter protein to transmit it from intracellular to extracellular.

T7 promoter is a dna sequence that can be recognized by T7 RNA polymerase and is mainly used to regulate gene expression of recombinant proteins,all of which allows it to be used in downstream research approaches.It is considered to be a constitutive promoter with high levels of transcription and expression in the presence of T7 RNAP and minimal interaction with bacterial RNAP.

---

The TyrR protein in Escherichia coli has the ability to both activate and repress the transcription operons necessary for the production and intake of aromatic amino acids (tyrosine, phenylalanine, and tryptophan). As an illustration, the TyrR dimer activates the promoter of the tyrP gene, which codes for a transporter specific to tyrosine, when tyrosine is present

---

ParoF promoter is a constituive promoter present in E. Coli which is sensitive to tyrosine and regulated by tyrR protein, it intitiates the synthesis of DAHP (3-deoxy-D-arabino-heptulosonate 7-phosphate synthase) and it is what makes it sensitive to phenylalanine as TyrR & Phenylalanine complex causes inhibition of the transcription of aroF

---

T2A: In order to mediate the "cleavage" of two proteins, 2A peptide sequences were discovered in Picornaviruses. Effective multiple protein expression from a single open reading frame (ORF) is achieved using 2A peptide-linked multicistronic vectors. Use alternative 2A peptide sequences when linking more than two genes to reduce the likelihood of homologous recombination. All eukaryotic systems studied to date, including mammalian cells, yeast, and plants, have effectively utilised the 2A peptide system.
L7ae: A protein called L7Ae that binds to RNA inhibits the targeted transcript's ability to be translated. Transcriptional repressors take longer to suppress the intended construct than L7Ae that regulate gene expression at the translational level. As it specifically targets a sequence on the 5' end of the RNA termed the 2x-kturn. L7Ae can be used to build more complicated genetic circuits that are regulated at both the translational and transcriptional levels

---

An exclusive transporter for tyrosine is encoded by the tyrP gene in Escherichia coli. Tyrosine inhibits it, whereas phenylalanine and, to a lesser extent, tryptophan activate it. Tyrosine also inhibits its production. The TyrR protein, which has two cognate binding sites (TyrR boxes) that span nucleotides 30 to 75, mediates both of these effects when it binds to one of both of these sites. A dimer that binds to the upstream box and interacts with the subunit (CTD) of RNA polymerase causes the activation in the presence of phenylalanine or tryptophan (RNAP). A hexamer binds to both TyrR boxes during repression in the presence of tyrosine.

---

This year, we enhanced the LacZ alpha gene by incorporating a peptide signal that controls the secretion of B galactosidase extracellularly in an effort to improve the cleavage capacity of the X gal, heighten the intensity of the dark blue color emitted by the X gal product, and reduce the amount of time required to receive the results after performing the test on the chip
KP-SP: is a secreted signal peptide at the N-terminus of the reporter protein that enables its secretion extracellularly with high efficiency which indicates the versatility of this signal peptide and its significant potential in heterologous protein expression. this peptide comes from E.Coli as well as actinomycetes and can be secreted under normal instances
The LacZ-alpha fragment: is encoded by this region and is derived from the pUC19 cloning vector. Because it is smaller than the LacZ-omega region, the LacZ-alpha fragment can be easily incorporated into a plasmid when the two non-functional LacZ gene fragments (alpha and omega) are co-expressed. X-gal (5-bromo-4-chloro-3-indoyl-d-galactopyranoside), a soluble colourless molecule that is a substrate of ß-galactosidase and generates a blue product when cleaved, is used to detect the alpha-complementation

---

Hu-6-Promotor: A proximal sequence element, which attracts the multisubunit component SNAPc, and a TATA box, which recruits the TATA box-binding protein, TBP, make up the human U6 small nuclear RNA core promoter. Two clearly identified factors are necessary for transcription from the human U6 promoter in addition to SNAPc and TBP. The first is the human homologue of the yeast TFIIIB subunit B" (hB), which is generally necessary for the transcription of RNA polymerase III genes, and the second is the human homologue of the TFIIIB subunit BRF (hBRFU), which is one of two human homologues and is specifically necessary for the transcription of U6-type RNA polymerase III promoters.

---

We carried out an improvement to its functionality limiting its off-targeting effect by adding a kink turn upstream to the 5’ end(translation initiation site) of its mRNA
k-turn: A common structural motif identified in functioning RNA species is the kink-turn (k-turn). It typically consists of a tandem trans sugar edge-Hoogsteen G followed by a three-nucleotide bulge: pair A bases. The minor grooves are juxtaposed and the axis of duplex RNA is given a strong bend. The conserved adenine nucleobases of the G:A basepairs accept the cross-strand H-bonds that form at the interface. The k-turns are split into two conformational classes, N3 and N1, by alternative acceptors for one of these. The conformation that a specific k-turn adopts is determined by the base pair (3b:3n) that follows the G:A pairings. K-turns typically bind proteins and mediate tertiary contacts in folded RNA species. Members of the L7Ae family of proteins are frequently found to bind k-turns
guide RNA (gRNA): is an RNA that is responsible for acting as a guide for enzymes that target DNA or RNA. Guide RNA targets their sequences by simple complementary base pairing of the target specific regions and it is used more specifically in applications that require targeted editing of DNA such as found and present in the CRISPR-Cas9 or Cas12 system.
Cas12g: is a ribonuclease. Comparing it to other Cas12 proteins that have been found so far, CRISPS-Cas12g selectively detects RNA substrates, making it a potentially useful platform for transcriptome editing and diagnostics. While guided RNAs fold into a "F" shape that is primarily identified by the Rec lobes, a bilobed structure of Cas12g displays a tiny NUC2 and REC2 domain. To change the conformation of the REC and NUC lobes and activate Cas12g, target RNA and crRNA guide combine to form a duplex that is inserted into the cavity in the middle of the structure.

---

Lac_Promotor: A CAP binding site and the lac operator sequence lacO are present in this lac promoter. Although the lac promoter is not as potent as the tac or trc promoters, it nevertheless provides for decent quantities of foreign protein production in high copy-number vectors. Expression from the lac promoter is activated in the absence of the lac repressor (LacI).
Permease: The bacterium Escherichia coli naturally transports phenylalanine across membranes via PheP (Phenylalanine-specific permease). PheP is a single, integral membrane transporter that uses the proton motive force to antiport L-phenylalanine and L-tyrosine. This transporter's activity under natural expression is known to range between 9 and 17,5.

---

We are developing a tunable therapeutic approach to give the body the ability to produce phenylalanine hydroxylase (PAH) by implementing our designed therapeutic circuit in a cell-based system that produces PAH according to phenylalanine and tyrosine levels as shown in figure 1. In the presence of phenylalanine (Phe), the TyrR will activate tyrP, accordingly tyrP will enhance the production of PAH gene. While the inhibitory effect of the ParoF will not be initiated, thus L7Ae will be expressed, preventing the expression of the dcas12g.
Our therapeutic design is working through sensing the concentration of Phe and Tyr that lead to expression of PAH enzyme so in the presence of phenylalanine (Phe), the TyrR will activate tyrP, accordingly tyrP will enhance the production of PAH gene. While the inhibitory effect of the ParoF will not be initiated, thus L7Ae will be expressed, preventing the expression of the dcas12g, and in the presence of tyrosine (Tyr), the TyrR will stimulate the inhibitory effect of the ParoF, therefore L7Ae will not be expressed. This will lead to free expression of the dcas12g to prevent the synthesis of the PAH enzyme. At the same time, tyrP will not be activated and PAH will not be expressed by its promoter.

---

Phenylalanine hydroxylase (PAH) gene: is the part responsible for providing instructions to make phenylalanine hydroxylase enzymes. This enzyme is responsible for hydroxylating phenylalanine and turning it to tyrosine in the presence of tetrahydrobiopterin (BH4). and as phenylalanine is present in almost all proteins and some artificial sweeteners this process of hydroxylation is really important to prevent its accumulation. also the product of tyrosine is used to make various hormones and neurotransmitters.

---

Synthetic biology was born out of the desire to construct biological circuits with engineering precision. That's why this year we will display our circuit from multiple perspectives in order to increase our control over the final outcome. There are several of them, one of which is logical engineering. This project combines logical engineering with cell biological functions through the use of logic gates. An input and output value are given as 0/1 in a truth table. In order to find a simple digital circuit scheme based on the availability of PHe or Tyr in the blood , we converted the truth table into a Boolean formula and applied relevant algorithms. Based on this, the inputs are either 0 or 1.
There are three main inputs in our design this year, each with a specific function. A tetracycline-based regulatory system for Crispr can be used by using anti-crispr proteins that bind to the CRISPR-Cas system at multiple sites, inhibiting a specific function of the system, inhibiting it under extreme conditions or unpredictable circumstances taking in consideration that the absence of tet will not affect the circuit.

Fig.1 shows the effect of Tetracycline on the circuit.

In addition, we have phenylalanine and tyrosine. It's obvious from their names that tyrosine is dependent on phenylalanine consumption, so they can't both be high in the blood at the same time. In other words there is no way for any patient to be suffering from both phenylketonuria (elevated phe-levels) and Tyrosinemia (excess Tyr-levels) at the same time. As a result, the one with the highest score dominates. In order to achieve this, we used a Nimply gate, which is activated when the phenylalanine value equals 1 and the tyrosine value equals 0. In the process of activation, L7ae is expressed, which inhibits the action of dCas12g, resulting in PAH expression.