1)Preparation of gold nanoparticles:

*We used the help of Nanogate company in Egypt to synthesis and characterize the gold nanoparticle
1) Prepare aqua regia by mixing 3:1 concentrated HCl:HNO3 in a large beaker in a fume hood. The capped aqua regia bottle may explode. Render it safe by dilution and neutralization.
2) Soak the 200 ml two-neck flask, magnetic stir bar, stopper and condenser in aqua regia for at least 15 min. Rinse the glassware with copious amounts of deionized water and then Millipore-filtered water21.
3) Load 98 ml of Millipore water into the two-neck flask. Add 2 ml of 50 mM HAuCl4 solution so that the final HAuCl4 concentration is 1 mM.
4) Connect the condenser to one neck of the flask, and place the stopper in the other neck. Put the flask on the hot plate to reflux while stirring.
5) When the solution begins to reflux, remove the stopper. Quickly add 10 ml of 38.8 mM sodium citrate, and replace the stopper. The color should change from pale yellow to deep red in 1 min. Allow the system to reflux for another 20 min.
6) Turn off heating and allow the system to cool to room temperature (23–25 °C) under stirring. The diameter of such prepared nanoparticles is ∼13 nm. The extinction value of the 520-nm plasmon peak is ∼2.4, and the nanoparticle concentration is ∼13 nM. The color should be burgundy red, and the nanoparticle shape should be spherical under transmission electron microscopy (TEM).

Absorption spectrum of nanoparticles:

This figure shows the surface plasmon resonance of the prepared AuNPs. UV-Vis absorption spectra were obtained on Cary series UV-Vis- NIR, Australia.

Transmission electron microscopy

This figure shows the TEM images of the prepared AuNPs. TEM were performed on JEOL JEM-2100 high resolution transmission electron microscope at an accelerating voltage of 200 kV.

Optimization the ratio of aptamer-AuNPs able to consume the normal level of phe:

Three methods were conducted
1. GNP +aptamer then phenylalanine then NaCl
2. Aptamer + phenylalanine then GNP then NaCl
3. Aptamer + phenylalanine then NaCl then gold In the method 3, Very weak aggregation was observed.

This figure shows colourimetric change based on the concentration of phenylalanine added at each trial, showing Red color reflecting maximum saturation of the 3ng of aptamers when phenylalanine is added at the given concentration of 20 mg/dL.

-This graph represents plotting both analyte in free state (A) and aptamer analyte complex (RA) after the binding occurs between them. It’s shown that the analyte (phenylalanine) is loaded to the test strip through the sample at the beginning. (A) shows gradual decrease of analyte over the time units. On the other hand, There’s a gradual increase in the aptamer-analyte complex over the time units via binding between the aptamer with its target. This model was beneficial in the optimization of aptamer to analyte concentration that is essential in constructing the consumption line in our lateral flow assay (LFA).

According to the single tube transformation protocol, which is provided on the Notebook page, we transformed the circuit to validate its function. In addition to providing X-gal media, which is the substrate of the beta-galactosidase enzyme, which is released as a reporter after sensing excess levels of phenylalanine. The transformation of the bacteria by plating them on LB-agar plates included negative and positive controls to validate the transferred circuits and the cell viability. Testing the plasmids and performing transformation protocols with them allowed us to determine whether these transformations were successful or not.

The figures display the chosen outcome of the transformation, providing the proof of concept and showing the different iterations as follows:

This figure on the left (Phe-ve/X-gal +ve) shows that the WCB emits a low signal of the blue color at 0 mg/dL of phenylalanine. The results are shown to the right (Phe-ve/X-gal +ve), which shows that the WCB emits a negligible signal of the blue color at 0 mg/dL of phenylalanine. which is also a background noise from the circuit but with a very low signal. This iteration was essential as it proved the concept of background noise without using the regulatory circuit unless the result after transforming it.

This figure on the left (Phe-ve/X-gal +ve) shows that the WCB emits a low signal of the blue color at 0 mg/dL of phenylalanine. The results are shown to the right (Phe-ve/X-gal +ve), which shows that the WCB emits a negligible signal of the blue color at 0 mg/dL of phenylalanine. which is also a background noise from the circuit but with a very low signal. This iteration was essential as it proved the concept of background noise without using the regulatory circuit unless the result after transforming it.

This graph illustrates the modeled results of riboswitch kinetics where L7Ae is expressed and bound to its kink-turns ,therefore inhibiting the expression of cas12g. However, M represents no expression of L7Ae in which cas12g would be expressed to control the circuit if the phenylalanine is absent.

For this plate, it showed an incomplete negative percentage of dominant control color in the test sample. To confirm the results, we plotted the absorbance (nM) of both the tested sample and the control sample as they showed slight overlapping ranges of wavelength (nM).

The graph on the left shows the absorbance of the test sample (nM) which shows a slight overlap between the absorbance range of the control sample (nM) which confirms the incomplete negativity.

This figure shows a very negligible signal of the blue color at the periphery of the plate at 0 mg/dL of phenylalanine. which is also a background noise from the circuit but with a very low signal after the usage of the regulatory circuit (kin-turn and cas12g). For this plate, it showed a negative percentage of dominant control color in the test sample. To confirm the results, we plotted the absorbance (nM) of both the tested sample and the control sample as they showed different ranges of wavelength (nM).

The graph on the left shows the absorbance of the test sample (nM) which shows different absorbance ranges of the control sample (nM) which confirms the negativity.

We enhanced the LacZ alpha gene by incorporating a peptide signal that controls the secretion of B-galactosidase extracellularly (KP-SP tagging), which acts as a reporter protein in our diagnostic circuit, 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.

This figure shows a characterization of KP-SP and lacZ alpha part as it's validated through gel electrophoresis as it is in lane 3.

This figure shows (Phe/X-gal with KP-SP) +ve, shows that the WCB emitted blue color with good intensity at a standard 20 mg/dL concentration of phenylalanine. The results shown to the right (Phe/X-gal) +ve, without the tag KP-SP, show that the WCB emitted a low level of blue color at the same concentration of phenylalanine.

Our genetic circuit activity was dependent on Phe concentration. The increase in Phe concentration leads to an increase in the color intensity due to increased TyrR and TyrP activities and LacZ expression.

This plate (Phe/X-gal) +ve shows that the WCB emitted a bright blue color at a phenylalanine concentration of 20mg/dL. This is the best outcome after the improvements and making good use of our iterations.

We interpreted the results using our own built software tool in which the user uses a reference image as a control to analyze the color of the other image according to it. This control image should show the optimum results.
For this plate, it showed a positive percentage of dominant control color in the test sample. To confirm the results, we plotted the absorbance (nM) of both the tested sample and the control sample as they showed overlapping within the same range of wavelength (nM)

The graph on the left shows the absorbance of the test sample (nM) which shows an overlap between the absorbance range of the control sample (nM) which confirms the positivity.

This graph illustrates the modeled results from the modeling of the direct relationship between the biomarker and beta-galactosidase. As the biomarker increases, the released amount of beta-galactosidase increases till it reaches a constant value after about 30 time units. Therefore, the maximum amount of the biomarker releases the maximum amount of beta-galactosidase, which is achieved from the test after iterations of troubleshooting.

This figure shows characterization of (T7P-TyrR RBS-TyrR-TyrPromoter), which is ordered from IDT as it's validated through gel electrophoresis as it is in lane 6 (the last one).

This figure shows characterization of the ParoF promoter-P2A-L7Ae, which is ordered from IDT as it's validated through gel electrophoresis as it is in lane 5.

This figure shows the lacZ alpha part as it's validated through gel electrophoresis as it is in lane 3, which is ordered from IDT.

To make sure that our approach goes well, we tested the WCB if it senses any aptamer-phenylalanine complex or not by using a different concentration of the aptamer binded with Phe, so by the advantage of the permease and TyrR parts that help the entry and sensing of Phe, there is no Apt-Phe complex that will pass to the cell to sense it.

This plate (Apt-Phe complex +ve/X-gal +ve) shows that the WCB emits a low signal of the blue color at 20 mg/dL of phenylalanine. which validates the specificity of the TyrR for Phe only, not the Apt-Phe complex.

The dissected hippocampi were transferred into 10 ml of preparation medium [Hank’s balanced salt solution (HBBS),1 mM sodium pyruvate, and 10 mM HEPES on a 35-mm Petri dish on ice. The hippocampi were transferred using a fire-polished glass Pasteur pipette into a 15-ml polypropylene Falcon tube with 5 ml of papain (0.5 mg papain,10 μg DNase I in 5 ml Papain Buffer) buffer. The tissue was incubated for 10 min at 37°C. After the tissue had sunk to the bottom, excess papain solution was discarded using a glass pipette.

Tissue is suspended in 10 mL of PBS pH 7.4, centrifugated at 900 rpm for 10 minutes, The clear supernatant was discarded. The cell pellet was resuspended in 1 ml DMEM/F12 with 10% FBS after the supernatant was discarded. Minced and digested hippocampal tissues were seeded into T25 flasks in DMEM/F12 medium (Gibco, Thermosientific, Germany) containing 10% foetal bovine serum (FBS) (Gibco, Thermosientific, Germany) and 1% penicillin G sodium (10.000 UI), streptomycin (10 mg), and amphotericin B (25 g) (PSA) (Invitrogen Life Technologies). Flasks were incubated at 37o C in a 5% CO2 environment. Non-adherent cells were eliminated by changing the media every two days after single cells had adhered to the plastic surface. Plastic adherent cells were multiplied until they reached a confluence of about 80%. During this time, phase-contrast microscopy was employed to examine cells, and cells from passages 1 to 5 were used in all experiments. Because it appears that asymmetrically dividing progenitor cells ratify after passing, early passages were employed.

this figure shows a microscopic image showed the cultured Hippocampi cells. The magnification power is 40X and the scale bar is 100µm. Images are captured with LABOMED inverted microscope, USA Labomed-USA

The hippocampi cells were equally distributed onto 6-well culture plate that are intended for the three different experimental groups including: (1) naïve cells (negative control), (2) cells transfected with empty vector (pcDNA3.1+), and (3): cells transfected with pcDNA3.1+ therapeutic circuit) (pcDNA3.1+). Transfected cells are incubated at 37o C in a 5% CO2 for 72 hours. One day before conducting the experiment, the Hippocampi cells were seeded in 6-well culture plate. An average 1x106 cells were seeded in 200 µL of Dulbecco's Modified Eagle Medium (DMEM) (Gibco, Thermosientific, Germany) containing 10% fetal bovine serum (FBS) (Gibco, Thermosientific, Germany) and 1% of penicillin G sodium (10.000 UI), streptomycin (10 mg) and amphotericin B (25 μg) (PSA) (Gibco, Thermosientific, Germany). Culture plates were incubated at 37 °C in an atmosphere of 5% CO2 for 24 hours to reach the 70% confluence. On the next day, cells are transfected with pcDNA3.1 vector + therapeutic circuit using Lipofectamine 3000 (Thermosientific, Germany). In addition, the carrier solvent (0.1% DMSO) was used for control cells. The treated Hippocampi cells were incubated at 37 °C in an atmosphere of 5% CO2 for 72 hours.

At the end of the incubation period, cells were harvested using 0.25% Trypsin EDTA Gibco, Thermosientific, Germany). The harvested cells washed three times with phosphate buffer saline (PBS) pH 7.4, then the cell pellet is suspended in 2.0 mL DMEM media and stored at -800 C until used for PCR analysis.

To validate the efficacy of the therapeutic circuit, the Phenyl alanine hydroxylase gene was measured in harvested cells using Syber-green-based Real time PCR. The following steps are performed:

Disruption and homogenization for harvested cells was performed using the Tissue Ruptor II (Qiagen, Hilden, Germany), a rotor–stator homogenizer that thoroughly disrupts and simultaneously homogenizes single tissue samples in the presence of lysis buffer in 15–90 seconds, depending on the toughness and size of the sample. Then the mixture is centrifugated for 20 minutes at 4000rpm. Finally, the cell supernatant is collected for RNA extraction.

Following disruption and homogenization of liver tissue by bead-milling in a guanidine-thiocyanate– containing lysis buffer. the sample is loaded onto RNeasy Mini spin column. Total RNA binds to the RNeasy silica-membrane, contaminants are efficiently washed away, and high-quality RNA is eluted in RNase-free water. The RNA extraction & purification was performed using RNeasy blood/ tissue Mini kit, Qiagen, Hilden, Germany. The process was conducted according to the manufacturer protocol.

The reverse transcription step was performed by the QuantiTect Reverse Transcription Kit , cat. No: 205310, (Qiagen, Hilden, Germany). The reverse-transcription master mix was prepared on ice in total volume of 20µL, which was composed of 1µL of Quantitect Reverse transcriptase enzyme, 4 µL of RT buffer, 1 µL of RT primer mix, and 14 µL of genomic DNA, the reaction mix was mixed and then kept on ice. The reverse-transcription master mix contains all components required for first-strand cDNA synthesis except template RNA. The reaction mix was incubated for 15 min at 42°C, then it was incubated for 3 min at 95°C to inactivate Quantiscript Reverse Transcriptase. The reverse-transcription reactions were placed on ice and then real-time PCR was proceeded directly.

The gene expression level was amplified from mRNA using specific primer sequences for PAH gene “the sequence is listed” and ACTB_1_SG QuantiTect Primer Assay (β-actin) cat no: 249900, as housekeeper gene. All samples were analyzed using the 5 plex Rotor Gene PCR Analyzer (Qiagen, Germany). The PCR reaction mix was prepared by adding as follow, 2x QuantiTect SYBR Green PCR Master Mix, 10x QuantiTect Primer Assay, template cDNA, and RNase-free water were thawed at room temperature (15–25ºC). Then, the reaction mix was prepared for a final volume 18 μl per well reaction volume as following: 10 μl of 2x QuantiTect SYBR Green PCR Master Mix, 2 μl 10x t Universal Primer, 2 μl 10x Quantitect Primer Assay and 4 μl RNase-free water. The reaction mix was mixed thoroughly but gently, and dispensed appropriate volumes into the Rotor-Disc wells then 2 μl template cDNA was added, to reach 20 μl as final volume. Carefully, tightly the disc was sealed with Rotor-Disc Heat-Sealing Film. Consequently, the real-time cycler Initial was programmed as: activation step for 15 minutes at 95ºC for Hot-Star Taq DNA Polymerase activation. Three-step cycling: denaturation for 15 seconds at 94ºC, annealing for 30 seconds at 55ºC, extension for 30 seconds at 70ºC, for 40 cycles. Moreover, the expression levels were normalized to β-actin levels as a reference gene. The relative gene expression level (fold change) for APP was normalized to an internal control (β-actin) and relative to the calibrator (negative control sample) were calculated using the equation 2-∆∆Ct test/ control.

TCt: cycle threshold, Δ: delta, FC: fold change of PAH gene after normalization to untreated Hippocampi cells, ACTB: β-actin housekeeper gene used for normalization