We design three plasmids: The DNA sequences of the tnaC-amilGFP was inserted into the pTrc99K vector, and the codon-optimized inaK-gldh and SLC7A5 were inserted into the HindIII and NcoI sites of the pET28a vector, respectively. These plasmids were shown in Figure1.

In order to build our plasmids, we let the synthetic company synthesize the codon-optimized inaK-gldh fused DNA fragment and integrate it into the pET28a vector (Figure 1C).
Next, we amplified the tnaC, amilGFP, and SLC7A5 DNA fragments by PCR. Then, we fused the tnaC and amilGFP through overlap-PCR (Figure 2). We extracted the target DNA fragments and digested the tnaC-amilGFP and pTrc99K with NcoI and HindIII, and ligated by T4 DNA ligase. The DNA fragment SLC7A5 and pET28a with EcoRI and XhoI, and ligated by T7 DNA ligase. The recombinant plasmids were transformed into E. coli Top10 competent cells and coated on LB medium plate with corresponding antibiotics.

We verified our recombinant plasmids by colony PCR (Figure 3), and we inoculated the correct strains and extracted plasmids.

We send the constructed recombinant plasmid to a sequencing company for sequencing. The returned sequencing comparison results showed that there were no mutations in the ORF region (Figure 4), and the plasmid was successfully constructed. So far, we have successfully obtained ASD-detection-related plasmids.

In order to obtain the inaK-gldh and SLC7A5 proteins, we transformed the recombinant plasmids into E. coli BL21(DE3), inoculated the recombinants and added different concentrations of IPTG to induce protein expression when the OD₆₀₀ reached 0.6. After overnight induce and culture, we collected the cells and ultrasonic fragmentation of cells to release the intracellular proteins. Next, we verified the expression level of the target protein by SDS-PAGE (Figure 5). As a result, the recombinants successfully expressed our target protein.

To confirm the ability of our ASD-detection-related biosensor, we used E. coli BL21(DE3) as host strains and detected the related proteins’ activity. There are several markers that could be chosen to detect, such as tryptophan and glutamic acid.
To better show the activity of our biosensor, we constructed a recombinant plasmid pTrc99k-tnaC-amilGFP as a reporter of tryptophan detection. We measured the relationship of the fluorescence intensity with the concentration of tryptophan and the incubation time.
What’s more, we also measured the activity of the glutamine dehydrogenase by constructing the plasmid pET28a-inaK-gldh and transforming it into BL21(DE3). We purified the protein and developed an in vitro reaction platform to detect its activity.
The results showed that the biosensor we developed could be used to detect ASD. A detailed analysis of the biosensor is given below.

Single colonies of the engineered strains containing the recombinant plasmid pTrc99K-tnaC-amilGFP were picked, placed in a triangular flask containing 10mL of fresh LB medium (containing 100mg/L Ampicillin), and incubated at 37°C, 220 rpm for 12h. 1 mL of bacterial cultured medium was added to a triangular flask containing 100mL of fresh LB medium (including 100mg/L Ampicillin) and incubated at 37°C, 220 rpm until the OD₆₀₀ was around 0.6. The solution was divided into conical flasks, adding 50ml of cultured medium and L-tryptophan to a final concentration of 0.75mmol/L, 1mmol/L, 1.5mmol/L, 2mmol/L, 2.5mmol/L, and 3mmol / L and cultured at 22°C, 220 rpm for 7 hours, and samples were taken at 1h, 2h, 4h, 5h, 6h, 7h. Fluorescence intensity was detected immediately by using 200 μL bacterial solution samples to a 96-well black microplate plate (Figure 6).

Glutamate dehydrogenase could use L-glutamate as a substrate, with reversible oxidation and deamination under the action of coenzyme (NAD + or NADP +), and the NADH or NADPH generated by the reaction has an obvious absorption peak at 340 nm. To detect glutamate dehydrogenase activity, the amount of NADPH generated by the hydrogenase catalytic reaction was measured spectrophotometrically.
We used the Glutamate Dehydrogenase (GDH) Activity Assay Kit to measure the protein activity. The bacteria cells were collected by centrifugation into centrifugal tubes, treated samples in the proportion of bacteria: extract volume (mL) (500-1000): 1 (1 mL of 5 million bacteria), crushed by ice bath sonicated (power 20% or 200 W, ultrasonic for 3s, 10s interval, 30 repeats), centrifuged at 4℃ 8000 g for 10 min, and the supernatant was placed on ice for testing. Absorption values at 340 nm were determined using a UV-visible spectrophotometer.
Cells containing the expression vector pET28a-inaK-gldh were centrifuged and washed twice with 100mM Tris-HCl (pH 8.0) buffer after induction. The standard reaction system contained bacterial cells (OD₆₀₀-1.0), 100mM Tris-HC1 buffer (pH 8.0), sodium L-glutamate (final concentration 2mM), and NADP + (final concentration 0.5mM). The reaction was performed at 60°C in a 1.5mL centrifuge tube for 2min and was terminated by centrifugation of the bacteria at 12,000 rpm for 1min. The absorptive values at 340 nm were measured using a UV-visible spectrophotometer. As a result, the enzyme activity of glutamine dehydrogenase is around 567.92975 U/mL. So that the biosensor we constructed worked well.

The enzyme activity calculation formula is as follows

* Vsample: 0.05 mL, V total: 1×10^-3 L, ε: NADH molar extinction coefficient, 6.22×10³ L/mol/cm, d: Cuvette light diameter, 1 cm, ∆T: Reaction time, 2 min.