We are the Guardians of the Star team dedicated to developing an L-glutamate detection biosensor for ASD diagnosis. In order to carry forward the spirit of iGEM, and inherit and spread the value of iGEM, we specially searched the iGEM Biological Parts library for related biosensors and picked BBa_K1025005, Tryptophan-sensor part. This is a biological part submitted by iGEM13_Tsinghua-E in 2013, which is composited by tnaC and Rho. In order to provide more tryptophan-detection biosensors, our team carried out a new composite part made up of tnaC and amilGFP in the laboratory, adding data from adding tryptophan in the reaction system to dedicate its function and properties. This information can be a good reference for future iGEM teams working on ASD detection.
In addition, through literature research, we found another ASD-related detection index, L-glutamate. We upload their DNA sequence information and basic introduction information in the registry of standard biological parts to provide more choices for ASD detection for future iGEM teams.

The existing tryptophan-sensor is composited by one regulation sequence upstream of the tryptophanase (tnaA) operon in wild-type E. coli, which encodes a 24-residue nascent peptide, and a transcription termination factor (Rho) recognition site. In our project, we developed another tryptophan-sensor with tnaC, and we fused it with the amilGFP gene to characterize our biosensor.

We amplified the DNA fragments of tnaC from the MG1655 genomic DNA and the amilGFP DNA fragment from a plasmid containing this fragment (Figure 2A). Then we overlapped these two fragments by PCR (Figure 2B) and inserted the fused gene fragment into the NcoI and HindIII sites of the pTrc99k vector.

We extracted the plasmid and send it to the company for Sanger sequencing. The certificate of recombinant plasmid sequencing results is as Figure3.

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 0D600 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 4).

The tnaC regulatory gene from the tna operon of E. coli controls the transcription of its own operon through an attenuation mechanism relying on the accumulation of arrested ribosomes during inhibition of its own translation termination. In this project, the expression of tnaC was regulated by tnaCAB operon in the pTrc99K vector. This mechanism requires ribosomal arrest induced by the regulatory nascent TnaC peptide in response to free L-tryptophan (L-Trp).

Glutamate dehydrogenase (gldh) is a mitochondrial enzyme that is involved in the metabolism of glutamate to 2-oxoglutarate, and reversibly converts glutamate to α-ketoglutarate as part of the urea cycle. The gldh enzyme is found primarily in the liver, kidney, and cardiac muscle, while the liver has the highest concentration of gldh activity and lower levels in the brain, skeletal muscle, and leukocytes. GLDH has a housekeeping role in cell metabolism.

SLC7A5, known as LAT1, belongs to the APC superfamily and forms a heterodimeric amino acid transporter interacting with the glycoprotein CD98 (SLC3A2) through a conserved disulfide. The complex is responsible for the uptake of essential amino acids in crucial body districts such as the placenta and blood-brain barrier. The SLC7A5 transports the so-called branched-chain amino acids (BCAA) into the brain. Mutation in this gene reduces branched-chain amino acid levels in the brain and interferes with neural cell protein synthesis. This shows reduced social interactions and other changes. There are many different genetic mutations causing autism, and this heterogeneity makes it difficult to develop effective treatments.

1. Deth R, Muratore C, Benzecry J, et al. How environmental and genetic factors combine to cause autism: a redox/methylation hypothesis. Neurotoxicology, 2008, 29: 190–201
2. Tomova A, Husarova V, Lakatosova S, et al. Gastrointestinal microbiota in children with autism in Slovakia. Physiol Behav, 2015, 138: 179-87
3. Dora C. Tărlungeanu, et al. Impaired Amino Acid Transport at the Blood Brain Barrier Is a Cause of Autism Spectrum Disorder.cell.2016
4. Chen L, Shi X-J, Liu H, Mao X, Gui L-N, Wang H, et al. Oxidative stress marker aberrations in children with autism spectrum disorder: a systematic review and meta-analysis of 87 studies (N = 9109). Transl Psychiatry. (2021) 11:15.