Autism Spectrum Disorder (ASD) is a neuro-developmental disorder characterized by social communication disorders. Our research aims at early diagnosis and accurately diagnosing and treatment.
In order to carry forward the spirit of iGEM, we specially searched the iGEM Biological Parts library for related projects and picked BBa_K302033, MazF. This is a biological part submitted by iGEM10_Newcastle in 2010, they developed an ASD diagnosis and treatment method, but didn’t totally achieve ASD treatment through the brain-gut-axis. So that it is really important to provide another candidate protein for ASD treatment.
In this project, our team carried out a related protein SLC7A4 for this part in the laboratory. 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 which reduced social interactions and other changes. Improving the SLC7A5 levels by building engineered probiotics may achieve the treatment of autism.
In order to verify if the new part SLC7A5 we developed worked well, we transformed the recombinant plasmid into E. coli BL21(DE3) to verify the protein expression level by SDS-PAGE. This protein may could be used in future research.

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.

In order to build our plasmids, we let the synthetic company synthesize the SLC7A5 DNA sequence and inserted it into the pUC57 vector. We amplified the SLC7A5 from the plasmid by PCR (Figure 1A) and inserted it into the EcoRI and XhoI sites of the pET28a vector. We transformed it into E. coli Top10 competent cells and screened the colonies by colony-PCR (Figure 1B). We inoculated the correct colonies in LB (Kan+) liquid medium and extracted the plasmids and sent them to the company for Sanger sequencing. The returned sequencing data showed that there were no mutations in the ORF region which means we successfully constructed the recombinant plasmid.

In order to verify if SLC7A5 expression in the E. coli host strain, we transformed the recombinant plasmids into E. coli BL21(DE3), inoculated the recombinants and added 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 2). As a result, the recombinants successfully expressed our target protein.