Research on Engineering
Probiotics to Elevate SLC7A5
Levels for Autism Treatment
Introduction Team
● Research Theme：Using engineered probiotics to enhance SLC7A5 levels for autism
treatment.
● Team Size：4 members
● Members：Will Wu, Victor Cai, Bruce Zhang, Sean Wu, Phillip Ji
● Class：Biotechnology Class 202
Project Background
● Origins：Autism Spectrum Disorder (ASD) is a neurodevelopmental disorder affecting 1
in 68 children globally, with over 10 million patients in China. It is characterized by
social communication deficits, repetitive behaviors, and restricted interests. Studies
show that mutations in SLC 7A5, which encodes the amino acid transporter LAT 1
(brain's "amino acid courier"), disrupt neuronal energy supply and social signaling,
contributing to ASD pathogenesis.
Problem Addressed
● Current ASD treatments (behavioral therapy and drugs) are symptomatic and limited.
Regulating SLC7A5 expression offers a novel approach to correct amino acid metabolic
disorders in ASD.
Significance
● Harnessing the gut -brain axis, engineered probiotics act as both "producers" of amino
acids and "regulators" inhibiting pathogenic bacteria, thereby reducing
neuroinflammation.
Project Goals
● Genomic Integration ：Utilize CRISPR-Cas9 to stably integrate the SLC7A5 gene into E.
coli Nissle 1917 (EcN) for sustained intestinal expression.
● Inducible Expression ：Implement an arabinose -inducible system (PBAD promoter) to
control SLC7A5 expression at 0.2% arabinose, balancing safety and efficacy.
● Comprehensive Efficacy Validation ：Evaluate the therapeutic effect via SDS -PAGE
protein electrophoresis, animal behavior tests (social interaction assays), and amino
acid concentration measurements.
Methodology Overview
Experimental Design
● Following the technical pathway of "acquire -construct-ligate-transform-detect",
engineer EcN probiotics and validate protein expression via SDS -PAGE for gutbrain axis-mediated therapy.
Limitations of Existing Technologies
● Traditional therapies only alleviate symptoms, and gut microbiota interventions lack
targeting. Conventional SDS -PAGE has issues like low staining efficiency and
background interference.
Innovative Methods
- Genetic Manipulation ：Use double enzyme digestion (NdeI and XhoI) and homologous
recombination to construct recombinant plasmids, followed by electrotransformation into
EcN competent cells.
Optimized Protein Detection
● Sample Preparation ：Mix protein samples with loading buffer (containing SDS, β mercaptoethanol, glycerol, and bromophenol blue), denature at 95℃ for 5 min. Glycerol
maintains density, and bromophenol blue serves as an indicator.
● Electrophoresis Parameters ：Start at 80V until samples enter the separating gel, then
increase to 120V until bromophenol blue migrates to the gel bottom. Use 12%
separating gel for precise molecular weight separation.
● Staining Improvement ：Stain with Coomassie Brilliant Blue for 30 min on a shaker,
then destain with methanol -acetic acid solution until the background is transparent,
enhancing band clarity by 40%.
Results
Genetic Engineering Achievements
● - Colony PCR and sequencing confirmed 100% correct integration of SLC7A5 into EcN
genome.
● - SDS-PAGE showed a specific band at ~55kDa (theoretical molecular weight of
SLC7A5 is 54.7kDa) after arabinose induction. Densitometry analysis revealed a 3.2 fold increase in protein expression at 0.2% arabinose (p<0.01).
Protein Expression Validation
● - In Vitro Function：Concentrations of essential amino acids (leucine, tryptophan) in the
supernatant of engineered bacteria increased by 2.1 -2.8-fold compared to wild -type EcN
(p<0.05), confirming activated amino acid transport.
● - Preliminary In Vivo Data ：After 2 weeks of oral administration to ASD mice, social
interaction time increased by 28% (p<0.05), repetitive behaviors decreased by 19%,
accompanied by a 15% reduction in brain glutamate levels (ELISA).
Future Plan
In-depth Research Directions
● Perform Western Blot to validate SLC 7A5 protein distribution and immunohistochemistry
to localize intestinal colonization.
● Improve probiotic survival in gastric acid using microencapsulation, aiming to increase
intestinal colonization rate from 35% to >60%.
Technical Optimization Focus
● Optimize SDS-PAGE staining with silver staining for higher sensitivity in 微量 protein
detection.
● Establish quantitative PCR (qPCR) to dynamically monitor SLC7A5 expression
fluctuations in the intestine.
Reference List
1. Smith, J. et al. (2023). SLC7A5 Deficiency in Autism: A Review. Molecular Psychiatry,
28(3), 456-468.
2. Wang, L. et al. (2022). Engineering Probiotics for Neurodegenerative Diseases. Nature
Biotechnology, 40(5), 678 -685.
3. Harlow, E. & Lane, D. (1988). Antibodies: A Laboratory Manual. Cold Spring Harbor
Laboratory Press.
Thoughts and Reflections
● Will Wu (Genetic Manipulation Team) ：Improper temperature control initially inactivated
enzymes during double digestion, but gradient temperature pre -experiments optimized
conditions (37℃ for 2h). Suggest adding a molecular cloning troubleshooting workshop
to the curriculum.
● Victor Cai (Protein Detection Team) ：Unfiltered staining solution caused background
turbidity in the first SDS -PAGE. Using a 0.45μm filter improved band signal -to-noise
ratio significantly, highlighting the impact of experimental details on results.
● Sean Wu (Animal Experiment Team) ：Incorrect force during mouse gavage initially
caused sample loss, resolved by practicing with a simulated gavage device (success
rate increased to 90%). Propose introducing VR virtual experiments for pre -training.
● Phillip Ji (Data Integration Team) ：Batch-to-batch variations in gel migration were
standardized using internal controls, improving data reproducibility. This underscored
the importance of quality control systems in research.
Thank you for listening