C7 - FRET-based Synthetic Biology Approach for SUMOylation Cascade in Bacterial Cell and Interaction with Influenza A Virus

Introduction: : The Post-translational modification, SUMOylation, plays critical roles for protein activities and half-life regulations in physiological and pathological processes, such as cancers, immune responses, diabetes, and neurodegenerative diseases. SUMOylation cascade, a process of SUMO peptide conjugation to a substrate involving activating enzyme E1, conjugating enzyme E2, and E3 ligase, regulates substrate interactions with other proteins. However, the study of SUMOylation faces difficulties due to low abundance and competing post-translational modifications in eukaryotic cells. Synthetic biology is a versatile field that uses large DNA synthesis and pathway reconstruction, to create pathways/organisms for applications not found in nature. This process involves design of regulatory circles and de novo DNA synthesis, which allows for the creation of novel DNA sequences from scratch. Using synthetic biology techniques, we reconstitute the full SUMOylation pathway, Polycistronic SUMO, in bacterial E.coli to constitute the full pathway to a substrate with the monitoring of qFRET technology.

Materials and

Methods: : The Polycistronic SUMO construct is synthesized and cloned by the company Genscript. This construct included Cypet-SUMO1 and SUMO enzymes (E1, E2, E3) linked by engineered ribosome binding sites and translation enhancers. We then transformed the construct into different bacterial E. Coli strains to determine optimal CyPet protein expression through FRET scans. The highest Polycistronic SUMO-expressing cells were double transformed with the YPet-IAV M1 plasmid to perform SUMO conjugation determined with the FRET of CyPet-YPet to YPet-M1. SUMOylation inhibition is tested by adding IPTG and SUMOylation inhibitor to the culture, followed by a FRET assay.

Results, Conclusions, and Discussions:: Having screened 13+ E.Coli strains, we determined that BL21(DE3) PlysS had the most optimal Polycistronic SUMO CyPet protein expression. We chose this strain to undergo qFRET analyses in SUMO conjugation to YPet-IAV M1 and SUMO inhibitor testing, which further validated the feasibility of our engineered cascade. The SUMO conjugation qFRET scans presented distinct YPet and CyPet peaks, indicating that Polycistronic SUMO successfully expressed SUMOylation, while the drug test confirmed functional activity of the reconstituted SUMOylation cascade. However, a primary concern about our SUMO construct is the gene order within the polycistronic vector, as genes positioned downstream of the promoter tend to exhibit reduced expression levels, requiring further validation by other methods such as Western Blot.

From qFRET measurement, the Polycistronic SUMO construct yielded high protein expression rates and reproduced the full activity of SUMOylation to conjugate CyPet-SUMO1 to IAV M1 in E.Coli. As SUMOylation plays critical roles in viral replication and host antiviral defense, these results hold potential for improved SUMOylation-virus research. The construct allows for convenient and efficient induction of SUMO protein expression, activating its cascade of supporting enzymes to express SUMO conjugation. This bacterial system is also more cost effective in scaling up for drug testing with different small molecule inhibitors, enhancing future drug discovery.

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