{"pk":47131,"title":"FRET-based Synthetic Biology Approach for SUMOylation Cascade in Bacterial Cell and Interaction with Influenza A Virus  ","subtitle":null,"abstract":"<p>Post-translational modifications, SUMOylation, and Ubiquitination are critical in protein activities and half-life regulations in physiological and pathological processes, such as cancers, immune responses, and virus infections. SUMOylation requires an activating enzyme E1, conjugating enzyme E2, and E3 ligase to catalyze the attachment of SUMO peptide to substrates. Using synthetic biology techniques, we reconstituted the SUMOylation cascade in bacterial cells, aiding future research to be more efficient. We determine the activities of the SUMOylation enzymes expressed in the polycistronic SUMO construct using the quantitative FRET assay developed in our lab for CyPet-SUMO1 conjugation to substrate YPet fused-influenza A virus (IAV) M1 protein. In this project, we screened the CyPet protein expression of Polycistronic SUMO in 13 E.Coli strains to determine optimal bacteria for the assay. From the best performing strain, BL21 (DE3) PlysS, we performed double transformation of PolycistronicSUMO and IAV M1 constructs into bacterial cell culture for FRET-based SUMOylation conjugation assay. We also applied this system for SUMOylation inhibitor testing. In summary, using qFRET technology, we developed a novel synthetic biology approach for the SUMOylation cascade in bacterial cells. The synthetic biology approach for polycistronic SUMO cascade is a novel strategy to reconstitute the cascade reaction in bacterial cells.</p>","language":"eng","license":{"name":"Creative Commons Attribution-NonCommercial  4.0","short_name":"CC BY-NC 4.0","text":"Attribution — You must give appropriate credit, provide a link to the license, and indicate if changes were made. You may do so in any reasonable manner, but not in any way that suggests the licensor endorses you or your use.\r\n\r\nNonCommercial — You may not use the material for commercial purposes.\r\n\r\nNo additional restrictions — You may not apply legal terms or technological measures that legally restrict others from doing anything the license permits.","url":"https://creativecommons.org/licenses/by-nc/4.0"},"keywords":[{"word":"SUMOylation"},{"word":" Quantitative Förster Resonance Energy Transfer"},{"word":" synthetic biology"},{"word":" influenza A virus"},{"word":" viral proteins"},{"word":" protein interactions"},{"word":"Quantitative Förster Resonance Energy Transfer"},{"word":"synthetic biology"},{"word":"influenza A virus"},{"word":"viral proteins"},{"word":"protein interactions"}],"section":"Articles","is_remote":true,"remote_url":"https://escholarship.org/uc/item/1rt5d6hm","frozenauthors":[{"first_name":"My Linh","middle_name":"","last_name":"Le","name_suffix":"","institution":"University of California, Riverside","department":"Bioengineering"}],"date_submitted":"2025-04-02T04:53:26.989000Z","date_accepted":"2025-05-20T03:54:56.424000Z","date_published":"2025-08-11T03:54:00Z","render_galley":{"label":"Le - FRET-based Synthetic Biology Approach for SUMOylation Cascade","type":"pdf","path":"https://journalpub.escholarship.org/ucr_undergrad_research_j/article/47131/galley/38606/download/"},"galleys":[{"label":"Le - FRET-based Synthetic Biology Approach for SUMOylation Cascade","type":"pdf","path":"https://journalpub.escholarship.org/ucr_undergrad_research_j/article/47131/galley/38606/download/"}]}