Protocol for the establishment of a serine integrase-based platform for functional validation of genetic switch controllers in eukaryotic cells

• DOI: 10.1371/journal.pone.0303999
• Publication Date: 2024-05-23T17:25:44Z
• Authors: Marco A. de Oliveira; Lilian H. Florentino; Thais T. Sales; Rayane N. Lima; Luciana R. C. Barros; Cintia G. Limia; Mariana S. M. Alm...; Maria L. Robledo; Leila M. G. Barros; Eduardo O. Melo; Daniela M. Bitten...; Stevens K. Rehen; Martín H. Bonamino; Elibio Rech
• Keywords: Integrases; Synthetic Biology; Recombineering

Serine integrases (Ints) are a family of site-specific recombinases (SSRs) encoded by some bacteriophages to integrate their genetic material into the genome host. Their ability rearrange DNA sequences in different ways including inversion, excision, or insertion with no help from endogenous molecular machinery, confers important biotechnological value as editing tools high host plasticity. Despite advances use prokaryotic cells, only few Ints currently used gene editors eukaryotes, partly due functional loss and cytotoxicity presented candidates more complex organisms. To expand number available for assembly multifunctional circuits eukaryotic this protocol describes platform screening serine-integrase-based switches designed control expression directional inversions sequence orientation. The system consists two sets plasmids, an effector module reporter module, both assembled regulatory components (as promoter terminator regions) appropriate mammals, humans, plants. complete method involves plasmid design, delivery, testing phenotypical assessment results. This presents suitable workflow identification validation new regulation reprogramming organisms importance fields, medical applications crop enhancement, shown initial results obtained. can be completed 4 weeks mammalian cells up 8 plant considering cell culture growth time.