Publicação
Optimization of intersectional genetics-based biosensors for human cell targeting purposes
| dc.contributor.advisor | Gontijo,Álisson Marques de Miranda Cabral | |
| dc.contributor.advisor | Menezes,Juliane | |
| dc.contributor.author | Félix,Sara Grilo | |
| dc.contributor.institution | Faculty of Sciences | |
| dc.contributor.institution | Department of Physics | |
| dc.date.accessioned | 2026-06-01T15:15:13Z | |
| dc.date.available | 2026-06-01T15:15:13Z | |
| dc.date.issued | 2025 | |
| dc.description | Tese de Mestrado, Engenharia Biomédica e Biofísica, 2026, Universidade de Lisboa, Faculdade de Ciências | |
| dc.description.abstract | Genetically targeting human cell types with precision is a central challenge in synthetic biology and biomedicine. Intersectional genetic circuits address this using multiple inputs, ensuring that only cells with a unique regulatory signature activate the circuit. A common implementation uses multi-split proteins that are reassembled in the target cell type by split-inteins. Here, we sought to improve the reconstitution of two split-proteins (the TetR*-KRAB transcriptional repressor and the Cre recombinase), and to create a stable human induced pluripotent stem cell (hiPSC) line to report Cre activity. Our lab had previously engineered a TetR*-KRAB that is splitable into 2 to 4 fragments using split-inteins. The 4x-split design, which had the highest potential for cell targeting, performed suboptimally likely due to one of the split-inteins used, NrdJ-1. To overcome this, we substituted NrdJ-1 by split-inteins IMPDH-1 or RmaDnaB, but found little improvement in performance, so further work, likely on split-site choice, is required to ameliorate TetR*-KRAB reconstitution. Regarding Cre, previous split-versions near its catalytic residue 173, rendered it non-self-complementing–a valuable trait for intersectional genetics–, but also non-functional, likely because of an extein scar left at the split site after reconstitution. Here, by modifying scar length, we found that this split site is extremely sensitive to even small (3-residue) extein scars, and that local endogenous aminoacids could not support reconstitution of a scarless split-Cre, suggesting a requirement for more context-independent splitinteins to overcome this limitation. Finally, we identified a hiPSC line carrying a stably-integrated Cresensitive fluorescent reporter cassette, which showed homogeneous reporter expression and robust responsiveness to Cre. Together, this work contributes to design principles for multi-split protein reconstitution in human cells and delivers an integrated Cre reporter hiPSC line that can accelerate the development of intersectional biosensors for precise cell-type targeting. | en |
| dc.format | application/pdf | |
| dc.identifier.uri | http://hdl.handle.net/10400.5/118867 | |
| dc.identifier.url | http://hdl.handle.net/10400.5/118867 | |
| dc.language.iso | eng | |
| dc.subject | split inteins | |
| dc.subject | protein trans-splicing | |
| dc.subject | TetR*-KRAB | |
| dc.subject | Cre recombinase | |
| dc.subject | hiPSC reporter line | |
| dc.title | Optimization of intersectional genetics-based biosensors for human cell targeting purposes | en |
| dc.type | master thesis | |
| dspace.entity.type | Publication | |
| rcaap.rights | embargoedAccess |
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