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Expression and bioconjugation of the fusion proteins via outer membrane translocation for bacterial cell surface display
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Kemian tekniikan korkeakoulu |
Master's thesis
Electronic archive copy is available via Aalto Thesis Database.
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CHEM3022
Language
en
Pages
84+3
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Abstract
Cell surface display systems, in which molecules with anchoring motifs are exposed on the outer membrane of cells, offer numerous biotechnological applications. These systems involve expressing or conjugating proteins to the cell membrane, facilitating interactions with the external environment, and enabling innovative explorations in cellular behaviour.
The aim of this thesis was to develop a functional cell surface display system to enable the conjugation of magnetic beads to cells, allowing for manipulated bacterial movement under an external magnetic field. Two different conjugation pairs, SpyCatcher003 (SC003) and monomeric Streptavidin2 (mSA2) proteins were genetically fused with anchoring motifs from outer membrane protein A (OmpA) and a truncated form of lipoprotein (lpp’), to be translocated and anchored to the outer membrane of Escherichia coli. SC003 and mSA2 proteins enable specific bioconjugation with molecules fused with their counterparts SpyTag and Biotin, respectively. This bioconjugation affinity facilitates the controlled cell manipulation by conjugating protein-coated magnetic beads to the cells.
The results demonstrated the expression of the lpp’-OmpA-SC003 and lpp’-OmpA-mSA2 fusion proteins, confirmed by SDS-PAGE analysis. The translocation of the lpp’-OmpA-SC003 fusion protein was verified with confocal microscopy through the specific bioconjugation between the SpyCatcher003 and SpyTag-GFP fusion protein. The green fluorescence signal from GFP confirmed the localization of the fusion proteins on the cell surface. Subsequently, the thesis investigated the conjugation of magnetic beads with cells, verifying the translocation of the lpp’-OmpA-mSA2 fusion protein. This was achieved through the bioconjugation affinity between the mSA2 protein and biotin-coated magnetic beads, which facilitated the conjugation and verification of the translocation. The results were promising highlighting opportunities for utilizing bioconjugation techniques in future studies of manipulated bacterial cell movement.
Description
Supervisor
Mangayil, RahulThesis advisor
Aranko, SesiljaShen, Mengjie