First Advisor

Michael S. Bartlett

Date of Award

Spring 6-13-2025

Document Type

Thesis

Degree Name

Bachelor of Science (B.S.) in Biochemistry and University Honors

Department

Chemistry

Language

English

Subjects

BioID, Pyrococcus furiosus, Proximity-dependent biotinylation

Abstract

Proximity-dependent biotinylation is an approach that involves the fusion of an enzyme capable of catalyzing the activation of biotin to a bait protein, allowing for the biotinylation of nearby proteins in living cells that can later be captured and identified to study in vivo protein-protein interactions. A commonly used enzyme involves a biotin ligase, and an improved biotin ligase with more-selective targeting of fusion proteins has previously been isolated from Aquifex aeolicus for expression in other biological systems, which has been termed "BioID2". This study aimed to express BioID2 in the hyperthermophilic archaeon Pyrococcus furiosus to gain further insight into the archaeal transcription machinery by fusing BioID2 to the RNA polymerase subunit RpoD, the transcription factor TFB1, and the transcription factor TFB2. Experimental DNA constructs expected to produce BioID2 and BioID2-protein fusions were used to transform COM1 Pyrococcus furiosus through PCR and selection via a pyrF gene. Post-transformation PCR suggested successful transformation for the strains with the BioID2 control and BioID2 fusions proteins. Biotinylation of proteins was checked through a procedure involving SDS-PAGE and probing of a membrane with fluorescent streptavidin. The results of these experiments suggested that biotinylation was occurring, but no novel biotinylation targets were identified when compared to the BioID2 control. This study was centered mainly around the feasibility of expressing proximity biotinylation in P. furiosus, and the results obtained suggest that with further development, BioID2 holds promise as a potentially robust and versatile tool for studying protein-protein interactions in Pyrococcus furiosus.

Persistent Identifier

https://archives.pdx.edu/ds/psu/43743

Available for download on Saturday, May 22, 2027

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