Ghitescu, Ecaterina (2026) Investigating the unfolding activity of E. coli ClpA using RepA-Tagged Fluorescent reporters, mPlum and mCherry. Bachelor's Thesis, Chemistry.
|
Text
BScthesisEcaterina-Ghitescu2026.pdf Download (1MB) | Preview |
Abstract
ClpA is a hexameric AAA+ motor in E. coli that uses energy from ATP hydrolysis to unfold and translocate substrates. This project tested the unfolding efficiency of ClpA and the ClpAP complex for potential application in nanopore based protein sequencing. Using RepA-tagged mCherry, mPlum, GFP and mNeonGreen, we analyzed how different substarte:unfoldase molar ratios and ionic strength influences the motor’s activity. Florescence assays revealed that ClpAP consistently outperforms ClpA alone and that ClpP acts as an allosteric modulator that reduces mechanical stalling in ClpA. The motor also showed a salt tolerance for 0.3M and 0.5M KCl which is likely due to hydrophobic RepA-ClpA binding interface. This is a promising trait for nanopore biosensing since high salt concentration is required for a good signal-to-noise ratios. While RepA-mCherry, mPlum and GFP were successfully processed, mNeonGreen proved highly resistant to N-terminal unfolding. A slight resistance to unfolding of RepA-mCherry was observed in 0.5M KCl, however, current findings are based on duplicate measurements, thus, the data lacks the statistical power required to conclude our observations.
| Item Type: | Thesis (Bachelor's Thesis) |
|---|---|
| Supervisor name: | Maglia, Giovanni and Tych, Katarzyna M. |
| Degree programme: | Chemistry |
| Thesis type: | Bachelor's Thesis |
| Language: | English |
| Date Deposited: | 27 Mar 2026 11:38 |
| Last Modified: | 27 Mar 2026 11:38 |
| URI: | https://fse.studenttheses.ub.rug.nl/id/eprint/37270 |
Actions (login required)
 |
View Item |