Single-molecule FRET studies in live bacteria
<p>Single-molecule fluorescence and single-molecule Förster resonance energy transfer (FRET) have proven enormously successful in understanding molecular and cellular processes over the last two decades. However, in vivo single-molecule FRET studies are still very difficult, mainly because the...
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| Format: | Thesis |
| Language: | English |
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2016
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| _version_ | 1797083717838045184 |
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| author | Plochowietz, A |
| author2 | Kapanidis, A |
| author_facet | Kapanidis, A Plochowietz, A |
| author_sort | Plochowietz, A |
| collection | OXFORD |
| description | <p>Single-molecule fluorescence and single-molecule Förster resonance energy transfer (FRET) have proven enormously successful in understanding molecular and cellular processes over the last two decades. However, in vivo single-molecule FRET studies are still very difficult, mainly because they require site-specifically labelled biomolecules with photostable organic dyes.</p> <p>In this work, I established single-molecule FRET studies in live bacteria and applied the developed method to study mechanisms of gene expression and gene regulation. To begin with, I optimised a recently developed internalisation method based on electroporation for the efficient loading of bacterial cells with organic dye-labelled nucleic acids and proteins while maintaining cell viability.</p> <p>Following these studies, I internalised labelled tRNA molecules, measured their diffusion coefficient, and showed that most tRNA molecules diffuse freely in live bacteria. I also showed that bound tRNA molecules are predominantly at the cell periphery and compete with the endogenous tRNA pool during translation using antibiotic controls and simulations.</p> <p>Finally, I studied transcription initiation in vivo by internalising promoter DNAs with different FRET labelling schemes reporting on individual steps in transcription initiation. Thus, I observed single-molecule FRET signatures attributed to open complex formation, DNA scrunching during initial transcription, and promoter escape, which were not present in null-promoter DNA and antibiotic controls.</p> <p>By carefully designing single-molecule FRET assays, I imagine single-molecule FRET studies to become a major tool in understanding protein dynamics, and elucidating mechanistic details of gene regulation processes in living cells.</p> |
| first_indexed | 2024-03-07T01:45:25Z |
| format | Thesis |
| id | oxford-uuid:9841649a-30e6-4237-ada2-1ceaba8d9a99 |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-03-07T01:45:25Z |
| publishDate | 2016 |
| record_format | dspace |
| spelling | oxford-uuid:9841649a-30e6-4237-ada2-1ceaba8d9a992022-03-27T00:05:42ZSingle-molecule FRET studies in live bacteriaThesishttp://purl.org/coar/resource_type/c_db06uuid:9841649a-30e6-4237-ada2-1ceaba8d9a99BiophysicsEnglishORA Deposit2016Plochowietz, AKapanidis, A<p>Single-molecule fluorescence and single-molecule Förster resonance energy transfer (FRET) have proven enormously successful in understanding molecular and cellular processes over the last two decades. However, in vivo single-molecule FRET studies are still very difficult, mainly because they require site-specifically labelled biomolecules with photostable organic dyes.</p> <p>In this work, I established single-molecule FRET studies in live bacteria and applied the developed method to study mechanisms of gene expression and gene regulation. To begin with, I optimised a recently developed internalisation method based on electroporation for the efficient loading of bacterial cells with organic dye-labelled nucleic acids and proteins while maintaining cell viability.</p> <p>Following these studies, I internalised labelled tRNA molecules, measured their diffusion coefficient, and showed that most tRNA molecules diffuse freely in live bacteria. I also showed that bound tRNA molecules are predominantly at the cell periphery and compete with the endogenous tRNA pool during translation using antibiotic controls and simulations.</p> <p>Finally, I studied transcription initiation in vivo by internalising promoter DNAs with different FRET labelling schemes reporting on individual steps in transcription initiation. Thus, I observed single-molecule FRET signatures attributed to open complex formation, DNA scrunching during initial transcription, and promoter escape, which were not present in null-promoter DNA and antibiotic controls.</p> <p>By carefully designing single-molecule FRET assays, I imagine single-molecule FRET studies to become a major tool in understanding protein dynamics, and elucidating mechanistic details of gene regulation processes in living cells.</p> |
| spellingShingle | Biophysics Plochowietz, A Single-molecule FRET studies in live bacteria |
| title | Single-molecule FRET studies in live bacteria |
| title_full | Single-molecule FRET studies in live bacteria |
| title_fullStr | Single-molecule FRET studies in live bacteria |
| title_full_unstemmed | Single-molecule FRET studies in live bacteria |
| title_short | Single-molecule FRET studies in live bacteria |
| title_sort | single molecule fret studies in live bacteria |
| topic | Biophysics |
| work_keys_str_mv | AT plochowietza singlemoleculefretstudiesinlivebacteria |