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Chemical Tools for Protein Imaging in Live Bacterial Cells

Citation

Ho, Samuel Hin-Yuen (2019) Chemical Tools for Protein Imaging in Live Bacterial Cells. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/KNWF-CZ96. https://resolver.caltech.edu/CaltechTHESIS:05312019-211114775

Abstract

Bacteria spatially and temporally localize their proteins to carry out fundamental cellular processes. Methods for visualizing protein subcellular localization have been critical to our understanding of prokaryotic cell biology. Fluorescent reporters have been instrumental for imaging bacterial proteins in live cells. Small-molecule fluorescent dyes, which have favorable spectral properties, including high brightness and photostability, are attractive in labeling proteins of interest. Here we present a method to site-specifically label the N-termini of bacterial protein targets in situ for fluorescence imaging in bacterial cells. The method uses the eukaryotic enzyme N-myristoyltransferase to ligate target proteins, bearing a nonapeptide recognition sequence, with an azide-bearing fatty acid. Subsequent strain-promoted azide–alkyne cycloaddition with fluorophores enable tagging of chemotaxis and cell division proteins in live cells. We describe using a reactive BODIPY fluorophore for visualization of the chemotaxis proteins Tar and CheA and the division proteins FtsZ and FtsA. Next we integrate a single copy of the gene encoding the protein target into the chromosome via Tn7 transposon mutagenesis and use the method to fluorescently label a bacterial chemoreceptor. Finally, we describe the preparation of photoswitchable rhodamine spirolactam dyes for super-resolution imaging in live bacterial cells. Our work highlights the utility of using photoswitchable molecules to label intracellular protein targets. The ability to tag proteins, perform super-resolution imaging, and visualize proteins in space and time will prove broadly useful.

Item Type:Thesis (Dissertation (Ph.D.))
Subject Keywords:bioorthogonal; fluorescence microscopy; N-myristoyltransferase; super-resolution microscopy; protein modification
Degree Grantor:California Institute of Technology
Division:Chemistry and Chemical Engineering
Major Option:Chemistry
Thesis Availability:Public (worldwide access)
Research Advisor(s):
  • Tirrell, David A.
Thesis Committee:
  • Dervan, Peter B. (chair)
  • Miller, Thomas F.
  • Jensen, Grant J.
  • Tirrell, David A.
Defense Date:3 May 2019
Funders:
Funding AgencyGrant Number
National Science Foundation Graduate Research FellowshipUNSPECIFIED
Center for Environmental Microbiology InteractionsUNSPECIFIED
Jacobs Institute for Molecular Engineering for MedicineUNSPECIFIED
Institute for Collaborative Biotechnologies W911NF-09-D-0001
Record Number:CaltechTHESIS:05312019-211114775
Persistent URL:https://resolver.caltech.edu/CaltechTHESIS:05312019-211114775
DOI:10.7907/KNWF-CZ96
Related URLs:
URLURL TypeDescription
https://doi.org/10.1021/jacs.6b07067DOIArticle adapted from Chapter 2
ORCID:
AuthorORCID
Ho, Samuel Hin-Yuen0000-0001-7647-0752
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:11592
Collection:CaltechTHESIS
Deposited By: Samuel Ho
Deposited On:04 Jun 2019 22:31
Last Modified:04 Dec 2019 20:22

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