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Visualization of RNA Transcripts Within Live Eukaryotic Cells

Williamson, Allen (2024) MOBN03 20232
Degree Projects in Molecular Biology
Popular Abstract
Making live, single-celled organisms fluoresce

Protist is a term for a type of organism that has a nucleus and is not an animal, plant or fungus. Most protists are single-celled and can be found in all types of environments across the planet. Protists are highly diverse and understudied, and researching protists directly from the environment has many challenges. One method for researching microbes is to fluorescently stain their DNA (or RNA, a molecule very similar to DNA) to help visualize them. The goal of my thesis is to attempt to do this on live protists.

The technique employed uses a DNA probe with a dye attached to it to cause fluorescence. When this carefully designed probe binds to RNA that matches its own sequence, it can... (More)
Making live, single-celled organisms fluoresce

Protist is a term for a type of organism that has a nucleus and is not an animal, plant or fungus. Most protists are single-celled and can be found in all types of environments across the planet. Protists are highly diverse and understudied, and researching protists directly from the environment has many challenges. One method for researching microbes is to fluorescently stain their DNA (or RNA, a molecule very similar to DNA) to help visualize them. The goal of my thesis is to attempt to do this on live protists.

The technique employed uses a DNA probe with a dye attached to it to cause fluorescence. When this carefully designed probe binds to RNA that matches its own sequence, it can fluoresce. If there is not a match in the cell to the probe, it remains dark and will not fluoresce. Normally, to get these probes into cells, the cell must be dead in order to create holes in the cell’s membrane to allow entry of the probes. To get the probes inside the cell without killing it, I used a bunch of very small proteins that can cross the cell membrane while also bringing the probe with them. Think of it like this, the proteins form a package with the probe inside of it. Just like an online order of pants might be shipped to your door in a package, the probe is shipped to the cell in proteins. You take the package inside your home and open it to get the pants out. The cell does a similar thing, and once the protein package is inside, the probe is free to search for RNA that it matches so it can bind and fluoresce.

To start, I needed to design these probes so they would match RNA specific to protists. Because there are so many protists, I could not design a probe that would match all of them, so instead I focused on a probe that would match to RNA inside of some amoeba (a type of protist). I made two probes, and then I confirmed they worked as intended in a tube (with no cells). Next, I figured out how many of these proteins I needed to build a package of the right size to hold the probe. Then I tested my probes with the old method, where the cells are killed so the probes get in. Confirming that they worked, I then packaged the probes with their proteins and tested them on some amoeba. The amoeba were alive and the probes got inside to fluoresce.

This technique will be helpful for all researchers of protists, allowing an easy way to visualize, sort and investigate these creatures while keeping them alive.

Master’s Degree Project in Molecular Biology 60 credits 2024
Department of Biology, Lund University

Advisor: Courtney Stairs
Department of Biology, Lund University (Less)
Please use this url to cite or link to this publication:
author
Williamson, Allen
supervisor
organization
course
MOBN03 20232
year
type
H2 - Master's Degree (Two Years)
subject
language
English
id
9176709
date added to LUP
2024-10-17 09:26:00
date last changed
2024-10-17 09:26:00
@misc{9176709,
  author       = {{Williamson, Allen}},
  language     = {{eng}},
  note         = {{Student Paper}},
  title        = {{Visualization of RNA Transcripts Within Live Eukaryotic Cells}},
  year         = {{2024}},
}