Assessment of the Fluorescent Activities of Different Fluorescent Proteins for Potential Use as Biosensors in Saccharomyces cerevisiae
(2020) KMBM05 20201Applied Microbiology
- Abstract
- Development of genetically modified microorganisms such as Saccharomyces cerevisiae for use as microbial cell factories to produce fine and bulk chemicals has lately proceeded very rapidly, much thanks to new gene editing methods based on a CRISPR/Cas9 system adapted from bacteria which increases the rate of genetic engineering. However, development of methods which enable fast and simple screening of the best performing strains with specific sought-after characteristics is not advancing at the same pace. Therefore, screening can be a bottleneck in the cell factory engineering process, slowing down the overall development time. In this context, biosensors where inducible promoters are coupled to reporter molecules are of interest because... (More)
- Development of genetically modified microorganisms such as Saccharomyces cerevisiae for use as microbial cell factories to produce fine and bulk chemicals has lately proceeded very rapidly, much thanks to new gene editing methods based on a CRISPR/Cas9 system adapted from bacteria which increases the rate of genetic engineering. However, development of methods which enable fast and simple screening of the best performing strains with specific sought-after characteristics is not advancing at the same pace. Therefore, screening can be a bottleneck in the cell factory engineering process, slowing down the overall development time. In this context, biosensors where inducible promoters are coupled to reporter molecules are of interest because they allow for high throughput screening. As reporter molecules, fluorescent proteins are good candidates because of their non- invasiveness and easy detection through, for example, fluorescence imaging or flow cytometry. Biosensors for analyzing multiple properties in parallel require bright fluorescent proteins with several different colors. In this project, the fluorescent activities of fluorescent protein candidates were investigated by integrating the genes encoding the proteins into the genome of S. cerevisiae and analyzing the fluorescent activities using flow cytometry. The candidates yEGFP, mEGFP, CyOFP1, yeast codon optimized CyOFP1 and yeast codon optimized mBeRFP were bright enough to be distinguished from the background fluorescence, whereas the strain with the gene encoding smURFP could not be distinguished from the background fluorescence, not even after attempting to increase the fluorescence signals by supplying the strain with a cofactor necessary for smURFP fluorescence. Not all proteins were suitable to be combined due to fluorescence spillover. Two strains were generated, one with codon optimized CyOFP1 and mEGFP and one with codon optimized mBeRFP and mEGFP. The data for these strains suggested that bright signals derived from each fluorescent protein could be detected, showing promise for use as dual biosensors. More work is required to find suitable combinations of three or more fluorescent proteins for biosensor applications, and it is recommended that the method of color compensation is investigated to handle unavoidable spillover fluorescence. (Less)
- Popular Abstract
- Using proteins as molecular light bulbs
For a very long time, humans have taken advantage of the microorganism Saccharomyces cerevisiae, a unicellular species of yeast, to produce food and beverages such as bread and beer. Thanks to scientific advances, we are now able to tailor microorganisms, such as the yeast we have used for thousands of years, to work as micro-scale factories for production of, for example, pharmaceuticals or biofuels. In this project I have worked on finding suitable parts for a tool which hopefully can help researchers figure out what processes are taking place in the microorganisms and how to manipulate them to create the best possible micro-scale factories. I found a small collection of molecular light bulbs... (More) - Using proteins as molecular light bulbs
For a very long time, humans have taken advantage of the microorganism Saccharomyces cerevisiae, a unicellular species of yeast, to produce food and beverages such as bread and beer. Thanks to scientific advances, we are now able to tailor microorganisms, such as the yeast we have used for thousands of years, to work as micro-scale factories for production of, for example, pharmaceuticals or biofuels. In this project I have worked on finding suitable parts for a tool which hopefully can help researchers figure out what processes are taking place in the microorganisms and how to manipulate them to create the best possible micro-scale factories. I found a small collection of molecular light bulbs which make yeast cells glow in different colors.
Many people likely associate proteins with something that we eat or a something that builds up the muscles in our bodies. While both these things are true, there is a massive range of different proteins which, thanks to differences in structure, have a variety of functions in all living beings. In this project I assessed proteins with specific structures which allow them to emit fluorescence,or simply put, shine in different colors. Most of the proteins that I assessed shone very brightly, and this opens possibilities to essentially use them as molecular light bulbs.
No light bulb starts to glow by itself, there’s always a person who switches it on. Likewise, the molecular light bulbs also need to be switched on to be able to glow. This is done by connecting them to molecular light switches, much like electric wires are connected between the lamps in your living room and the switches on your walls. The molecular light switches can be turned on by specific molecules which attach to the switches. So, the idea is that when a specific process occurs inside a microorganism, this will trigger the activation of a specific light switch, resulting in a brightly glowing molecular light bulb and thereby also a brightly glowing microorganism. Thus, researchers can figure out what process is going on in the microorganism based on which colored light bulb has been turned on. In addition, researchers can figure out how intensely a process is being carried out based on how strongly the microorganism is glowing.
The process of tailoring a microorganism to produce a compound of human interest is very complex – essentially it comes down to “hacking” the intricate metabolic system and reprogramming it. Hacking is done by performing many precise edits to the DNA of the microorganism. It is crucial that the best edits are made to generate an optimal micro-scale factory out of the microorganism. Currently, the various ways for researchers to assess and screen for the best edits are complex and require a lot of time. This is where the molecular light switches and light bulbs can come in handy.
In this project, I found several proteins which have potential to be used as molecular light bulbs in the yeast species called S. cerevisiae. It is hoped that these proteins will be of use to facilitate the process of engineering S. cerevisiae to produce both fine chemicals (e.g. pharmaceuticals) and bulk chemicals (e.g. biofuel). One may wonder why researchers would go through such a long and complicated process to produce these commodities when there are already chemical processes in place for such production. The reason is that taking advantage of microorganisms has the potential to be a much more sustainable production method, for example by reducing reliance on fossil resources, since the microorganisms can be fed with renewable resources. (Less)
Please use this url to cite or link to this publication:
http://lup.lub.lu.se/student-papers/record/9018904
- author
- Davidsson, Johan LU
- supervisor
- organization
- course
- KMBM05 20201
- year
- 2020
- type
- H2 - Master's Degree (Two Years)
- subject
- keywords
- Fluorescent proteins, Saccharomyces cerevisiae, Flow cytometry, CRISPR/Cas9, Biosensors, Applied microbiology, Tillämpad mikrobiologi
- language
- English
- id
- 9018904
- date added to LUP
- 2020-06-18 08:58:02
- date last changed
- 2020-06-18 08:58:02
@misc{9018904,
abstract = {{Development of genetically modified microorganisms such as Saccharomyces cerevisiae for use as microbial cell factories to produce fine and bulk chemicals has lately proceeded very rapidly, much thanks to new gene editing methods based on a CRISPR/Cas9 system adapted from bacteria which increases the rate of genetic engineering. However, development of methods which enable fast and simple screening of the best performing strains with specific sought-after characteristics is not advancing at the same pace. Therefore, screening can be a bottleneck in the cell factory engineering process, slowing down the overall development time. In this context, biosensors where inducible promoters are coupled to reporter molecules are of interest because they allow for high throughput screening. As reporter molecules, fluorescent proteins are good candidates because of their non- invasiveness and easy detection through, for example, fluorescence imaging or flow cytometry. Biosensors for analyzing multiple properties in parallel require bright fluorescent proteins with several different colors. In this project, the fluorescent activities of fluorescent protein candidates were investigated by integrating the genes encoding the proteins into the genome of S. cerevisiae and analyzing the fluorescent activities using flow cytometry. The candidates yEGFP, mEGFP, CyOFP1, yeast codon optimized CyOFP1 and yeast codon optimized mBeRFP were bright enough to be distinguished from the background fluorescence, whereas the strain with the gene encoding smURFP could not be distinguished from the background fluorescence, not even after attempting to increase the fluorescence signals by supplying the strain with a cofactor necessary for smURFP fluorescence. Not all proteins were suitable to be combined due to fluorescence spillover. Two strains were generated, one with codon optimized CyOFP1 and mEGFP and one with codon optimized mBeRFP and mEGFP. The data for these strains suggested that bright signals derived from each fluorescent protein could be detected, showing promise for use as dual biosensors. More work is required to find suitable combinations of three or more fluorescent proteins for biosensor applications, and it is recommended that the method of color compensation is investigated to handle unavoidable spillover fluorescence.}},
author = {{Davidsson, Johan}},
language = {{eng}},
note = {{Student Paper}},
title = {{Assessment of the Fluorescent Activities of Different Fluorescent Proteins for Potential Use as Biosensors in Saccharomyces cerevisiae}},
year = {{2020}},
}