Modifying Fletchervirus with CRISPRi in Campylobacter jejuni and using PCR to create a synthetic genome
(2023) KBKM05 20231Pure and Applied Biochemistry
Computational Chemistry
- Abstract
- Campylobacter jejuni causes around 100 million cases of gastroenteritis each year and are often transmitted to humans through the consumption of contaminated food, especially poultry. One of the natural enemies of C. jejuni is the bacteriophage Fletchervirus. In this master thesis I have been working on developing and evaluating different tools to modify Fletchervirus which could in the future help with decreasing the burden of diseases. The primary aim was to use a CRISPRi system developed for C. jejuni to suppress the expression of the receptor binding protein 2 (RBP2) used by Fletchervirus to infect C. jejuni. The suppression of RBP2 would lead to a change in plaque morphology which would be visible. The secondary aim was to create a... (More)
- Campylobacter jejuni causes around 100 million cases of gastroenteritis each year and are often transmitted to humans through the consumption of contaminated food, especially poultry. One of the natural enemies of C. jejuni is the bacteriophage Fletchervirus. In this master thesis I have been working on developing and evaluating different tools to modify Fletchervirus which could in the future help with decreasing the burden of diseases. The primary aim was to use a CRISPRi system developed for C. jejuni to suppress the expression of the receptor binding protein 2 (RBP2) used by Fletchervirus to infect C. jejuni. The suppression of RBP2 would lead to a change in plaque morphology which would be visible. The secondary aim was to create a synthetic Fletchervirus genome through creating fragments of the genome through PCR and then assembling the fragments using Gibson assembly. This could make large modifications simpler to do in the future. The results were that a single transformant with the CRISPRi system was tested using a plaque assay but no change in plaque morphology was observed. Further tests are needed to fully evaluate if the CRISPRi system works on Fletchervirus while in C. jejuni. The synthetic genome was not assembled but two fragments were created and the primers that were made could be used as a starting point for future work. (Less)
- Popular Abstract
- The genetic modification of Fletchervirus bacteriophages
There is right now a lot of ongoing research concerning the use of bacteriophages (viruses that prey on bacteria) since antibiotic resistance is on the rise.
In my master thesis I have been testing different ways of modifying the bacteriophage Fletchervirus that prey on the common food pathogen Campylobacter Jejuni. Campylobacter causes around 100 million cases of gastroenteritis each year, it is therefore highly interesting to combat, and one way to do that is with bacteriophages. To use Fletchervirus against C. jejuni there is more research needed, research that could be done faster with better tools for modifying Fletchervirus. Therefore, I have tested a new system that... (More) - The genetic modification of Fletchervirus bacteriophages
There is right now a lot of ongoing research concerning the use of bacteriophages (viruses that prey on bacteria) since antibiotic resistance is on the rise.
In my master thesis I have been testing different ways of modifying the bacteriophage Fletchervirus that prey on the common food pathogen Campylobacter Jejuni. Campylobacter causes around 100 million cases of gastroenteritis each year, it is therefore highly interesting to combat, and one way to do that is with bacteriophages. To use Fletchervirus against C. jejuni there is more research needed, research that could be done faster with better tools for modifying Fletchervirus. Therefore, I have tested a new system that should be able to suppress one protein at a time and I have also tested a way to create a synthetic Fletchervirus genome. The system that can suppress one protein at a time is a CRISPRi system that interferes with protein transcription (the reading of DNA to create messenger RNA which is used to create proteins). A group in the UK has created this CRISPRi system and has used it in C. jejuni before, but not to work on Fletchervirus while inside C. jejuni.
The CRISPR-Cas system is a naturally occurring mechanism that bacteria use to defend against viral infections. The system consists of two components: a guideRNA that binds to a specific DNA sequence and the Cas protein that cuts the DNA at that site. CRISPRi stands for "CRISPR interference", and a deactivated Cas protein is used, that can bind to the DNA but does not cut it. Instead, it blocks some part of the transcription of the gene, preventing the whole protein from being expressed.
The group from the UK made four plasmids which expressed a CRISPRi system that could block the transcription of receptor binding protein 2 (RBP2) that Fletchervirus uses to infect C. jejuni. If successful, blocking this protein could hopefully be detected in a plaque assay, since RBP2 creates a unique plaque morphology. The results were that a single transformant with the CRISPRi system was tested using a plaque assay but that there was no change in plaque morphology, further tests are however needed to conclude if the system works or not.
The secondary aim was to create a synthetic Fletchervirus genome through creating fragments of the genome through PCR and then assembling the fragments using Gibson assembly. This could make future large modifications simpler to do. The synthetic genome was not assembled but two fragments were created and the primers made could be used in the future, a lot was also learned about how to build a synthetic Fletchervirus genome. (Less)
Please use this url to cite or link to this publication:
http://lup.lub.lu.se/student-papers/record/9128947
- author
- Miefalk, Jacob LU
- supervisor
- organization
- course
- KBKM05 20231
- year
- 2023
- type
- H2 - Master's Degree (Two Years)
- subject
- keywords
- applied biochemistry, genetic engineering, bacteriophages, CRISPRi
- language
- English
- id
- 9128947
- date added to LUP
- 2023-06-26 08:57:31
- date last changed
- 2023-06-26 08:57:31
@misc{9128947,
abstract = {{Campylobacter jejuni causes around 100 million cases of gastroenteritis each year and are often transmitted to humans through the consumption of contaminated food, especially poultry. One of the natural enemies of C. jejuni is the bacteriophage Fletchervirus. In this master thesis I have been working on developing and evaluating different tools to modify Fletchervirus which could in the future help with decreasing the burden of diseases. The primary aim was to use a CRISPRi system developed for C. jejuni to suppress the expression of the receptor binding protein 2 (RBP2) used by Fletchervirus to infect C. jejuni. The suppression of RBP2 would lead to a change in plaque morphology which would be visible. The secondary aim was to create a synthetic Fletchervirus genome through creating fragments of the genome through PCR and then assembling the fragments using Gibson assembly. This could make large modifications simpler to do in the future. The results were that a single transformant with the CRISPRi system was tested using a plaque assay but no change in plaque morphology was observed. Further tests are needed to fully evaluate if the CRISPRi system works on Fletchervirus while in C. jejuni. The synthetic genome was not assembled but two fragments were created and the primers that were made could be used as a starting point for future work.}},
author = {{Miefalk, Jacob}},
language = {{eng}},
note = {{Student Paper}},
title = {{Modifying Fletchervirus with CRISPRi in Campylobacter jejuni and using PCR to create a synthetic genome}},
year = {{2023}},
}