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Improving the efficiency of transposon library preparation in Bacillus subtilis

Ghazi Karim, Heran (2025) MOBM02 20251
Degree Projects in Molecular Biology
Popular Abstract
Peeking under the hood of bacteria

Imagine you want to figure out what the different parts of your car do and essentially how important they are for it to function properly. The way to answer these questions could be to remove one part at a time and see what happens. In a similar way, whenever scientists want to better understand bacteria, they have learned different ways or techniques of doing so, involving removing or disrupting one gene at a time to see how it affects the organism.

Background
My project is looking at one such technique called transposon mutagenesis, which uses transposons, “jumping DNA”, that can jump from one place to another. Once in a while, they jump into a gene and stop it from working, creating a mutant... (More)
Peeking under the hood of bacteria

Imagine you want to figure out what the different parts of your car do and essentially how important they are for it to function properly. The way to answer these questions could be to remove one part at a time and see what happens. In a similar way, whenever scientists want to better understand bacteria, they have learned different ways or techniques of doing so, involving removing or disrupting one gene at a time to see how it affects the organism.

Background
My project is looking at one such technique called transposon mutagenesis, which uses transposons, “jumping DNA”, that can jump from one place to another. Once in a while, they jump into a gene and stop it from working, creating a mutant (fig 1.). By having a huge number of bacteria, each containing a different gene disruption, we say that we have created a transposon mutant library. A final piece of the technology is sequencing, which helps us identify the specific gene disrupted in each mutant. We use this information to see how each mutant behaves and to better understand the genes involved. Essentially, this technique enables us to look under the hood of life’s machinery by generating a whole library of mutants in one go.

Project’s aim & method
Generally, these jumping DNAs are introduced into the bacteria via a plasmid. This is like a delivery truck carrying a cargo – in our case the transposon. However, this “truck” needs to be removed after it has done its job, otherwise it can cause issues later on. Traditional methods for removing the “truck” have proven to be time consuming and not applicable in all experiments, so my project investigated new ways of removing it. I explored two unique and non-traditional methods: (i) using the technology of CRISPR-Cas, “genetic scissors” to remove the plasmid by cutting it away and (ii) directly introducing the transposon into the DNA of the bacteria without the delivery system. The systems were designed using standard DNA cloning tools, in the well-established model organism, Bacillus subtilis.

Results & discussion
Due to the project’s limited timeline and cloning difficulties, the CRISPR-based system for removing the plasmid after transposon delivery was never fully finished. However, I managed to create the guide RNA construct needed for CRISPR scissors to find and later on cut the plasmid. Further work is required to complete this system. The second method, which involved directly introducing the transposon system into the DNA of B. subtilis at a specific site, was initially verified but further work is also needed to fully confirm a stable integration.

Conclusion & applications
My project laid the foundational work for new ways of generating bacterial transposon mutant libraries, which are more straightforward, less time-consuming and lastly more applicable. Although more research is needed to refine and complete these new methods, they are worth the while, since they show potential for future applications, especially in understanding how bacteria works. This is interesting in terms of finding new genes that could, for example, be targets for antibiotics or serve industrial benefits.

Master’s Degree Project in Molecular Biology 30 credits 2025
Department of Biology, Lund University
Advisor: Fabia Koch & Leendert Hamoen
Group Bacterial and Cell Biology, University of Amsterdam
Swammerdam Institute for Life Sciences, Amsterdam, Netherlands (Less)
Please use this url to cite or link to this publication:
author
Ghazi Karim, Heran
supervisor
organization
course
MOBM02 20251
year
type
H2 - Master's Degree (Two Years)
subject
language
English
id
9214497
date added to LUP
2025-10-24 14:05:05
date last changed
2025-10-24 14:05:05
@misc{9214497,
  author       = {{Ghazi Karim, Heran}},
  language     = {{eng}},
  note         = {{Student Paper}},
  title        = {{Improving the efficiency of transposon library preparation in Bacillus subtilis}},
  year         = {{2025}},
}