Related Experiment Video
Updated: May 11, 2026
08:19
Generating Transposon Insertion Libraries in Gram-Negative Bacteria for High-Throughput Sequencing
Published on: July 7, 2020
Mariner-based transposon mutagenesis for Bacteroides species
Minoru Ichimura1, Keiko Uchida, Haruyuki Nakayama-Imaohji
1Department of Microbiology, Faculty of Medicine, Kagawa University, Miki, Kagawa, Japan; Department of Immunology and Parasitology, Institute of Health Biosciences, The University of Tokushima Graduate School, Kuramoto-cho, Tokushima, Japan.
Journal of Basic Microbiology
|May 21, 2013
Summary
Researchers developed a novel transposon mutagenesis method for Bacteroides species. This technique efficiently generates insertion mutants, aiding the discovery of genes crucial for human-Bacteroides symbiosis and gut health.
Area of Science:
- Microbiology
- Genetics
- Gastroenterology
Background:
- Bacteroides are key human gut commensals influencing host health.
- Understanding Bacteroides' symbiotic roles requires genetic tools.
- Mariner transposons offer random mutagenesis potential.
Purpose of the Study:
- To develop a mariner transposon-based mutagenesis system for Bacteroides.
- To enable the identification of genes involved in Bacteroides-host interactions.
Main Methods:
- Construction of the pMI07 plasmid carrying ermF/ITR gene cassette.
- Electrotransformation of pMI07 into multiple Bacteroides species.
- Analysis of insertion sites in B. thetaiotaomicron and B. vulgatus genomes.
Main Results:
- Successful delivery and genome integration of ermF/ITR in B. thetaiotaomicron, B. fragilis, B. ovatus, and B. vulgatus.
- Generation of thousands of insertion mutants per μg of pMI07.
- Unbiased integration targeting the TA dinucleotide in B. thetaiotaomicron and B. vulgatus.
Conclusions:
- The pMI07 plasmid is an effective tool for mariner transposon mutagenesis in Bacteroides.
- This method facilitates large-scale genetic screening in Bacteroides species.
- Provides a foundation for identifying genes essential for Bacteroides' symbiotic functions.
More Related Videos
Related Concept Videos
01:24Transposons
Transposons, or "jumping genes," are small mobile genetic elements (MGEs) that range from 700 to 40,000 base pairs in length. They are found in all organisms and can move within the same chromosome or transfer to different chromosomes. In some cases, transposons can also jump between different host DNA molecules, such as plasmids or viruses, contributing to genetic variability.Barbara McClintock first discovered these mobile genetic elements in the 1940s while studying maize genetics, and she...
01:33Bacterial Transformation
In 1928, bacteriologist Frederick Griffith worked on a vaccine for pneumonia, which is caused by Streptococcus pneumoniae bacteria. Griffith studied two pneumonia strains in mice: one pathogenic and one non-pathogenic. Only the pathogenic strain killed host mice.
Griffith made an unexpected discovery when he killed the pathogenic strain and mixed its remains with the live, non-pathogenic strain. Not only did the mixture kill host mice, but it also contained living pathogenic bacteria that...
Griffith made an unexpected discovery when he killed the pathogenic strain and mixed its remains with the live, non-pathogenic strain. Not only did the mixture kill host mice, but it also contained living pathogenic bacteria that...
01:26Bacterial Phylum Bacteroidota
The phylum Bacteroidota includes over 700 species classified into four primary orders: Bacteroidales, Cytophagales, Flavobacteriales, and Sphingobacteriales. These gram-negative, non-sporulating rods exhibit saccharolytic capabilities and can be aerobic or fermentative, encompassing obligate aerobes, facultative aerobes, and obligate anaerobes. Many species display gliding motility, though some are nonmotile or use flagella. The genus Bacteroides is well-studied due to its significant role in...
02:57DNA-only Transposons
DNA-only transposons are called autonomous transposons since they code for the enzyme transposase that is required for the transposition mechanism. Insertion of transposons can alter gene functions in multiple ways. They can mutate the gene, alter gene expression by introducing a novel promoter or insulator sequence, introduce new splice sites, and change the mRNA transcripts produced, or remodel chromatin structure.
The donor site from where the transposon is excised is either degraded or...
The donor site from where the transposon is excised is either degraded or...
01:26DNA Bacteriophages
Bacteriophages, or phages, are viruses that specifically infect bacteria, utilizing their genetic material to hijack host cellular machinery for replication. DNA bacteriophages employ single-stranded DNA (ssDNA) or double-stranded DNA (dsDNA) genomes. These phages exhibit diverse replication strategies and host interactions, influencing their ecological roles and applications in biotechnology and medicine.ssDNA BacteriophagesssDNA phages, with their small genomes, utilize unique strategies to...