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In-vitro Mutagenesis

In-vitro Mutagenesis

To learn more about the function of a gene, researchers can observe what happens when the gene is inactivated or “knocked out,” by creating genetically engineered knockout animals. Knockout mice have been particularly useful as models for human diseases such as cancer, Parkinson’s disease, and diabetes.

DNA-only Transposons

DNA-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...

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Related Experiment Video

Updated: Aug 6, 2025

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Published on: May 13, 2015

High-Throughput Mutant Screening via Transposon Sequencing.

Jacob Bourgeois¹, Andrew Camilli²

¹Department of Molecular Biology and Microbiology, Tufts University, School of Medicine, Boston, Massachusetts 02067, USA.

Cold Spring Harbor Protocols

|March 17, 2023

Summary

This summary is machine-generated.

Transposon sequencing (Tn-seq) enables rapid identification of essential and conditional genes in bacteria. This high-throughput method analyzes transposon insertion libraries to understand gene function and bacterial fitness.

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Generation of Enterobacter sp. YSU Auxotrophs Using Transposon Mutagenesis

Generation of Enterobacter sp. YSU Auxotrophs Using Transposon Mutagenesis

Published on: October 31, 2014

Area of Science:

Bacteriology
Genetics
Molecular Biology

Background:

Transposon mutagenesis is a key tool for bacterial genetic analysis.
Transposon sequencing (Tn-seq) offers high-throughput screening of mutant libraries.
Identifying gene essentiality and conditional roles is crucial for understanding bacterial physiology.

Purpose of the Study:

To introduce a method for constructing saturated transposon insertion libraries in Gram-negative bacteria.
To capture transposon junctions efficiently for large-scale analysis.
To identify essential and conditional genes using high-throughput sequencing.

Main Methods:

Construction of a saturating transposon insertion library.
High-throughput sequencing of transposon junctions.
Analysis of mutant fitness across different conditions.

Main Results:

Successful generation of a comprehensive transposon insertion library.
Identification of essential genes required for bacterial survival.
Discovery of genes with conditional roles in fitness.

Conclusions:

The presented method facilitates large-scale genetic screens in bacteria.
Tn-seq is a powerful approach for dissecting bacterial gene essentiality and function.
This protocol aids in understanding bacterial adaptation and survival mechanisms.