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Related Concept Videos

Transposons01:24

Transposons

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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...
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DNA-only Transposons02:57

DNA-only Transposons

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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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Overview of Transposition and Recombination02:13

Overview of Transposition and Recombination

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Transposons make up a significant part of genomes of various organisms. Therefore, it is believed that transposition played a major evolutionary role in speciation by changing genome sizes and modifying gene expression patterns. For example, in bacteria, transposition can lead to conferring antibiotic resistance. Movement of transposable elements within the genetic pool of pathogenic bacteria can aid in transfer of antibiotic-resistant genetic elements. In eukaryotes, transposons can carry out...
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In-vitro Mutagenesis01:16

In-vitro Mutagenesis

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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.
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Tagging and Fusion Proteins01:24

Tagging and Fusion Proteins

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Proteins are involved in several cellular processes and biochemical reactions. Analyzing a specific protein of interest requires it to be isolated from the other proteins in the cell. This is achieved by overexpressing the specific gene in a suitable host to produce large quantities of the target protein. A tag or label is recombined with the gene to produce a fusion protein containing the target protein and the tag. The tags on these fusion proteins can then be used for easy detection and...
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LTR Retrotransposons03:08

LTR Retrotransposons

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LTR retrotransposons are class I transposable elements with long terminal repeats flanking an internal coding region. These elements are less abundant in mammals compared to other class I transposable elements. About 8 percent of human genomic DNA comprises LTR retrotransposons. Some of the common examples of LTR retrotransposons are Ty elements in yeast and Copia elements in Drosophila.
The internal coding region of LTR retrotransposons and their mechanism of transposition closely resembles a...
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Related Experiment Video

Updated: Jan 1, 2026

Generating Transposon Insertion Libraries in Gram-Negative Bacteria for High-Throughput Sequencing
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Random tag insertions by Transposon Integration mediated Mutagenesis (TIM).

Brigitte M Hoeller1, Birgit Reiter, Sandra Abad

  • 1Research Centre Applied Biocatalysis, Petersgasse 14, 8010 Graz, Austria.

Journal of Microbiological Methods
|July 17, 2008
PubMed
Summary

Transposon Integration mediated Mutagenesis (TIM) allows random protein modification for engineering. This method efficiently isolates active enzymes with random tags in a single experiment.

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Area of Science:

  • Biotechnology
  • Molecular Biology
  • Protein Engineering

Background:

  • Transposon-based mutagenesis is a powerful tool for genetic manipulation.
  • Efficient protein engineering requires methods for targeted and random modifications.
  • Existing methods may lack the flexibility for diverse applications like random tagging.

Purpose of the Study:

  • To develop and validate a novel method called Transposon Integration mediated Mutagenesis (TIM) for protein engineering.
  • To demonstrate the capability of TIM for various genetic modifications, including random insertions and site-saturation mutagenesis.
  • To showcase the application of TIM for isolating functional, randomly tagged proteins.

Main Methods:

  • TIM combines random integration of modified bacteriophage Mu transposons with defined excision using AarI restriction endonuclease.
  • A transposon, GeneOpenerAarIKan, was designed for introducing 6xHis tags.
  • A library of Burkholderia gladioli esterase EstC was generated and screened using colony-based assays.

Main Results:

  • TIM successfully enabled random insertion of functional sequence tags (6xHis) into the esterase EstC.
  • The method allowed for the screening of a library for both the presence of the tag and enzyme activity.
  • Proof-of-concept demonstrated the isolation of active enzymes with randomly integrated 6xHis tags.

Conclusions:

  • Transposon Integration mediated Mutagenesis (TIM) is a versatile tool for protein engineering.
  • TIM facilitates the random insertion of functional tags and enables the isolation of active, tagged enzymes.
  • This strategy streamlines the process of generating and screening modified proteins for desired functions.