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GENETIC ENGINEERING
pUT mini-Tn5 System
The mini-Tn5 are mobile elements able to transpose to the chromosome from a delivery plasmid, by means of the transposase activity encoded by the tnp* gene, which is present in cis in the plasmid but external to the mini-Tn5 element. pUT mini-Tn5 vectors can be used to insert any DNA fragment in the bacterial chromosome, by cloning it within the mini-Tn5 region. Due to the loss of the tnp* gene after insertion, minitransposons are stably integrated into the chromosome and inherited. The different pUTmini-Tn5 vectors can then be used for repeated insertion events and therefore multiple insertions in the same strain are only limited by the availability of distinct selection markers.
Applications:
Some of the most common applications of pUTmini-Tn5 vectors are:
- - Cloning.
- - Insertional mutagenesis.
- - Promoter probing.
- - Generation of conditional phenotypes and non-polar mutations.
- - Analysis of transcriptional terminators.
- - Environmental applications: tagging with marker genes for monitoring of released microorganisms and/or transcriptional regulation in the environment.
Advantages:
- - Easy and stable insertion of heterologous DNA in the chromosome of the strain of interest, after few simple genetic manipulations.
- - Wide range of inserted fragment sizes.
- - Possibility of multiple insertions in the same strain.
- - Easy modification of the cloning vector.
- - Use of cleavage sites of the rare cutters NotI and SfiI.
- - Wide range of Gram negative recipient strains.