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Updated: Feb 23, 2026
Generating Transposon Insertion Libraries in Gram-Negative Bacteria for High-Throughput Sequencing
Published on: July 7, 2020
Modification of the Method of Receiving of Insertion Mutants with the EZ::TN5 System
E F Avdyusheva1, Ya A Lopasteyska1,2, T N Sharov2
1Department of Molecular Biology and Genetics, Volgograd State Medical University, Ministry of Health of the Russian Federation, Volgograd, Russia.
Abstract:
We demonstrated the possibility of obtaining insertion mutants by a modified technique using EZ::TN5 system during culturing of the recipient strain on a dense nutrient medium and exclusion of the centrifugation stage. The frequency of transposon mutants of E. coli 10979/EZ::TN5 was 2×10-6. Genetically modified strains were characterized by kanamycin resistance, inability to L-malate assimilation, changes in the expression of individual proteins of protein mass-spectra (5096.3, 6252.9, and 9067.7 Da), and the presence of fragments in genomic DNA amplified by specific forward and reverse primers that were homologous to Tn5 transposon insertion sites. The modified procedure for obtaining insertion mutants by using EZ::TN5 system was not inferior by the efficiency to the standard procedure, but shortens experiment duration, simplifies it, and reduces the risks related to working with group 2 pathogenicity microorganisms.
