Modification of the Method of Receiving of Insertion Mutants with the EZ::TN5 System


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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.

作者简介

E. Avdyusheva

Department of Molecular Biology and Genetics, Volgograd State Medical University, Ministry of Health of the Russian Federation

Email: elenakalinki@yandex.ru
俄罗斯联邦, Volgograd

Ya. Lopasteyska

Department of Molecular Biology and Genetics, Volgograd State Medical University, Ministry of Health of the Russian Federation; Volgograd Research Anti-Plague Institute, Federal Service for Supervision of Consumer Right Protection and Human Welfare

Email: elenakalinki@yandex.ru
俄罗斯联邦, Volgograd; Volgograd

T. Sharov

Volgograd Research Anti-Plague Institute, Federal Service for Supervision of Consumer Right Protection and Human Welfare

Email: elenakalinki@yandex.ru
俄罗斯联邦, Volgograd

N. Teteryatnikova

Department of Molecular Biology and Genetics, Volgograd State Medical University, Ministry of Health of the Russian Federation; Volgograd Research Anti-Plague Institute, Federal Service for Supervision of Consumer Right Protection and Human Welfare

Email: elenakalinki@yandex.ru
俄罗斯联邦, Volgograd; Volgograd

E. Molchanova

Department of Molecular Biology and Genetics, Volgograd State Medical University, Ministry of Health of the Russian Federation; Volgograd Research Anti-Plague Institute, Federal Service for Supervision of Consumer Right Protection and Human Welfare

编辑信件的主要联系方式.
Email: elenakalinki@yandex.ru
俄罗斯联邦, Volgograd; Volgograd

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