Skip to main content
SpringerLink
Log in

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

  • METHODS
  • Published:
Bulletin of Experimental Biology and Medicine Aims and scope

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Challacombe JF, Duncan AJ, Brettin TS, Bruce D, Chertkov O, Detter JC, Han CS, Misra M, Richardson P, Tapia R, Thayer N, Xie G, Inzana TJ. Complete genome sequence of Haemophilus somnus (Histophilus somni) strain 129Pt and comparison to Haemophilus ducreyi 35000HP and Haemophilus influenzae Rd. J. Bacteriol. 2007;189(5):1890-1898.

    Article  CAS  PubMed  Google Scholar 

  2. Dawoud TM, Jiang T, Mandal RK, Ricke SC, Kwon YM. Improving the efficiency of transposon mutagenesis in Salmonella enteritidis by overcoming host-restriction barriers. Mol. Biotechnol. 2014;56(11):1004-1010.

    Article  CAS  PubMed  Google Scholar 

  3. Gonzales MF, Brooks T, Pukatzki SU, Provenzano D. Rapid protocol for preparation of electrocompetent Escherichia coli and Vibrio cholerae. J. Vis. Exp. 2013(80):doi: 10.3791/50684.

  4. Goryshin IY, Jendrisak J, Hoffman LM, Meis R, Reznikoff WS. Insertion transposon mutagenesis by electroporation of released Tn5 transposition complexes. Nat. Biotechnol. 2000;18(1):97-100.

    Article  CAS  PubMed  Google Scholar 

  5. Goryshin IY, Reznikoff WS. Tn5 in vitro transposition. J. Biol. Chem. 1998;273(13):7367-7374.

    Article  CAS  PubMed  Google Scholar 

  6. Lennen RM, Herrgård MJ. Combinatorial strategies for improving multiple-stress resistance in industrially relevant Escherichia coli strains. Appl. Environ. Microbiol. 2014;80(19):6223-6242.

    Article  PubMed  PubMed Central  Google Scholar 

  7. Maeder DL, Anderson I, Brettin TS, Bruce DC, Gilna P, Han CS, Lapidus A, Metcalf WW, Saunders E, Tapia R, Sowers KR. The Methanosarcina barkeri genome: comparative analysis with Methanosarcina acetivorans and Methanosarcina mazei reveals extensive rearrangement within Methanosarcinal genomes. J. Bacteriol. 2006;188(22):7922-7931.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Nakamura H, Funahashi J. Electroporation: past, present and future. Dev. Growth Differ. 2013;55(1):15-19.

    Article  PubMed  Google Scholar 

  9. O’Mullan PJ. Direct sequencing of BAC clones without subcloning or primer walking using EZ::TN™ transposon tools. Epicentre Forum. 2000;7(4): [URL: http://www.epibio.com/docs/default-source/forum-archive/forum-07-4---direct-sequencing-of-bac-clones-without-subcloning-or-primer-walking-using-ez-tn-transposon-tools.pdf?sfvrsn=6].

  10. O’Reilly EK, Kreuzer KN. Isolation of SOS constitutive mutants of Escherichia coli. J. Bacteriol. 2004;186(21):7149-7160.

    Article  PubMed  PubMed Central  Google Scholar 

  11. Shi H, Djikeng A, Mark T, Wirtz E, Tschudi C, Ullu E. Genetic interference in Trypanosoma brucei by heritable and inducible double-stranded RNA. RNA. 2000;6(7):1069-1076.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Author information

Authors and Affiliations

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

    E. F. Avdyusheva, Ya. A. Lopasteyska, N. N. Teteryatnikova & E. V. Molchanova

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

    Ya. A. Lopasteyska, T. N. Sharov, N. N. Teteryatnikova & E. V. Molchanova

Authors
  1. E. F. Avdyusheva
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ya. A. Lopasteyska
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. T. N. Sharov
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. N. N. Teteryatnikova
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. E. V. Molchanova
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to E. V. Molchanova.

Additional information

Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 163, No. 4, pp. 524-528, April, 2017

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Avdyusheva, E.F., Lopasteyska, Y.A., Sharov, T.N. et al. Modification of the Method of Receiving of Insertion Mutants with the EZ::TN5 System. Bull Exp Biol Med 163, 519–522 (2017). https://doi.org/10.1007/s10517-017-3842-7

Download citation

  • Received:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10517-017-3842-7

Key Words

Search

Navigation