Advertisement

Journal of Microbiology

, Volume 57, Issue 12, pp 1041–1047 | Cite as

Construction of a multicopy genomic DNA library and its application for suppression analysis

  • Hongbaek ChoEmail author
Protocol
  • 34 Downloads

Abstract

Suppression analysis is used for the identification of new genes and genetic interactions when there is a notable phenotype available for genetic selection or screening. A random genomic DNA library constructed on a multi-copy plasmid is a useful tool for suppression analysis when one expects that an overdose of a few genes will suppress the phenotype. These libraries have been successfully used to determine the function of a gene by revealing genes whose functions are related to the gene of interest. They have also been used to identify the targets of chemical or biological agents by increasing the number of unaffected target gene products in a cell. In this article, I will discuss important considerations for constructing multicopy genomic DNA libraries. The protocol provided in this paper should be a useful guide for constructing genomic DNA libraries in many bacterial species for which multi-copy plasmids are available.

Keywords

genomic DNA multicopy plasmid library construction suppression analysis genetic selection 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

Notes

Acknowledgements

This work was supported by the National Research Foundation of Korea (NRF) Grant (NRF-2019R1A2C1002648).

References

  1. Bae, S., Mueller, O., Wong, S., Rawls, J.F., and Valdivia, R.H. 2016. Genomic sequencing-based mutational enrichment analysis identifies motility genes in a genetically intractable gut microbe. Proc. Natl. Acad. Sci. USA113, 14127–14132.CrossRefGoogle Scholar
  2. Bola, G. 2005. Evaluating the role of G, C-nucleotides and length of overhangs in T4 DNA ligase efficiency. J. Exp. Microbiol. Immunol.8, 1–7Google Scholar
  3. Chamakura, K.R., Sham, L.T., Davis, R.M., Min, L., Cho, H., Ruiz, N., Bernhardt, T.G., and Young, R. 2017. A viral protein antibiotic inhibits lipid II flippase activity. Nat. Microbiol.2, 1480–1484.CrossRefGoogle Scholar
  4. Cho, S.H., Szewczyk, J., Pesavento, C., Zietek, M., Banzhaf, M., Roszczenko, P., Asmar, A., Laloux, G., Hov, A.K., and Leverrier, P., et al. 2014a. Detecting envelope stress by monitoring β-barrel assembly. Cell159, 1652–1664.CrossRefGoogle Scholar
  5. Cho, H., Uehara, T., and Bernhardt, T.G. 2014b. Beta-lactam antibiotics induce a lethal malfunctioning of the bacterial cell wall synthesis machinery. Cell159, 1300–1311.CrossRefGoogle Scholar
  6. Gardner, K.A.J.A., Osawa, M., and Erickson, H.P. 2017. Whole genome re-sequencing to identify suppressor mutations of mutant and foreign Escherichia coli FtsZ. PLoS One12, e0176643.CrossRefGoogle Scholar
  7. Gropp, M., Strausz, Y., Gross, M., and Glaser, G. 2001. Regulation of Escherichia coli RelA requires oligomerization of the C-terminal domain. J. Bacteriol.183, 570–579.CrossRefGoogle Scholar
  8. Hamer, L., DeZwaan, T.M., Montenegro-Chamorro, M.V., Frank, S.A., and Hamer, J.E. 2001. Recent advances in large-scale transposon mutagenesis. Curr. Opin. Chem. Biol.5, 67–73.CrossRefGoogle Scholar
  9. Koo, J.T., Choe, J., and Moseley, S.L. 2004. HrpA, a DEAH-box RNA helicase, is involved in mRNA processing of a fimbrial operon in Escherichia coli. Mol. Microbiol.52, 1813–1826.CrossRefGoogle Scholar
  10. Lai, G.C., Cho, H., and Bernhardt, T.G. 2017. The mecillinam resistome reveals a role for peptidoglycan endopeptidases in stimulating cell wall synthesis in Escherichia coli. PLoS Genet.13, e1006934.CrossRefGoogle Scholar
  11. Li, X., Zolli-Juran, M., Cechetto, J.D., Daigle, D.M., Wright, G.D., and Brown, E.D. 2004. Multicopy suppressors for novel antibacterial compounds reveal targets and drug efflux susceptibility. Chem. Biol.11, 1423–1430.CrossRefGoogle Scholar
  12. Nichols, B.P., Shafiq, O., and Meiners, V. 1998. Sequence analysis of Tn10 insertion sites in a collection of Escherichia coli strains used for genetic mapping and strain construction. J. Bacteriol.180, 6408–6411.PubMedPubMedCentralGoogle Scholar
  13. Rockabrand, D. and Blum, P. 1995. Multicopy plasmid suppression of stationary phase chaperone toxicity in Escherichia coli by phosphogluconate dehydratase and the N-terminus of DnaK. Mol. Gen. Genet.249, 498–506.CrossRefGoogle Scholar
  14. Singer, M., Baker, T.A., Schnitzler, G., Deischel, S.M., Goel, M., Dove, W., Jaacks, K.J., Grossman, A.D., Erickson, J.W., and Gross C.A. 1989. A collection of strains containing genetically linked alternating antibiotic resistance elements for genetic mapping of Escherichia coli. Microbiol. Rev.53, 1–24.PubMedPubMedCentralGoogle Scholar
  15. Singh, S.K., SaiSree, L., Amrutha, R.N., and Reddy, M. 2012. Three redundant murein endopeptidases catalyse an essential cleavage step in peptidoglycan synthesis of Escherichia coli K12. Mol. Microbiol.86, 1036–1051.CrossRefGoogle Scholar
  16. Ueguchi, C. and Ito, K. 1992. Multicopy suppression: an approach to understanding intracellular functioning of the protein export system. J. Bacteriol.174, 1454–1461.CrossRefGoogle Scholar
  17. Yunck, R., Cho, H., and Bernhardt, T.G. 2015. Identification of MltG as a potential terminase for peptidoglycan polymerization in bacteria. Mol. Microbiol.99, 700–718.CrossRefGoogle Scholar

Copyright information

© The Microbiological Society of Korea 2019

Authors and Affiliations

  1. 1.Department of Biological Sciences, College of Natural SciencesSungkyunkwan UniversitySuwonRepublic of Korea

Personalised recommendations