Bioluminescent Imaging of Single Bacterial Cells Using an Enhanced ilux Operon

  • Carola GregorEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 2081)


The lux operon is a useful reporter for bioluminescence imaging due to its independence of exogenous luciferin supply, but its relatively low brightness hampers the imaging of single cells. This chapter describes a procedure for the imaging of individual Escherichia coli cells using an improved ilux operon. The enhanced brightness of ilux enables long-term bioluminescence imaging of single bacteria with high sensitivity without the requirement for an external luciferin.

Key words

Bioluminescence Luciferase Bacteria Microscopy Imaging lux 



I thank Prof. Stefan W. Hell for the opportunity to carry out this work in his department.


  1. 1.
    Kotlobay AA et al (2018) Genetically encodable bioluminescent system from fungi. Proc Natl Acad Sci U S A 115(50):12728–12732CrossRefGoogle Scholar
  2. 2.
    Karsi A, Menanteau-Ledouble S, Lawrence ML (2006) Development of bioluminescent Edwardsiella ictaluri for noninvasive disease monitoring. FEMS Microbiol Lett 260:216–223CrossRefGoogle Scholar
  3. 3.
    Gonzalez RJ, Weening EH, Frothingham R, Sempowski GD, Miller VL (2012) Bioluminescence imaging to track bacterial dissemination of Yersinia pestis using different routes of infection in mice. BMC Microbiol 12:147CrossRefGoogle Scholar
  4. 4.
    Huang YK, Chu C, Wu CH, Chen CL, Chiu CH (2014) Evaluation of Gram-negative bacterial infection by a stable and conjugative bioluminescence plasmid in a mouse model. J Biomed Sci 21:78CrossRefGoogle Scholar
  5. 5.
    Nguyen VH, Kim HS, Ha JM, Hong Y, Choy HE et al (2010) Genetically engineered Salmonella typhimurium as an imageable therapeutic probe for cancer. Cancer Res 70:18–23CrossRefGoogle Scholar
  6. 6.
    Cronin M, Akin AR, Collins SA, Meganck J, Kim JB et al (2012) High resolution in vivo bioluminescent imaging for the study of bacterial tumour targeting. PLoS One 7:e30940CrossRefGoogle Scholar
  7. 7.
    Engelbrecht J, Simon M, Silverman M (1985) Measuring gene expression with light. Science 227:1345–1347CrossRefGoogle Scholar
  8. 8.
    Shen H, Gold SE, Tamaki SJ, Keen NT (1992) Construction of a Tn7-lux system for gene expression studies in gram-negative bacteria. Gene 122:27–34CrossRefGoogle Scholar
  9. 9.
    Kondo T, Strayer CA, Kulkarni RD, Taylor W, Ishiura M et al (1993) Circadian rhythms in prokaryotes: luciferase as a reporter of circadian gene expression in cyanobacteria. Proc Natl Acad Sci U S A 90:5672–5676CrossRefGoogle Scholar
  10. 10.
    Sayler GS, Fleming JT, Nivens DE (2001) Gene expression monitoring in soils by mRNA analysis and gene lux fusions. Curr Opin Biotechnol 12:455–460CrossRefGoogle Scholar
  11. 11.
    Shimizu T, Ohta Y, Tsutsuki H, Noda M (2011) Construction of a novel bioluminescent reporter system for investigating Shiga toxin expression of enterohemorrhagic Escherichia coli. Gene 478:1–10CrossRefGoogle Scholar
  12. 12.
    Vannini A, Agriesti F, Mosca F, Roncarati D, Scarlato V et al (2012) A convenient and robust in vivo reporter system to monitor gene expression in the human pathogen Helicobacter pylori. Appl Environ Microbiol 78:6524–6533CrossRefGoogle Scholar
  13. 13.
    Ulitzur S, Lahav T, Ulitzur N (2002) A novel and sensitive test for rapid determination of water toxicity. Environ Toxicol 17:291–296CrossRefGoogle Scholar
  14. 14.
    Min J, Chang YS, Gu MB (2003) Bacterial detection of the toxicity of dioxins, polychlorinated diphenyls, and polybrominated diphenyl ethers. Environ Toxicol Chem 22:2238–2242CrossRefGoogle Scholar
  15. 15.
    Kelly CJ, Tumsaroj N, Lajoie CA (2004) Assessing wastewater metal toxicity with bacterial bioluminescence in a bench-scale wastewater treatment system. Water Res 38:423–431CrossRefGoogle Scholar
  16. 16.
    Ivask A, Rõlova T, Kahru A (2009) A suite of recombinant luminescent bacterial strains for the quantification of bioavailable heavy metals and toxicity testing. BMC Biotechnol 9:41CrossRefGoogle Scholar
  17. 17.
    Gregor C, Gwosch KC, Sahl SJ, Hell SW (2018) Strongly enhanced bacterial bioluminescence with the ilux operon for single-cell imaging. Proc Natl Acad Sci U S A 115:962–967CrossRefGoogle Scholar

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© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  1. 1.Department of NanoBiophotonicsMax Planck Institute for Biophysical ChemistryGöttingenGermany

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