Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Review Article
  • Published:

Beyond toothpicks: new methods for isolating mutant bacteria

Nature Reviews Microbiology volume 5, pages 680–688 (2007)Cite this article

Key Points

  • New single-cell screening technologies have revolutionized research into the genetics, evolution and biotechnological applications of bacteria.

  • This Review focuses on the uses of autofluorescent proteins (AFPs) and fluorescence techniques such as Förster resonance energy transfer (FRET) by microbiologists.

  • Flow cytometry, which is a staple tool of eukaryotic cell biologists, is gaining popularity among microbiologists and the use of this technique is described here.

  • The applications of microfluidic cell sorters and microfluidics, which enable mutant cells to be examined in isolation rather than as a population, in microbiology are described.

  • The application of AFP transcriptional fusions to probe the interactions between hosts and microorganisms is also discussed.

  • The suitability of AFPs for studying the subcellular localization of proteins is described.

  • The identification and characterization of protein–protein interactions using AFPs is discussed.

  • The use of water–oil emulsions in enzymatic screening are highlighted.

  • Fluorescent screening for ligand-binding proteins is a useful technology that can be scaled up for high throughput.

Abstract

Over the past 50 years genetic analysis in microbiology has relied predominantly on selections and plate assays using chromogenic enzyme substrates — for example, X-gal assays for the detection of β-galactosidase activity. Recent advances in fluorescent assays and high throughput screening technologies have paved the way for the rapid isolation of mutants that confer complex phenotypes and for the quantitative analysis of the evolution of new traits in bacterial populations. This Review highlights the power of novel single-cell screening technologies and their applications to genetics, evolution and the biotechnological uses of bacteria.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: A microfluidic cell sorter based on dielectrophoresis.
Figure 2: A schematic of a spotted-cell array.
Figure 3: Fluorescence-based techniques for analysing protein–protein interactions.
Figure 4: Microdroplet compartmentalization technologies.
Figure 5: Different strategies for the isolation of polypeptides that bind to the surface of mammalian cells.

Similar content being viewed by others

References

  1. Shuman, H. A. Just toothpicks and logic: how some labs succeed at solving complex problems. J. Bacteriol. 185, 387–390 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Shuman, H. A. & Silhavy, T. J. The art and design of genetic screens: Escherichia coli. Nature Rev. Genet. 4, 419–431 (2003).

    Article  CAS  PubMed  Google Scholar 

  3. Silhavy, T. J. Gene fusions — guest commentary. J. Bacteriol. 182, 5935–5938 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Georgopoulos, C. Toothpicks, serendipity and the emergence of the Escherichia coli DnaK (Hsp70) and GroEL (Hsp60) chaperone machines. Genetics 174, 1699–1707 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Miller, J. H. et al. Fusions of the lac and trp regions of the Escherichia coli chromosome. J. Bacteriol. 104, 1273–1279 (1970).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Johnsson, N. & Johnsson, K. Chemical tools for biomolecular imaging. ACS Chem. Biol. 2, 31–38 (2007).

    Article  CAS  PubMed  Google Scholar 

  7. Cormack, B. P., Valdivia, R. H. & Falkow, S. FACS-optimized mutants of the green fluorescent protein (GFP). Gene 173, 33–38 (1996).

    Article  CAS  PubMed  Google Scholar 

  8. Plovins, A., Alvarez, A. M., Ibanez, M., Molina, M. & Nombela, C. Use of fluorescein-di-Β-D-galactopyranoside (Fdg) and C-12-Fdg as substrates for β-galactosidase detection by flow-cytometry in animal, bacterial, and yeast-cells. Appl. Environ. Microbiol. 60, 4638–4641 (1994).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Georgiou, G. Analysis of large libraries of protein mutants using flow cytometry. Adv. Protein Chem. 55, 293–316 (2000).

    Article  CAS  PubMed  Google Scholar 

  10. Griswold, K. E. et al. Evolution of highly active enzymes by homology-independent recombination. Proc. Natl Acad. Sci. USA 102, 10082–10087 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Masip, L., Veeravalli, K. & Georgiou, G. The many faces of glutathione in bacteria. Antioxid. Redox Signal. 8, 753–762 (2006).

    Article  CAS  PubMed  Google Scholar 

  12. Belousov, V. V. et al. Genetically encoded fluorescent indicator for intracellular hydrogen peroxide. Nature Methods 3, 281–286 (2006).

    Article  CAS  PubMed  Google Scholar 

  13. Miesenbock, G., De Angelis, D. A. & Rothman, J. E. Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins. Nature 394, 192–195 (1998).

    Article  CAS  PubMed  Google Scholar 

  14. Olsen, K. N. et al. Noninvasive measurement of bacterial intracellular pH on a single-cell level with green fluorescent protein and fluorescence ratio imaging microscopy. Appl. Environ. Microbiol. 68, 4145–4147 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Schuster, S., Enzelberger, M., Trauthwein, H., Schmid, R. D. & Urlacher, V. B. pHluorin-based in vivo assay for hydrolase screening. Anal. Chem. 77, 2727–2732 (2005).

    Article  CAS  PubMed  Google Scholar 

  16. Olsen, K. N. et al. Noninvasive measurement of bacterial intracellular pH on a single-cell level with green fluorescent protein and fluorescence ratio imaging microscopy. Appl. Environ. Microbiol. 68, 4145–4147 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Lewenza, S., Vidal-Ingigliardi, D. & Pugsley, A. P. Direct visualization of red fluorescent lipoproteins indicates conservation of the membrane sorting rules in the family Enterobacteriaceae. J. Bacteriol. 188, 3516–3524 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Looser, V. et al. Flow-cytometric detection of changes in the physiological state of E. coli expressing a heterologous membrane protein during carbon-limited fedbatch cultivation. Biotechnol. Bioeng. 92, 69–78 (2005).

    Article  CAS  PubMed  Google Scholar 

  19. Hewitt, C. J. & Nebe-Von-Caron, G. An industrial application of multiparameter flow cytometry: assessment of cell physiological state and its application to the study of microbial fermentations. Cytometry 44, 179–187 (2001).

    Article  CAS  PubMed  Google Scholar 

  20. Fu, A. Y., Spence, C., Scherer, A., Arnold, F. H. & Quake, S. R. A microfabricated fluorescence-activated cell sorter. Nature Biotechnol. 17, 1109–1111 (1999). This article describes the first μFACS system for cell sorting.

    Article  CAS  Google Scholar 

  21. Fu, A. Y., Chou, H. P., Spence, C., Arnold, F. H. & Quake, S. R. An integrated microfabricated cell sorter. Anal. Chem. 74, 2451–2457 (2002).

    Article  CAS  PubMed  Google Scholar 

  22. Wolff, A. et al. Integrating advanced functionality in a microfabricated high-throughput fluorescent-activated cell sorter. Lab Chip 3, 22–27 (2003).

    Article  CAS  PubMed  Google Scholar 

  23. Hu, X. Y. et al. Marker-specific sorting of rare cells using dielectrophoresis. Proc. Natl Acad. Sci. USA 102, 15757–15761 (2005). This article describes the first successful screening of a bacterial library by the μFACS system.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Bessette, P. H., Hu, X. Y., Soh, H. T. & Daugherty, P. S. Microfluidic library screening for mapping antibody epitopes. Anal. Chem. 79, 2174–2178 (2007).

    Article  CAS  PubMed  Google Scholar 

  25. Ottesen, E. A., Hong, J. W., Quake, S. R. & Leadbetter, J. R. Microfluidic digital PCR enables multigene analysis of individual environmental bacteria. Science 314, 1464–1467 (2006).

    Article  CAS  PubMed  Google Scholar 

  26. Chalfie, M., Tu, Y., Euskirchen, G., Ward, W. W. & Prasher, D. C. Green fluorescent protein as a marker for gene expression. Science 263, 802–805 (1994).

    Article  CAS  PubMed  Google Scholar 

  27. Shaner, N. C., Steinbach, P. A. & Tsien, R. Y. A guide to choosing fluorescent proteins. Nature Methods 2, 905–909 (2005).

    Article  CAS  PubMed  Google Scholar 

  28. Nagai, T. et al. A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications. Nature Biotechnol. 20, 87–90 (2002).

    Article  CAS  Google Scholar 

  29. Griesbeck, O., Baird, G. S., Campbell, R. E., Zacharias, D. A. & Tsien, R. Y. Reducing the environmental sensitivity of yellow fluorescent protein. Mechanism and applications. J. Biol. Chem. 276, 29188–29194 (2001).

    Article  CAS  PubMed  Google Scholar 

  30. Mena, M. A., Treynor, T. P., Mayo, S. L. & Daugherty, P. S. Blue fluorescent proteins with enhanced brightness and photostability from a structurally targeted library. Nature Biotechnol. 24, 1569–1571 (2006).

    Article  CAS  Google Scholar 

  31. Matz, M. V. et al. Fluorescent proteins from nonbioluminescent Anthozoa species. Nature Biotechnol. 17, 969–973 (1999).

    Article  CAS  Google Scholar 

  32. Campbell, R. E. et al. A monomeric red fluorescent protein. Proc. Natl Acad. Sci. USA 99, 7877–7882 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Shaner, N. C. et al. Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nature Biotechnol. 22, 1567–1572 (2004). Together with reference 32, this article describes the engineering of red fluorescent protein.

    Article  CAS  Google Scholar 

  34. Zaslaver, A. et al. A comprehensive library of fluorescent transcriptional reporters for Escherichia coli. Nature Methods 3, 623–628 (2006).

    Article  CAS  PubMed  Google Scholar 

  35. Zaslaver, A. et al. Just-in-time transcription program in metabolic pathways. Nature Genet. 36, 486–491 (2004). Together with reference 34, this article describes the application of a fluorescent system (GFP) to the systems biology of E. coli.

    Article  CAS  PubMed  Google Scholar 

  36. Valdivia, R. H. & Falkow, S. Flow cytometry and bacterial pathogenesis. Curr. Opin. Microbiol. 1, 359–363 (1998).

    Article  CAS  PubMed  Google Scholar 

  37. Valdivia, R. H. & Falkow, S. Fluorescence-based isolation of bacterial genes expressed within host cells. Science 277, 2007–2011 (1997).

    Article  CAS  PubMed  Google Scholar 

  38. Yang, S. H. et al. Genome-wide identification of plant-upregulated genes of Erwinia chrysanthemi 3937 using a GFP-based IVET leaf array. Mol. Plant Microbe Interact. 17, 999–1008 (2004).

    Article  CAS  PubMed  Google Scholar 

  39. Kumar, Y., Cocchiaro, J. & Valdivia, R. H. The obligate intracellular pathogen Chlamydia trachomatis targets host lipid droplets. Curr. Biol. 16, 1646–1651 (2006).

    Article  CAS  PubMed  Google Scholar 

  40. Knodler, L. A. et al. Cloning vectors and fluorescent proteins can significantly inhibit Salmonella enterica virulence in both epithelial cells and macrophages: implications for bacterial pathogenesis studies. Infect. Immun. 73, 7027–7031 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Narayanaswamy, R. et al. Systematic profiling of cellular phenotypes with spotted-cell microarrays reveals mating-pheromone response genes. Genome Biol. 7, R6 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Huh, W. K. et al. Global analysis of protein localization in budding yeast. Nature 425, 686–691 (2003).

    Article  CAS  PubMed  Google Scholar 

  43. Newman, J. R. S. et al. Single-cell proteomic analysis of S. cerevisiae reveals the architecture of biological noise. Nature 441, 840–846 (2006).

    Article  CAS  PubMed  Google Scholar 

  44. DeLisa, M. P., Lee, P., Palmer, T. & Georgiou, G. Phage shock protein PspA of Escherichia coli relieves saturation of protein export via the Tat pathway. J. Bacteriol. 186, 366–373 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. DeLisa, M. P., Samuelson, P., Palmer, T. & Georgiou, G. Genetic analysis of the twin arginine translocator secretion pathway in bacteria. J. Biol. Chem. 277, 29825–29831 (2002). This article describes the development of the GFP-fused periplasmic reporter system that was used as a genetic analysis tool for the elucidation of the Tat pathway of E. coli.

    Article  CAS  PubMed  Google Scholar 

  46. Tullman-Ercek, D. et al. Export pathway selectivity of Escherichia coli twin arginine translocation signal peptides. J. Biol. Chem. 282, 8309–8316 (2007).

    Article  CAS  PubMed  Google Scholar 

  47. Heinrich, J. & Wiegert, T. YpdC determines site-1 degradation in regulated intramembrane proteolysis of the RsiW anti-σ factor of Bacillus subtilis. Mol. Microbiol. 62, 566–579 (2006).

    Article  CAS  PubMed  Google Scholar 

  48. Nguyen, A. W. & Daugherty, P. S. Evolutionary optimization of fluorescent proteins for intracellular FRET. Nature Biotechnol. 23, 355–360 (2005).

    Article  CAS  Google Scholar 

  49. Sourjik, V. & Berg, H. C. Receptor sensitivity in bacterial chemotaxis. Proc. Natl Acad. Sci. USA 99, 123–127 (2002).

    Article  CAS  PubMed  Google Scholar 

  50. Nagai, T., Yamada, S., Tominaga, T., Ichikawa, M. & Miyawaki, A. Expanded dynamic range of fluorescent indicators for Ca2+ by circularly permuted yellow fluorescent proteins. Proc. Natl Acad. Sci. USA 101, 10554–10559 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. You, X. et al. Intracellular protein interaction mapping with FRET hybrids. Proc. Natl Acad. Sci. USA 103, 18458–18463 (2006). This article demonstrated the first quantitative analysis of the protein–protein interaction in the E. coli cytoplasm and the screening of interacting proteins by the use of new FRET probes.

    Google Scholar 

  52. Ghosh, I., Hamilton, A. D., Regan, L. Antiparallel leucine zipper-directed protein reassembly: application to the green fluorescent protein. J. Am. Chem. Soc. 122, 5658–5659 (2000).

    Article  CAS  Google Scholar 

  53. Zhang, S., Ma, C. & Chalfie, M. Combinatorial marking of cells and organelles with reconstituted fluorescent proteins. Cell 119, 137–144 (2004).

    Article  CAS  PubMed  Google Scholar 

  54. Cabantous, S., Terwilliger, T. C. & Waldo, G. S. Protein tagging and detection with engineered self-assembling fragments of green fluorescent protein. Nature Biotechnol. 23, 102–107 (2005).

    Article  CAS  Google Scholar 

  55. Jach, G., Pesch, M., Richter, K., Frings, S. & Uhrig, J. F. An improved mRFP1 adds red to bimolecular fluorescence complementation. Nature Methods 3, 597–600 (2006).

    Article  CAS  PubMed  Google Scholar 

  56. Jeong, J. et al. Monitoring of conformational change in maltose binding protein using split green fluorescent protein. Biochem. Biophys. Res. Commun. 339, 647–651 (2006).

    Article  CAS  PubMed  Google Scholar 

  57. Jeong, K. J., Seo, M. J., Iverson, B. L. & Georgiou, G. APEx 2-hybrid, a quantitative protein–protein interaction assay for antibody discovery and engineering. Proc. Natl Acad. Sci. USA 104, 8247–8252 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Olsen, M. J. et al. Function-based isolation of novel enzymes from a large library. Nature Biotechnol. 18, 1071–1074 (2000).

    Article  CAS  Google Scholar 

  59. Varadarajan, N., Gam, J., Olsen, M. J., Georgiou, G. & Iverson, B. L. Engineering of protease variants exhibiting high catalytic activity and exquisite substrate selectivity. Proc. Natl Acad. Sci. USA 102, 6855–6860 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Santoro, S. W., Wang, L., Herberich, B., King, D. S. & Schultz, P. G. An efficient system for the evolution of amino-acyl-tRNA synthetase specificity. Nature Biotechnol. 20, 1044–1048 (2002).

    Article  CAS  Google Scholar 

  61. Link, A. J. et al. Discovery of aminoacyl-tRNA synthetase activity through cell-surface display of noncanonical amino acids. Proc. Natl Acad. Sci. USA 103, 10180–10185 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Santoro, S. W. & Schultz, P. G. Directed evolution of the site specificity of Cre recombinase. Proc. Natl Acad. Sci. USA 99, 4185–4190 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Eklund, B. I., Edalat, M., Stenberg, G. & Mannervik, B. Screening for recombinant glutathione transferases active with monochlorobimane. Anal. Biochem. 309, 102–108 (2002).

    Article  CAS  PubMed  Google Scholar 

  64. Aharoni, A. et al. High-throughput screening methodology for the directed evolution of glycosyltransferases. Nature Methods 3, 609–614 (2006).

    Article  CAS  PubMed  Google Scholar 

  65. Becker, S., Schmoldt, H. U., Adams, T. M., Wilhelm, S. & Kolmar, H. Ultra-high-throughput screening based on cell-surface display and fluorescence-activated cell sorting for the identification of novel biocatalysts. Curr. Opin. Biotechnol. 15, 323–329 (2004).

    Article  CAS  PubMed  Google Scholar 

  66. Tawfik, D. S. & Griffiths, A. D. Man-made cell-like compartments for molecular evolution. Nature Biotechnol. 16, 652–656 (1998). The first demonstration of the screening of protein libraries encapsulated in oil–water emulsions.

    Article  CAS  Google Scholar 

  67. Aharoni, A., Amitai, G., Bernath, K., Magdassi, S. & Tawfik, D. S. High-throughput screening of enzyme libraries: thiolactonases evolved by fluorescence-activated sorting of single cells in emulsion compartments. Chem. Biol. 12, 1281–1289 (2005). An important extension of microcompartment screening to include single cells in oil–water emulsions.

    Article  CAS  PubMed  Google Scholar 

  68. Levin, A. M. & Weiss, G. A. Optimizing the affinity and specificity of proteins with molecular display. Mol. Biosyst. 2, 49–57 (2006).

    Article  CAS  PubMed  Google Scholar 

  69. Georgiou, G. et al. Display of heterologous proteins on the surface of microorganisms: from the screening of combinatorial libraries to live recombinant vaccines. Nature Biotechnol. 15, 29–34 (1997).

    Article  CAS  Google Scholar 

  70. Chen, G. et al. Isolation of high-affinity ligand-binding proteins by periplasmic expression with cytometric screening (PECS). Nature Biotechnol. 19, 537–542 (2001).

    Article  CAS  Google Scholar 

  71. Harvey, B. R. et al. Anchored periplasmic expression, a versatile technology for the isolation of high-affinity antibodies from Escherichia coli-expressed libraries. Proc. Natl Acad. Sci. USA 101, 9193–9198 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  72. Boder, E. T., Midelfort, K. S. & Wittrup, K. D. Directed evolution of antibody fragments with monovalent femtomolar antigen-binding affinity. Proc. Natl Acad. Sci. USA 97, 10701–10705 (2000). An extraordinary example of affinity maturation of an immunological molecule by cell-surface display on yeast. Antibodies with femtomolar binding affinity to the small molecule fluorescein were isolated.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  73. Daugherty, P. S., Iverson, B. L. & Georgiou, G. Flow cytometric screening of cell-based libraries. J. Immunol. Methods 243, 211–227 (2000).

    Article  CAS  PubMed  Google Scholar 

  74. Hoogenboom, H. R. Selecting and screening recombinant antibody libraries. Nature Biotechnol. 23, 1105–1116 (2005).

    Article  CAS  Google Scholar 

  75. Feldhaus, M. J. & Siegel, R. W. Yeast display of antibody fragments: a discovery and characterization platform. J. Immunol. Methods 290, 69–80 (2004).

    Article  CAS  PubMed  Google Scholar 

  76. Garcia-Rodriguez, C. et al. Molecular evolution of antibody crossreactivity for two subtypes of type A botulinum neurotoxin. Nature Biotechnol. 25, 107–116 (2007).

    Article  CAS  Google Scholar 

  77. Kieke, M. C. et al. Selection of functional T-cell receptor mutants from a yeast surface-display library. Proc. Natl Acad. Sci. USA 96, 5651–5656 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  78. Chao, G., Cochran, J. R. & Wittrup, K. D. Fine epitope mapping anti-epidermal growth factor receptor antibodies through random mutagenesis and yeast surface display. J. Mol. Biol. 342, 539–550 (2004).

    Article  CAS  PubMed  Google Scholar 

  79. Starwalt, S. E., Masteller, E. L., Bluestone, J. A. & Kranz, D. M. Directed evolution of a single-chain class II MHC product by yeast display. Protein Eng. 16, 147–156 (2003).

    Article  CAS  PubMed  Google Scholar 

  80. Esteban, O. & Zhao, H. M. Directed evolution of soluble single-chain human class II MHC molecules. J. Mol. Biol. 340, 81–95 (2004).

    Article  CAS  PubMed  Google Scholar 

  81. Taschner, S., Meinke, A., Von Gabain, A. & Boyd, A. P. Selection of peptide entry motifs by bacterial surface display. Biochem. J. 367, 393–402 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  82. Dane, K. Y., Chan, L. A., Rice, J. J. & Daugherty, P. S. Isolation of cell specific peptide ligands using fluorescent bacterial display libraries. J. Immunol. Methods 309, 120–129 (2006).

    Article  CAS  PubMed  Google Scholar 

  83. Wang, X. X., Cho, Y. K. & Shusta, E. V. Mining a yeast library for brain endothelial cell-binding antibodies. Nature Methods 4, 143–145 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  84. Bessette, P. H., Rice, J. J. & Daugherty, P. S. Rapid isolation of high-affinity protein binding peptides using bacterial display. Protein Eng. Des. Sel. 17, 731–739 (2004).

    Article  CAS  PubMed  Google Scholar 

  85. Rice, J. J., Schohn, A., Bessette, P. H., Boulware, K. T. & Daugherty, P. S. Bacterial display using circularly permuted outer membrane protein OmpX yields high affinity peptide ligands. Protein Sci. 15, 825–836 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  86. Boulware, K. T. & Daugherty, P. S. Protease specificity determination by using cellular libraries of peptide substrates (CLiPS). Proc. Natl Acad. Sci. USA 103, 7583–7588 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

Work in the Georgiou laboratory is supported by National Institutes of Health (NIH) grants GM069872, GM073089 and by the Foundation for Research. A. J. L. was supported by a NIH Kirschstein National Research Service Award (NRSA) fellowship (GM078908).

Author information

Authors and Affiliations

  1. Department of Chemical Engineering, University of Texas, 1 University Station, Austin, 78712, Texas, USA

    A. James Link, Ki Jun Jeong & George Georgiou

  2. Institute for Cellular and Molecular Biology, University of Texas, 1 University Station, Austin, 78712, Texas, USA

    A. James Link, Ki Jun Jeong & George Georgiou

  3. Department of Biomedical Engineering, University of Texas, 1 University Station, Austin, 78712, Texas, USA

    George Georgiou

  4. Department of Molecular Genetics and Microbiology, University of Texas, 1 University Station, Austin, 78712, Texas, USA

    George Georgiou

Authors
  1. A. James Link
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ki Jun Jeong
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. George Georgiou
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to George Georgiou.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Related links

Related links

DATABASES

Entrez Genome Project

Bacillus subtilis

Chlamydia trachomatis

Erwinia chrysanthemi

Escherichia coli

Saccharomyces cerevisiae

Salmonella typhimurium

Entrez Protein

DsRed

RsiW

FURTHER INFORMATION

George Georgiou's homepage

Glossary

Flow cytometry

The direction of a light source (typically emitted by one or more lasers) onto cells that are moving in single file in a stream of fluid. Several parameters are measured simultaneously for each cell, including forward light scatter, right-angle light scatter and multiple fluorescence emissions.

Microfluidic

A miniaturized (micron-scale) device, which is often fabricated by soft lithography, for the manipulation of small volumes of liquid under laminar-flow regimes.

Sheath fluid

The carrier fluid that surrounds the focused stream of cells in conventional flow cytometry.

Hydrodynamic focusing

The mechanism by which cells are arranged into single file in conventional flow cytometry. An outer annulus of sheath fluid maintains the inner stream, which contains the cells, in a laminar-flow regime.

Dampers

A relatively large fluid reservoir that is used to eliminate pulsatile fluid-flow effects from upstream pumps in microfluidics.

Dielectrophoresis

The translational motion of uncharged matter owing to changes in polarization of the matter in an electric field.

Spheroplasting

The removal or disruption of the outer membrane, while retaining inner-membrane integrity, in Gram-negative bacteria.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Link, A., Jeong, K. & Georgiou, G. Beyond toothpicks: new methods for isolating mutant bacteria. Nat Rev Microbiol 5, 680–688 (2007). https://doi.org/10.1038/nrmicro1715

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1038/nrmicro1715

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing