Monitoring dynamic protein expression in living E. coli. Bacterial cells by laser tweezers raman spectroscopy

James W Chan, Heiko Winhold, Michele H. Corzett, Joshua M. Ulloa, Monique Cosman, Rod Balhorn, Thomas R Huser

Research output: Contribution to journalArticle

48 Citations (Scopus)

Abstract

Background: Laser tweezers Raman spectroscopy (LTRS) is a novel, nondestructive, and label-free method that can be used to quantitatively measure changes in cellular activity in single living cells. Here, we demonstrate its use to monitor changes in a population of E. coli cells that occur during overexpression of a protein, the extracellular domain of myelin oligodendrocyte glycoprotein [MOG(1-120)]. Methods: Raman spectra were acquired from individual E. coli cells suspended in solution and trapped by a single tightly focused laser beam. Overexpression of MOG(1-120) in transformed E. coli Rosetta-Gami (DE3)pLysS cells was induced by addition of isopropyl thiogalactoside (IPTG). Changes in the peak intensities of the Raman spectra from a population of cells were monitored and analyzed over a total duration of 3 h. Data were also collected for concentrated purified MOG(1-120) protein in solution, and the spectra compared with that obtained for the MOG(1-120) expressing cells. Results: Raman spectra of individual, living E. coli cells exhibit signatures due to DNA and protein molecular vibrations. Characteristic Raman markers associated with protein vibrations, such as 1,257, 1,340, 1,453, and 1,660 cm-1, are shown to increase as a function of time following the addition of IPTG. Comparison of these spectra and the spectra of purified MOG protein indicates that the changes are predominantly due to the induction of MOG protein expression. Protein expression was found to occur mostly within the second hour, with a 470% increase relative to the protein expressed in the first hour. A 230% relative increase between the second and third hour indicates that protein expression begins to level off within the third hour. Conclusion: It is demonstrated that LTRS has sufficient sensitivity for real-time, nondestructive, and quantitative monitoring of biological processes, such as protein expression, in single living cells. Such capabilities, which are not currently available in flow cytometry, open up new possibilities for analyzing cellular processes occurring in single microbial and eukaryotic cells.

Original languageEnglish (US)
Pages (from-to)468-474
Number of pages7
JournalCytometry Part A
Volume71
Issue number7
DOIs
StatePublished - Jul 2007

Fingerprint

Optical Tweezers
Raman Spectrum Analysis
Escherichia coli
Proteins
Isopropyl Thiogalactoside
Vibration
Myelin-Oligodendrocyte Glycoprotein
Biological Phenomena
Eukaryotic Cells
Population
Flow Cytometry
Lasers

Keywords

  • E. coli
  • Glycoprotein
  • Laser trapping
  • Optical tweezers
  • Protein expression
  • Raman spectroscopy
  • Single cells

ASJC Scopus subject areas

  • Hematology
  • Cell Biology
  • Pathology and Forensic Medicine
  • Biophysics
  • Endocrinology

Cite this

Monitoring dynamic protein expression in living E. coli. Bacterial cells by laser tweezers raman spectroscopy. / Chan, James W; Winhold, Heiko; Corzett, Michele H.; Ulloa, Joshua M.; Cosman, Monique; Balhorn, Rod; Huser, Thomas R.

In: Cytometry Part A, Vol. 71, No. 7, 07.2007, p. 468-474.

Research output: Contribution to journalArticle

Chan, JW, Winhold, H, Corzett, MH, Ulloa, JM, Cosman, M, Balhorn, R & Huser, TR 2007, 'Monitoring dynamic protein expression in living E. coli. Bacterial cells by laser tweezers raman spectroscopy', Cytometry Part A, vol. 71, no. 7, pp. 468-474. https://doi.org/10.1002/cyto.a.20407
Chan, James W ; Winhold, Heiko ; Corzett, Michele H. ; Ulloa, Joshua M. ; Cosman, Monique ; Balhorn, Rod ; Huser, Thomas R. / Monitoring dynamic protein expression in living E. coli. Bacterial cells by laser tweezers raman spectroscopy. In: Cytometry Part A. 2007 ; Vol. 71, No. 7. pp. 468-474.
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abstract = "Background: Laser tweezers Raman spectroscopy (LTRS) is a novel, nondestructive, and label-free method that can be used to quantitatively measure changes in cellular activity in single living cells. Here, we demonstrate its use to monitor changes in a population of E. coli cells that occur during overexpression of a protein, the extracellular domain of myelin oligodendrocyte glycoprotein [MOG(1-120)]. Methods: Raman spectra were acquired from individual E. coli cells suspended in solution and trapped by a single tightly focused laser beam. Overexpression of MOG(1-120) in transformed E. coli Rosetta-Gami (DE3)pLysS cells was induced by addition of isopropyl thiogalactoside (IPTG). Changes in the peak intensities of the Raman spectra from a population of cells were monitored and analyzed over a total duration of 3 h. Data were also collected for concentrated purified MOG(1-120) protein in solution, and the spectra compared with that obtained for the MOG(1-120) expressing cells. Results: Raman spectra of individual, living E. coli cells exhibit signatures due to DNA and protein molecular vibrations. Characteristic Raman markers associated with protein vibrations, such as 1,257, 1,340, 1,453, and 1,660 cm-1, are shown to increase as a function of time following the addition of IPTG. Comparison of these spectra and the spectra of purified MOG protein indicates that the changes are predominantly due to the induction of MOG protein expression. Protein expression was found to occur mostly within the second hour, with a 470{\%} increase relative to the protein expressed in the first hour. A 230{\%} relative increase between the second and third hour indicates that protein expression begins to level off within the third hour. Conclusion: It is demonstrated that LTRS has sufficient sensitivity for real-time, nondestructive, and quantitative monitoring of biological processes, such as protein expression, in single living cells. Such capabilities, which are not currently available in flow cytometry, open up new possibilities for analyzing cellular processes occurring in single microbial and eukaryotic cells.",
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AU - Cosman, Monique

AU - Balhorn, Rod

AU - Huser, Thomas R

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N2 - Background: Laser tweezers Raman spectroscopy (LTRS) is a novel, nondestructive, and label-free method that can be used to quantitatively measure changes in cellular activity in single living cells. Here, we demonstrate its use to monitor changes in a population of E. coli cells that occur during overexpression of a protein, the extracellular domain of myelin oligodendrocyte glycoprotein [MOG(1-120)]. Methods: Raman spectra were acquired from individual E. coli cells suspended in solution and trapped by a single tightly focused laser beam. Overexpression of MOG(1-120) in transformed E. coli Rosetta-Gami (DE3)pLysS cells was induced by addition of isopropyl thiogalactoside (IPTG). Changes in the peak intensities of the Raman spectra from a population of cells were monitored and analyzed over a total duration of 3 h. Data were also collected for concentrated purified MOG(1-120) protein in solution, and the spectra compared with that obtained for the MOG(1-120) expressing cells. Results: Raman spectra of individual, living E. coli cells exhibit signatures due to DNA and protein molecular vibrations. Characteristic Raman markers associated with protein vibrations, such as 1,257, 1,340, 1,453, and 1,660 cm-1, are shown to increase as a function of time following the addition of IPTG. Comparison of these spectra and the spectra of purified MOG protein indicates that the changes are predominantly due to the induction of MOG protein expression. Protein expression was found to occur mostly within the second hour, with a 470% increase relative to the protein expressed in the first hour. A 230% relative increase between the second and third hour indicates that protein expression begins to level off within the third hour. Conclusion: It is demonstrated that LTRS has sufficient sensitivity for real-time, nondestructive, and quantitative monitoring of biological processes, such as protein expression, in single living cells. Such capabilities, which are not currently available in flow cytometry, open up new possibilities for analyzing cellular processes occurring in single microbial and eukaryotic cells.

AB - Background: Laser tweezers Raman spectroscopy (LTRS) is a novel, nondestructive, and label-free method that can be used to quantitatively measure changes in cellular activity in single living cells. Here, we demonstrate its use to monitor changes in a population of E. coli cells that occur during overexpression of a protein, the extracellular domain of myelin oligodendrocyte glycoprotein [MOG(1-120)]. Methods: Raman spectra were acquired from individual E. coli cells suspended in solution and trapped by a single tightly focused laser beam. Overexpression of MOG(1-120) in transformed E. coli Rosetta-Gami (DE3)pLysS cells was induced by addition of isopropyl thiogalactoside (IPTG). Changes in the peak intensities of the Raman spectra from a population of cells were monitored and analyzed over a total duration of 3 h. Data were also collected for concentrated purified MOG(1-120) protein in solution, and the spectra compared with that obtained for the MOG(1-120) expressing cells. Results: Raman spectra of individual, living E. coli cells exhibit signatures due to DNA and protein molecular vibrations. Characteristic Raman markers associated with protein vibrations, such as 1,257, 1,340, 1,453, and 1,660 cm-1, are shown to increase as a function of time following the addition of IPTG. Comparison of these spectra and the spectra of purified MOG protein indicates that the changes are predominantly due to the induction of MOG protein expression. Protein expression was found to occur mostly within the second hour, with a 470% increase relative to the protein expressed in the first hour. A 230% relative increase between the second and third hour indicates that protein expression begins to level off within the third hour. Conclusion: It is demonstrated that LTRS has sufficient sensitivity for real-time, nondestructive, and quantitative monitoring of biological processes, such as protein expression, in single living cells. Such capabilities, which are not currently available in flow cytometry, open up new possibilities for analyzing cellular processes occurring in single microbial and eukaryotic cells.

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KW - Glycoprotein

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KW - Protein expression

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