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Title: Monitoring Dynamic Protein Expression in Single Living E. Coli. Bacterial Cells by Laser Tweezers Raman Spectroscopy

Abstract

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)) Raman spectra were acquired of 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 three hours. Data was 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. 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 1257 cm{sup -1}, 1340 cm{sup -1}, 1453 cm{sup -1} and 1660 cm{sup -1}, are shown to increase as a function of time following the addition of IPTG. Comparison of thesemore » 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. 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.« less

Authors:
; ; ; ; ; ;
Publication Date:
Research Org.:
Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States)
Sponsoring Org.:
USDOE
OSTI Identifier:
920849
Report Number(s):
UCRL-JRNL-227444
TRN: US200805%%200
DOE Contract Number:  
W-7405-ENG-48
Resource Type:
Journal Article
Resource Relation:
Journal Name: Cytometry Part A, n/a, n/a, April 25, 2007, pp. 468-474
Country of Publication:
United States
Language:
English
Subject:
71 CLASSICAL AND QUANTUMM MECHANICS, GENERAL PHYSICS; 59 BASIC BIOLOGICAL SCIENCES; 42 ENGINEERING; DNA; GLYCOPROTEINS; INDUCTION; LASERS; MONITORING; MONITORS; MYELIN; PROTEINS; RAMAN SPECTRA; RAMAN SPECTROSCOPY; SENSITIVITY; SPECTRA

Citation Formats

Chan, J W, Winhold, H, Corzett, M H, Ulloa, J M, Cosman, M, Balhorn, R, and Huser, T. Monitoring Dynamic Protein Expression in Single Living E. Coli. Bacterial Cells by Laser Tweezers Raman Spectroscopy. United States: N. p., 2007. Web.
Chan, J W, Winhold, H, Corzett, M H, Ulloa, J M, Cosman, M, Balhorn, R, & Huser, T. Monitoring Dynamic Protein Expression in Single Living E. Coli. Bacterial Cells by Laser Tweezers Raman Spectroscopy. United States.
Chan, J W, Winhold, H, Corzett, M H, Ulloa, J M, Cosman, M, Balhorn, R, and Huser, T. Tue . "Monitoring Dynamic Protein Expression in Single Living E. Coli. Bacterial Cells by Laser Tweezers Raman Spectroscopy". United States. doi:. https://www.osti.gov/servlets/purl/920849.
@article{osti_920849,
title = {Monitoring Dynamic Protein Expression in Single Living E. Coli. Bacterial Cells by Laser Tweezers Raman Spectroscopy},
author = {Chan, J W and Winhold, H and Corzett, M H and Ulloa, J M and Cosman, M and Balhorn, R and Huser, T},
abstractNote = {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)) Raman spectra were acquired of 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 three hours. Data was 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. 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 1257 cm{sup -1}, 1340 cm{sup -1}, 1453 cm{sup -1} and 1660 cm{sup -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. 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.},
doi = {},
journal = {Cytometry Part A, n/a, n/a, April 25, 2007, pp. 468-474},
number = ,
volume = ,
place = {United States},
year = {Tue Jan 09 00:00:00 EST 2007},
month = {Tue Jan 09 00:00:00 EST 2007}
}