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


CONCLUSIONS
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.
Chan
James W
JW
Applied Physics and Biophysics Division, Physics and Advanced Technologies Directorate, Lawrence Livermore National Laboratory, Livermore, California 94551, USA. chan19@llnl.gov
Winhold
Heiko
H
Corzett
Michele H
MH
Ulloa
Joshua M
JM
Cosman
Monique
M
Balhorn
Rod
R
Huser
Thomas
T
D000818:Animals; D004926:Escherichia coli / Q000235:genetics*; D020869:Gene Expression Profiling / Q000295:instrumentation / Q000379:methods*; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D008954:Models, Biological; D009185:Myelin Proteins; D018992:Myelin-Associated Glycoprotein / Q000235:genetics; D063308:Myelin-Oligodendrocyte Glycoprotein; D052898:Optical Tweezers; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics; D013059:Spectrum Analysis, Raman / Q000295:instrumentation / Q000379:methods*
C571328:Mog protein, rat; D009185:Myelin Proteins; D018992:Myelin-Associated Glycoprotein; D063308:Myelin-Oligodendrocyte Glycoprotein; D011994:Recombinant Proteins; D007544:Isopropyl Thiogalactoside