Micro-resonance Raman study of optically trapped Escherichia coli cells overexpressing human neuroglobin.
We describe the possibility of using a microresonance Raman spectrometer combined with a microfluidic system and optical tweezers to study Escherichia coli (E. coli) overexpressing wild type (wt) neuroglobin (NGB) and its E7Leu mutant, respectively. NGB is a recently discovered heme protein and its function still is a matter of debate. So far, the protein has been studied in its purified form, and in vivo measurements on the single cell level could give more information. To study the feasibility of the combined techniques, the possibilities of the setup are investigated by taking spectra from single cells and clusters of cells. We find that the microresonance Raman technique enables studies of the wt NGB protein in a living cell under fluctuating aerobic and anaerobic conditions. E. coli cells overexpressing wt NGB are stable, and the reversible oxygenation-deoxygenation can be studied over a long period of time. Further, the experiment indicates the presence of an enzymatic system in the bacteria reducing the ferric form NGB. The study of E. coli cells overexpressing E7Leu NGB, on the other hand, gives insight into limiting factors of the setup, such as cell lysis, photoinduced chemistry, and protein concentrations.
Ramser
Kerstin
K
Göteborgs University, Department of Physics, SE-412 96 Göteborg, Sweden. kerstin.ramser@ltu.se
Wenseleers
Wim
W
Dewilde
Sylvia
S
Van Doorslaer
Sabine
S
Moens
Luc
L
Hanstorp
Dag
D
D004926:Escherichia coli / Q000502:physiology*; D005914:Globins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D046210:Microfluidic Analytical Techniques / Q000379:methods*; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D000078787:Neuroglobin; D052898:Optical Tweezers; D015202:Protein Engineering / Q000379:methods; D013059:Spectrum Analysis, Raman / Q000379:methods*
D009419:Nerve Tissue Proteins; D000078787:Neuroglobin; D005914:Globins