Green Fluorescent Protein as a Reporter for Macromolecular Localization in Bacterial Cells

Methods ◽  
2000 ◽  
Vol 20 (1) ◽  
pp. 62-72 ◽  
Author(s):  
William Margolin
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Bacterial Cells ◽  
Green Fluorescent

scholarly journals Predictive and Interpretive Simulation of Green Fluorescent Protein Expression in Reporter Bacteria

2001 ◽  
Vol 183 (23) ◽  
pp. 6752-6762 ◽  
Author(s):  
Johan H. J. Leveau ◽  
Steven E. Lindow
Keyword(s):  
Growth Rate ◽  
Green Fluorescent Protein ◽  
Green Fluorescent Protein Expression ◽  
Fluorescent Protein ◽  
Proteolytic Degradation ◽  
Bacterial Cells ◽  
Prior Testing ◽  
Gfp Fluorescence ◽  
Green Fluorescent ◽  
Best Fit

ABSTRACT We have formulated a numerical model that simulates the accumulation of green fluorescent protein (GFP) in bacterial cells from a generic promoter-gfp fusion. The model takes into account the activity of the promoter, the time it takes GFP to mature into its fluorescent form, the susceptibility of GFP to proteolytic degradation, and the growth rate of the bacteria. From the model, we derived a simple formula with which promoter activity can be inferred easily and quantitatively from actual measurements of GFP fluorescence in growing bacterial cultures. To test the usefulness of the formula, we determined the activity of the LacI-repressible promoter P A1/O4/O3 in response to increasing concentrations of the inducer IPTG (isopropyl-β-d-thiogalactopyranoside) and were able to predict cooperativity between the LacI repressors on each of the two operator sites within P A1/O4/O3 . Aided by the model, we also quantified the proteolytic degradation of GFP[AAV], GFP[ASV], and GFP[LVA], which are popular variants of GFP with reduced stability in bacteria. Best described by Michaelis-Menten kinetics, the rate at which these variants were degraded was a function of the activity of the promoter that drives their synthesis: a weak promoter yielded proportionally less GFP fluorescence than a strong one. The degree of disproportionality is species dependent: the effect was more pronounced in Erwinia herbicola than in Escherichia coli. This phenomenon has important implications for the interpretation of fluorescence from bacterial reporters based on these GFP variants. The model furthermore predicted a significant effect of growth rate on the GFP content of individual bacteria, which if not accounted for might lead to misinterpretation of GFP data. In practice, our model will be helpful for prior testing of different combinations of promoter-gfpfusions that best fit the application of a particular bacterial reporter strain, and also for the interpretation of actual GFP fluorescence data that are obtained with that reporter.

scholarly journals Noninvasive Measurement of Bacterial Intracellular pH on a Single-Cell Level with Green Fluorescent Protein and Fluorescence Ratio Imaging Microscopy

2002 ◽  
Vol 68 (8) ◽  
pp. 4145-4147 ◽  
Author(s):  
Katja N. Olsen ◽  
Birgitte B. Budde ◽  
Henrik Siegumfeldt ◽  
K. Björn Rechinger ◽  
Mogens Jakobsen ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Intracellular Ph ◽  
Fluorescent Protein ◽  
Direct Analysis ◽  
Bacterial Cells ◽  
Complex Environments ◽  
Fluorescence Ratio Imaging ◽  
Ratio Imaging ◽  
Green Fluorescent

ABSTRACT We show that a pH-sensitive derivative of the green fluorescent protein, designated ratiometric GFP, can be used to measure intracellular pH (pHi) in both gram-positive and gram-negative bacterial cells. In cells expressing ratiometric GFP, the excitation ratio (fluorescence intensity at 410 and 430 nm) is correlated to the pHi, allowing fast and noninvasive determination of pHi that is ideally suited for direct analysis of individual bacterial cells present in complex environments.

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