Cell sorting of formalin-treated pathogenic Mycobacterium paratuberculosis expressing GFP
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Published on: March 1, 2024
N B Harris1, D K Zinniel, M K Hsieh
1University of Nebraska, Lincoln, NE 68583-0905, USA.
Abstract:
GFP is widely used as a molecular tool for the study of microbial pathogens. However, the manipulation of these pathogenic microorganisms poses a health threat to the laboratory worker, requiring biosafety level II or III containment. Although the GFPfluorophore is tolerant toformalin, a thorough analysis of this treatment on fluorescent output in prokaryotic systems has not been described. In addition, the analysis of microorganisms expressing GFP often depends on specialized equipment, which may not be housed in biosafety level II or III laboratories. Therefore, we sought to develop a safe and effective method for manipulating the GFP-expressing pathogenic bacterium Mycobacterium avium subsp, paratuberculosis (M. paratuberculosis) utilizing a formalin treatment that would permit the analysis of GFP fluorescence without requiring stringent biosafety containment. We demonstrate that formalin-treated M. paratuberculosis expresses 50% less fluorescence than viable cells, but this reduction is still compatible with spectrofluorometry and cell sorting. Furthermore, plasmid DNA that expresses GFP can be recovered efficiently from nonviable, sorted fluorescent cells. This approach is flexible, provides an additional margin of safety for laboratory personnel, and can be easily applied to other pathogenic microorganisms expressing GFP.
Insights
Formalin treatment inactivates pathogenic bacteria expressing Green Fluorescent Protein (GFP), enabling safer lab analysis. This method allows GFP fluorescence detection in nonviable cells, reducing biosafety risks.
Area of Science:
- Microbiology
- Molecular Biology
- Biotechnology
Background:
- Green Fluorescent Protein (GFP) is a vital tool for studying microbial pathogens.
- Handling pathogenic microorganisms expressing GFP necessitates high biosafety containment (BSL-2/3).
- Limited data exists on formalin's effect on GFP fluorescence in prokaryotes and its utility for safe analysis.
Purpose of the Study:
- To develop a safe and effective method for manipulating GFP-expressing Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis).
- To enable GFP fluorescence analysis of M. paratuberculosis without stringent biosafety containment using formalin treatment.
Main Methods:
- Application of formalin treatment to GFP-expressing M. paratuberculosis.
- Analysis of GFP fluorescence in treated and untreated cells using spectrofluorometry.
- Assessment of cell sorting capabilities on nonviable, fluorescent cells.
- Recovery of plasmid DNA from sorted cells.
Main Results:
- Formalin treatment reduced GFP fluorescence by 50% compared to viable cells.
- Reduced fluorescence remained compatible with spectrofluorometry and cell sorting.
- Efficient recovery of GFP-expressing plasmid DNA from nonviable, sorted cells was achieved.
Conclusions:
- Formalin treatment offers a safe method for handling and analyzing GFP-expressing M. paratuberculosis.
- This approach enhances laboratory safety by reducing containment requirements.
- The method is adaptable for other GFP-expressing pathogenic microorganisms.
