Novel reporter gene in a fluorescent‐based whole cell sensing system

Abstract

A common problem encountered when using fluorescence detection in real samples analysis is that the matrix may contain compounds that autofluorescence or that can be excited at the wavelengths of commonly employed fluorescent reporter molecules. This causes an increase in background fluorescence, which in turn tends to compromise the detection limits of the system. To address this issue, we investigated the use of a reporter enzyme that produces fluorescent compounds, which can be excited at wavelengths that are not commonly encountered in compounds present in real samples. For that, a whole cell-based sensing system for arsenite that employs cobA as the reporter gene was developed. The system utilizes genetically engineered bacteria that incorporate the specificity of the ars operon with the sensitivity of the cobA gene. The cobA gene codes for uroporphyrinogen III methyltransferase that converts the substrate uroporphyrinogen (urogen) III into two fluorescent compounds sirohydrochlorin and trimethylpyrrocorphin. Urogen III is ubiquitous within the cell, however, because the cells use it for vitamin B12 and siroheme biosynthesis, this sensing system is limited by substrate availability. By supplementing the media with ALA, a precursor of urogen III, a more stable and reproducible response was obtained. We observed three excitation maxima at 357, 378, and 498 nm, with a single emission maximum at 605 nm. Excitation at 498 nm was selected because it results in less background interference as most endogenous substances are not active at this wavelength. Advantages and limitations of using the cobA gene in whole-cell sensing applications are presented.

Coupled selection of protein solubility in E. coli using uroporphyrinogen III methyltransferase as red fluorescent reporter

“…The maximum absorption at 410 nm was examined for cells expressing UMT fused to ALAS or UROD. Expression of the GLTR, CPOX, or SCFC partner resulted in a minor absorption peak at 375 nm and major one at 350 nm, which represent sirohydrochrolin and trimethylpyrrocorphin respectively (Feliciano et al, 2006), similar to that of MBP-UMT (Fig. 6B).…”

“…Additional ALA can increase cell fluorescence in the UMT reporter system (Feliciano et al, 2006;Roessner, 2002), resulted from the transformation of ALA into Uro'gen III in E. coli by the chromosome-encoded enzymes. In the current study, we identified that the supplementary ALA declined sensitivity of UMT as the solubility reporter for monitoring TEVp variants.…”

“…This character allows the UMT gene from Propionibacterium freudenreichii as a fluorescent reporter for screening recombinant plasmid (Roessner, 2002), denoting transcription in E. coli, yeast and mammalian cells (Wildt and Deuschle, 1999), and detecting arsenite and antimonite in a whole-cell sensing system (Feliciano et al, 2006). The UMT reporter provides a signal-to-noise ratio better than that of GFP, because the red wavelength reduces autofluorescence and light scattering of the cells, media, and plastic materials (Wildt and Deuschle, 1999).…”

“…Because most organisms contain the cellular level of uro'gen III, additional substrate is not essential for the UMT reporter (Wildt and Deuschle, 1999). In a whole-cell sensing system, supplemented ALA enhances fluorescence intensities of cells expressing the UMT and results in a more reproducible assay (Feliciano et al, 2006).…”

Characterization of a Gene Conferring Red Fluorescence Isolated from an Environmental DNA Library Constructed from Soil Bacteria

“…23,24) When this occurs, an excess of UPMTase also catalyzes a third methylation of uroporphyrinogen III at position 12 to give an overmethylated trimethylpyrrocorphin, which has no obvious physiological role. 13,25) These two products, sirohydrochrolin and trimethylpyrrocorphin, are known to emit strong red fluorescence when illuminated by UV, and their spectra patterns are in good agreement with those observed in cell extracts of E. coli JM109/pRF1.5 (Fig.…”