Green Fluorescent Protein

Abstracts

Several types of promoter-probe vectors are available for use in mycobacteria. The majority of such tools are based on the classical reporter genes lacZ (1–3), cat (41, xylE (5), and luc (6,7). Owing to the importance of the intracellular lifestyle for a number of pathogenic mycobacterial species, it seemed desirable to develop approaches for visualization of mycobacteria inside the eukaryotic cell (most commonly the macrophage) and for monitoring their gene expression in intracellular environments. Such concepts stimulated the recent development of reporter systems with the green fluorescent protein (GFP) (8) from the jellyfish Aequorea victoria as their centerpiece. The GFP fluorophore is generated via an autocatalytic reaction with the amino-acid backbone as the substrate and mvolves formation of a cyclic tripeptide (9). The GFP chromophore is formed in several heterologous cellular environments (8,10,12) and has been shown to emit green fluorescence (emission maximum at 509 nm) when irradiated with light of the appropriate wavelength (excitation peaks at 395 and 470 nm) (8). GFP variants with improved brightness and with shifted excitation and emission peaks are available (12). Of critical importance for meeting the standards expected of“in vivo monitoring technology,” GFP fluorescence is highly sensitive and occurs without exogenously added substrates or cofactors (8,13) (Fig. 1A). Although the quantum yield of GFP is high (11), the fluorescence brightness of each GFP molecule is one tenth that of fluorescein