A bacterial cloning vector using a mutated Aequorea green fluorescent protein as an indicator
Introduction
At the present time, one of the most widely used reporter/marker proteins is β-galactosidase (lacZ′ gene). Detection of β-galactosidase activity requires the addition of X-gal as a substrate for subsequent blue color production. The use of various cloning vectors in E. coli, such as pUC (Yanisch-Perron et al., 1985) and pBluescript (Short et al., 1988), is based on insertional inactivation of β-galactosidase α-complementation which allows for distinguishing vectors containing a foreign DNA insert. However, a simpler method of detecting vectors containing foreign inserts would be to use green fluorescent protein (GFP) as a marker, because of the sensitivity and convenience with which its fluorescence can be detected in intact cells (Chalfie et al., 1994; Inouye and Tsuji, 1994a).
GFP isolated from the luminescent jellyfish, Aequorea victoria, emits a greenish fluorescence when irradiated with long-wave ultraviolet light. The protein (27 kDa) is made up of 238 amino acid residues and contains a fluorescent chromophore formed by an intramolecular cyclization of a tripeptide segment within the monomeric chain (Prasher et al., 1992; Cody et al., 1993; Inouye and Tsuji, 1994a, Inouye and Tsuji, 1994b). GFP has been employed as a reporter/marker in eukaryotic and prokaryotic cells (Chalfie et al., 1994; Inouye and Tsuji, 1994a). When GFP is expressed in a cell, the detection of the green fluorescence in long-wave ultraviolet light does not require any exogenous cofactor or substrate. Recent studies have shown that, when GFP is mutated at serine-65, the fluorescence intensity is increased and the excitation wavelength is shifted to the long wavelength (Delagrave et al., 1995; Heim et al., 1995). The properties of Aequorea GFP make it highly suitable for use in living cells as a reporter/marker in gene expression studies, even though the mechanism of the formation of the chromophore has yet to be elucidated.
We describe here a new cloning vector, which takes advantage of the green fluorescence of GFP and is designated `pGreenscript A', for gene cloning in the E. coli system.
Section snippets
GFP-S65A expression in E. coli cells
Recent studies have shown that the formation of the chromophore of GFP may be dependent on molecular oxygen, temperature and incubation time (Inouye and Tsuji, 1994b; Heim et al., 1994, Heim et al., 1995; Ogawa et al., 1995; Aoki et al., 1996). Various chromophore mutants of GFP have been reported (Delagrave et al., 1995; Heim et al., 1994, Heim et al., 1995), but the mutant we found superior as a reporter is GFP with serine-65 replaced with alanine (GFP-S65A). As shown in Table 1, E. coli
Conclusions
The cloning vector, pGreenscript A, whose product can be detected instantly without addition of substrate, was found to be a highly useful vector for gene cloning and sequencing analysis with E. coli. The cells expressing GFP-S65A exhibited an intense green fluorescence and a characteristic yellow color readily distinguishable under daylight.
References (15)
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