Simultaneous detection of bacteria expressing GFP and DsRed genes with a flow cytometer

BACKGROUND: In this study, Escherichia coli cells producing red fluorescent protein of Discosoma sp. (drFP583 DsRed) were investigated with flow cytometry by using 488 nm excitation. We also studied whether green fluorescent protein (GFP) and drFP583 could be detected simultaneously from a single bacterial cell. METHODS: Plasmids pDsRed and pEGFP were used for the production of drFP583 and enhanced GFP, respectively, in E. coli MC1061 cells. To produce enhanced GFP and drFP583 simultaneously, plasmids pG9R and pG19R were constructed. These encode tandem fusions of enhanced GFP and drFP583 to ensure similar production levels for both proteins. RESULTS: Bacteria producing enhanced GFP and drFP583 were found to be brightly green and red fluorescent, respectively. Production of enhanced GFP and drFP583 fusion proteins resulted in bacteria that emitted both green and red fluorescence, which was detected easily by a flow cytometer using single laser excitation. Previously reported tetramerization of drFP583 did not restrict its use as a reporter gene, although it maturated significantly slower than enhanced GFP. CONCLUSIONS: The results show that enhanced GFP and drFP583 proteins can be detected simultaneously from single bacteria with a standard flow cytometer with simple optical configuration.     

Monitoring promoter activity in a single bacterial cell by using green and red fluorescent proteins

We investigated the possibility of monitoring promoter activity with flow cytometry by using green fluorescent protein (GFPmut2) and red fluorescent protein (drFP583) in a single bacterial cell. The drFP583 was used as an intrinsic marker of the bacterial cells, because it was expressed constantly in Escherichia coli MC1061 strain. The GFPmut2 expressed under the control of the Hg(2+) ion inducible mer promoter/operator, was used to study promoter activity. Over 75% of the cells were positive for red and green fluorescence in flow cytometric analysis. The average green fluorescence of the whole population increased from 6.7 to 1700 when the mercury concentration was increased from 0 to 1 x 10(-4) M, while the red fluorescence was unaffected by the mercury concentration. These results show that gfpmut2 and drFP583 could be expressed under different promoters in one bacterial cell and measured independently with a flow cytometer.     

Mutants of Discosoma red fluorescent protein with a GFP-like chromophore

The green fluorescent protein (GFP)-homologous red fluorescent protein (RFP) from Discosoma (drFP583) which emits bright red fluorescence peaking at 583 nm is an interesting novel genetic marker. We show here that RFP maturation involves a GFP-like fluorophore which can be stabilized by point mutations selected from a randomly mutated expression library. By homology modeling, these point mutations cluster near the imidazolidinone ring of the chromophore. Exciting the GFP-like absorption band in the mutant proteins produces both green and red fluorescence. Upon unfolding and heating, the absorption spectrum of the RFP chromophore slowly becomes similar to that of the GFP chromophore. This can be interpreted as a covalent modification of the GFP chromophore in RFP that appears to occur in the final maturation step.     

EFGP and DsRed expressing cultures of Escherichia coli imaged by confocal, two-photon and fluorescence lifetime microscopy

The green fluorescent protein (GFP) has become an invaluable marker for monitoring protein localization and gene expression in vivo. Recently a new red fluorescent protein (drFP583 or DsRed), isolated from tropical corals, has been described [Matz, M.V. et al. (1999) Nature Biotech. 17, 969-973]. With emission maxima at 509 and 583 nm respectively, EGFP and DsRed are suited for almost crossover free dual color labeling upon simultaneous excitation. We imaged mixed populations of Escherichia coli expressing either EGFP or DsRed by one-photon confocal and by two-photon microscopy. Both excitation modes proved to be suitable for imaging cells expressing either of the fluorescent proteins. DsRed had an extended maturation time and E. coli expressing this fluorescent protein were significantly smaller than those expressing EGFP. In aging bacterial cultures DsRed appeared to aggregate within the cells, accompanied by a strong reduction in its fluorescence lifetime as determined by fluorescence lifetime imaging microscopy.     

Variants of DsRed fluorescent protein: Development of a copper sensor

The fluorescence quenching of drFP583 (DsRed) protein by metal ions was investigated. CuSO4 reversibly and pH dependently quenched the red emission at 583 nm of drFP583. The copper binding constant was 15 mM. Following random mutagenesis, blue- and red-shifted mutants of drFP583 were generated, and their metal sensing properties were examined. Mutant gRF possessed properties similar to green fluorescent protein and had a 18 mM copper binding constant. Mutant Rmu162 had an extraordinary red-shifted emission peak at 620 nm. A third mutant, Rmu13, had dual emission peaks at 500 nm and 583 nm and possessed the properties of a copper sensor with a binding constant of 11 mM.     

Orexin signaling in recombinant neuron-like cells

Recently, we cloned several fluorescent proteins of different colors homologous to Aequorea victoria green fluorescent protein, which have great biotechnological potential as in vivo markers of gene expression. However, later investigations revealed severe drawbacks in the use of novel fluorescent proteins (FPs), in particular, the formation of tetramers (tetramerization) and high molecular weight aggregates (aggregation). In this report, we employ a mutagenic approach to resolve the problem of aggregation. The elimination of basic residues located near the N-termini of FPs results in the generation of non-aggregating versions of several FPs, specifically, drFP583 (DsRed), DsRed-Timer, ds/drFP616, zFP506, zFP538, amFP486, and asFP595.     

Novel fluorescent protein from Discosoma coral and its mutants possesses a unique far-red fluorescence

A novel gene for advanced red-shifted protein with an emission maximum at 593 nm was cloned from Discosoma coral. The protein, named dsFP593, is highly homologous to the recently described GFP-like protein drFP583 with an emission maximum at 583 nm. Using the remarkable similarity of the drFP583 and dsFP593 genes, we performed a 'shuffling' procedure to generate a pool of mutants consisting of various combinations of parts of both genes. One 'hybrid gene' was chosen for subsequent random mutagenesis, which resulted in a mutant variant with a uniquely red-shifted emission maximum at 616 nm.     

Genetic tags for labelling live cells: gap junctions and beyond

The availability of green fluorescent protein (GFP) as a tracer for observing proteins in living cells has revolutionized cell biology and spurred an intensive search for GFP variants with novel characteristics, additional autofluorescent proteins and alternative techniques of protein labelling. Two recent studies - one on tagging with tetracysteine motifs and labelling with biarsenic fluorophores of different colours, and the other on GFP tagging and fluorescence recovery after photobleaching (FRAP) - show how membrane channels are added and removed from gap junctions by using different fluorescent tags to distinguish between newly synthesized and older protein populations.     

绿色荧光蛋白——照亮生命科学的一盏明灯

绿色荧光蛋白的发现及应用具有划时代的重要意义,它不仅为当代生物学研究提供了极为实用的基本研究手段,并且在此基础上改造发展和发现了一系列荧光蛋白,拓展了应用范围。这使得对微观生物学的研究也可以进入一个时空结合,研究鲜活动态过程的新时代。本文主要回顾总结了绿色荧光蛋白的发现、优化改造及其应用。     

The dynamic microbe: green fluorescent protein brings bacteria to light

The demonstration that the green fluorescent protein (GFP) from the jellyfish Aequorea victoria required no jellyfish-specific cofactors and could be expressed as a fluorescent protein in heterologous hosts including both prokaryotes and eukaryotes sparked the development of GFP as one of the most common reporters in use today. Over the past several years, the utility of GFP as a reporter has been optimized through the isolation and engineering of variants with increased folding rates, different in vivo stabilities and colour variants with altered excitation and emission spectral properties. One of the great utilities of GFP is as a probe for characterizing spatial and temporal dynamics of gene expression, protein localization and protein-protein interactions in living cells. The innovative application of GFP as a reporter in bacteria has made a significant contribution to microbial cell biology. This review will highlight recent studies that demonstrate the potential of GFP for real-time analysis of gene expression, protein localization and the dynamics of signalling transduction pathways through protein-protein interactions.     

Dual-colour imaging with GFP variants

Green fluorescent protein (GFP) has become an important tool in cell biology and is widely used as a reporter for imaging intracellular proteins and structures in live cells. Recently, spectral variants of GFP with red- and blue-shifted fluorescence emissions have been characterized, opening the possibility of double labelling with two different-coloured GFP fusion proteins. This article reviews recent advances in this technique, with special emphasis on time-lapse imaging applications in living cells.     

A toolkit for graded expression of green fluorescent protein fusion proteins in mammalian cells

Green fluorescent protein (GFP) and GFP-like proteins of different colors are important tools in cell biology. In many studies, the intracellular targeting of proteins has been determined by transiently expressing GFP fusion proteins and analyzing their intracellular localization by fluorescence microscopy. In most vectors, expression of GFP is driven by the enhancer/promoter cassette of the immediate early gene of human cytomegalovirus (hCMV). This cassette generates high levels of protein expression in most mammalian cell lines. Unfortunately, these nonphysiologically high protein levels have been repeatedly reported to artificially alter the intracellular targeting of proteins fused to GFP. To cope with this problem, we generated a multitude of attenuated GFP expression vectors by modifying the hCMV enhancer/promoter cassette. These modified vectors were transiently expressed, and the expression levels of enhanced green fluorescent protein (EGFP) alone and enhanced yellow fluorescent protein (EYFP) fused to another protein were determined by fluorescence microscopy and/or Western blotting. As shown in this study, we were able to (i) clearly reduce the expression of EGFP alone and (ii) reduce expression of an EYFP fusion protein down to the level of the endogenous protein, both in a graded manner.     

Green fluorescent protein expression triggers proteome changes in breast cancer cells

Green fluorescent protein (GFP) is the most commonly used reporter of expression in cell biology despite evidence that it affects the cell physiology. The molecular mechanism of GFP-associated modifications has been largely unexplored. In this paper we investigated the proteome modifications following stable expression of GFP in breast cancer cells (MDA-MB-231). A combination of three different proteome analysis methods (2-DE, iTRAQ, label-free) was used to maximise proteome coverage. We found that GFP expression induces changes in expression of proteins that are associated with protein folding, cytoskeletal organisation and cellular immune response. In view of these findings, the use of GFP as a cell reporter should be carefully monitored.     

Green fluorescent protein (GFP) fusion constructs in gene therapy research

The history of green fluorescent protein (GFP) as a marker is less than 10 years old, but it has already made a major impact on many areas of natural sciences, especially on cell biology and histochemistry. GFP can be detected in living cells without selection or staining and it can be fused to other proteins to yield fluorescent chimeras. The potential of GFP has also been recognised by gene therapy researchers and various GFP-tagged therapeutic proteins have been constructed. These chimeric proteins have been used to determine the expression level, site and time course of the therapeutic gene, or the correlation between gene transfer rate and therapeutic outcome. This review summarises the status of the applications of GFP fusions in gene therapy research.     

绿荧光蛋白及其在转基因动物研究中的应用

绿荧光蛋白（ｇｒｅｅｎｆｌｕｏｒｅｓｃｅｎｔｐｒｏｔｅｉｎ,ＧＦＰ）是源于水母（Ｊｅｌｙｆｉｓｈ）、海笔（ＳｅａＰｅｎ,ＳｅａＰａｎｓｙ）等海洋无脊椎动物的一种蛋白质,这种蛋白质在体外经适当波长的光激发便可发出绿光,所发出的绿光用普通荧光显微镜或荧光激活细胞分拣器（ＦＡＣＳ）均可检测到。ＧＦＰ作为动、植物以及微生物基因工程研究上的一种选择标记具有检测灵敏度高,操作简便,对机体毒副作用小且不需要添加任何底物或辅助因子等优点,更重要的是检测ＧＦＰ无损于细胞或胚胎的完整性及活力。本文概括介绍ＧＦＰ的生化、发光光谱及遗传学特征及其在转基因动物研究上的应用。     

Green Fluorescent Protein Purification by Organic Extraction

Green fluorescent protein (GFP) is widely used as an excellent reporter molecule in biochemistry and cell biology. Some biochemical and immunological assays require high-purity GFP. However, the majority of current procedures for GFP purification include multiple time-consuming chromatography steps with a low yield of the desired product or require tag-containing proteins. An alternative method is described for the GFP purification without affinity extensions using organic extraction yielding a highly homogeneous protein indistinguishable in spectroscopic properties from that purified by previous methods.     

GFP expression alters osteosarcoma cell biology

Green fluorescent protein (GFP) expression is widely used for tracking of cells in vivo. Three human osteosarcoma cell lines were transfected with GFP and analyzed for expression of tissue markers and physiology. GFP-mediated increased tumor aggression in vivo in one cell line was corroborated by altered expression of bone markers and osteoblast-like cell biology in vitro as a result of transfection. The use of GFP for cell tracking in osteosarcoma animal models should therefore be used with caution.     

Expression of green or red fluorescent protein (GFP or DsRed) linked proteins in nonmuscle and muscle cells

The introduction of the green fluorescent protein (GFP) plasmids that allow proteins and peptides to be expressed with a fluorescent tag has had a major impact on the field of cell biology. It has enabled the dynamics of a wide variety of proteins to be analyzed that could not otherwise be detected in live cells. Transient transfections of muscle and nonmuscle cells with plasmids encoding various cytoskeletal proteins ligated to green fluorescent protein or Ds red protein allow changes in the cytoskeletal network to be studied in the same cell for time periods up to several days. With this approach, proteins that could not be purified and directly labeled with fluorescent dyes and microinjected into cells can now be expressed and visualized in a wide variety of cells. Procedures are presented for transfection of the nonmuscle cell, PtK2, and primary cultures of embryonic chick myocytes, and for studying the live transfected cells.     

Green fluorescent protein causes mitochondria to aggregate in the presence of the Bcl-2 family proteins

Green fluorescent protein (GFP) has been widely used in a variety of experiments in cell biology. When cells were co-transfected with the GFP gene and the bcl-2 family genes bcl-2, bcl-x(L), and bax, mitochondria appeared to aggregate at the periphery of the nucleus specifically where GFP was expressed. Little aggregation was seen in the presence of other members of the GFP family, EGFP (enhanced GFP), ECFP (enhanced cyan variant), and EYFP (enhanced yellow-green variant). GFP but not EGFP seemed to promote cell death induced by pro-apoptotic Bax. Thus, GFP specifically promotes the aggregation of mitochondria when co-expressed with a member of the Bcl-2 family in association with apoptosis.     

Photodynamics of Red Fluorescent Proteins Studied by Fluorescence Correlation Spectroscopy

Red fluorescent proteins are important tools in fluorescence-based life science research. Recently, we have introduced eqFP611, a red fluorescent protein with advantageous properties from the sea anemone Entacmaea quadricolor. Here, we have studied the submillisecond light-driven intramolecular dynamics between bright and dark states of eqFP611 and, for comparison, drFP583 (DsRed) by using fluorescence correlation spectroscopy on protein solutions. A three-state model with one dark and two fluorescent states describes the power-dependence of the flickering dynamics of both proteins at different excitation wavelengths. It involves two light-driven conformational transitions. We have also studied the photodynamics of individual (monomeric) eqFP611 molecules immobilized on surfaces. The flickering rates and dark state fractions of eqFP611 bound to polyethylene glycol-covered glass surfaces were identical to those measured in solution, showing that the bound FPs behaved identically. A second, much slower flickering process was observed on the 10-ms timescale. Deposition of eqFP611 molecules on bare glass surfaces yielded bright fluorescence without any detectable flickering and a >10-fold decreased photobleaching yield. These observations underscore the intimate connection between protein motions and photophysical processes in fluorescent proteins.