Green Fluorescent Protein Concentration Research Articles

Ultralow-threshold green fluorescent protein laser based on high Q microbubble resonators.

Biological lasers have attracted vast attention because of their potential medical application prospects, especially the low threshold biological laser that can be used for ultrasensitive biological detection while leaving the luminous gain medium undamaged by the pump light. By coupling the low concentration green fluorescent protein (GFP) solution with a high Q whispering gallery mode microbubble resonator, we managed to fabricate a miniature GFP laser with the lowest threshold and highest Q value compared to any known type of the GFP laser. The threshold energy is as low as 380 fJ, two orders of magnitude lower than any type of GFP laser at present. The Q value of the optical cavity in this biological laser is 5.3 × 107, two orders higher than the highest Q value of GFP lasers. We further confirmed the long-term stability of the working characteristics of GFP laser. It can work well nearly a month in temperature 3-4°C. Finally, we measured the effects of different concentrations of fluorescent protein on laser threshold. The data show that this biological laser can be used for highly sensitive detection of GFP concentration, which is particularly useful when the GFP is used as tracers.

Silica microspheres functionalized with the iminodiacetic acid/copper(II) complex as a peroxidase mimic for use in metal affinity chromatography-based colorimetric determination of histidine-tagged proteins.

Monodisperse porous silica microspheres were functionalized with the iminodiacetic acid/copper(II) complex and then evaluated as a group-specific peroxidase-mimicking nanozyme for colorimetric determination of histidine-tagged (His-tagged) proteins. The green fluorescent protein (GFP) was selected as a typical His-tagged protein. The specificity for GFP and the peroxidase-like activity for the selected substrate were obtained by immobilizing the complex on the porous microspheres. The modified microspheres were also evaluated as a group specific immobilized metal affinity chromatography (IMAC) sorbent for the purification of GFP from Escherichia coli extract. The peroxidase-like activity of the microspheres was inhibited by the GFP adsorbed onto the microspheres due to the interaction of His-tagged protein with the immobilized Cu(II) complex. Ortho-phenylenediamine is used as a substrate for the enzyme mimic. The photometric response (measured at 416nm) is linear in the 9.0-92μg·mL-1 GFP concentration range in E. coli lysate. The limit of detection is 6.9μg·mL-1. Graphical abstractSchematic representation of metal affinity chromatography-based colorimetric determination of histidine-tagged proteins using silica microspheres functionalized with iminodiacteic acid/copper (II) complex as a peroxidase mimic.

A novel RNA aptamer-modified riboswitch as chemical sensor

In this study, a novel label- and immobilization-free RNA aptamer-modified riboswitch-based biosensor was developed by using RNA aptamer modified secondary-structural scaffolds to control the identity of the ribosomal binding sequence (RBS). In the developed sensor, the duplex RNA aptamers-modified cis-repressor sequence is introduced upstream to the RBS of the indicating gene (gfp gene), leading to formatting an RNA bubble due to the none-complementary state of the RNA aptamers in the hairpin structure of the cis-repressor sequence. Without the presence of the target molecule, the ribosome cannot identify the RBS of the indicating gene as the RBS is hidden by the introduced cis-repressor, consequently, the indicating gene in the sensor would not be expressed, demonstrating the absence of the target. On the contrary, with the presence of the target molecule, the binding of aptamer with the target would induce the enlargement of the RNA bubble, leading to the separation of the cis-repressor sequence and RBS. Hence, the indicating gene would be expressed, manifesting the existence of the target. In addition, the developed sensor can quantitatively report the target concentrations by measuring the gfp gene-encoded GFP (green fluorescent protein) concentration. The approach proposed in this study can be used to construct sensors for detecting various chemicals by introducing the corresponding aptamers, therefore, this strategy can potentially provide a new set of analytical tools in the field of analytical chemistry.

Air Plasma-Enhanced Covalent Functionalization of Poly(methyl methacrylate): High-Throughput Protein Immobilization for Miniaturized Bioassays.

Miniaturized systems, such as integrated microarray and microfluidic devices, are constantly being developed to satisfy the growing demand for sensitive and high-throughput biochemical screening platforms. Owing to its recyclability, and robust mechanical and optical properties, poly(methyl methacrylate) (PMMA) has become the most sought after material for the large-scale fabrication of these platforms. However, the chemical inertness of PMMA entails the use of complex chemical surface treatments for covalent immobilization of proteins. In addition to being hazardous and incompatible for large-scale operations, conventional biofunctionalization strategies pose high risks of compromising the biomolecular conformations, as well as the stability of PMMA. By exploiting radio frequency (RF) air plasma and standard 1-ethyl-3-(3-(dimethylamino)propyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) chemistry in tandem, we demonstrate a simple yet scalable PMMA functionalization strategy for covalent immobilization (chemisorption) of proteins, such as green fluorescent protein (GFP), while preserving the structural integrities of the proteins and PMMA. The surface density of chemisorbed GFP is shown to be highly dependent on the air plasma energy, initial GFP concentration, and buffer pH, where a maximum GFP surface density of 4 × 10-7 mol/m2 is obtained, when chemisorbed on EDC-NHS-activated PMMA exposed to 27 kJ of air plasma, at pH 7.4. Furthermore, antibody-binding studies validate the preserved biofunctionality of the chemisorbed GFP molecules. Finally, the coupled air plasma and EDC-NHS PMMA biofunctionalization strategy is used to fabricate microfluidic antibody assay devices to detect clinically significant concentrations of Chlamydia trachomatis specific antibodies. By coupling our scalable and tailored air plasma-enhanced PMMA biofunctionalization strategy with microfluidics, we elucidate the potential of fabricating sensitive, reproducible, and sustainable high-throughput protein screening systems, without the need for harsh chemicals and complex instrumentation.

Cochlea-Specific Deletion of Cav1.3 Calcium Channels Arrests Inner Hair Cell Differentiation and Unravels Pitfalls of Conditional Mouse Models.

Inner hair cell (IHC) Cav1.3 Ca2+ channels are multifunctional channels mediating Ca2+ influx for exocytosis at ribbon synapses, the generation of Ca2+ action potentials in pre-hearing IHCs and gene expression. IHCs of deaf systemic Cav1.3-deficient (Cav1.3-/-) mice stay immature because they fail to up-regulate voltage- and Ca2+-activated K+ (BK) channels but persistently express small conductance Ca2+-activated K+ (SK2) channels. In pre-hearing wildtype mice, cholinergic neurons from the superior olivary complex (SOC) exert efferent inhibition onto spontaneously active immature IHCs by activating their SK2 channels. Because Cav1.3 plays an important role for survival, health and function of SOC neurons, SK2 channel persistence and lack of BK channels in systemic Cav1.3-/- IHCs may result from malfunctioning neurons of the SOC. Here we analyze cochlea-specific Cav1.3 knockout mice with green fluorescent protein (GFP) switch reporter function, Pax2::cre;Cacna1d-eGFPflex/flex and Pax2::cre;Cacna1d-eGFPflex/-. Profound hearing loss, lack of BK channels and persistence of SK2 channels in Pax2::cre;Cacna1d-eGFPflex/- mice recapitulated the phenotype of systemic Cav1.3-/- mice, indicating that in wildtype mice, regulation of SK2 and BK channel expression is independent of Cav1.3 expression in SOC neurons. In addition, we noticed dose-dependent GFP toxicity leading to death of basal coil IHCs of Pax2::cre;Cacna1d-eGFPflex/flex mice, likely because of high GFP concentration and small repair capacity. This and the slower time course of Pax2-driven Cre recombinase in switching two rather than one Cacna1d-eGFPflex allele lead us to study Pax2::cre;Cacna1d-eGFPflex/- mice. Notably, control Cacna1d-eGFPflex/- IHCs showed a significant reduction in Cav1.3 channel cluster sizes and currents, suggesting that the intronic construct interfered with gene translation or splicing. These pitfalls are likely to be a frequent problem of many genetically modified mice with complex or multiple gene-targeting constructs or fluorescent proteins. Great caution and appropriate controls are therefore required.

PURIFICATION OF RECOMBINANT GREEN FLUORESCENT PROTEIN FROM ESCHERICHIA COLI USING HYDROPHOBIC INTERACTION CHROMATOGRAPHY

□ In this study, an efficient purification system was developed using a packed-bed column for the purification of recombinant green fluorescent protein (GFP) from clarified feedstock. A series of hydrophobic ligands [phenyl (low sub), phenyl (high sub), butyl, and octyl] was screened at bench scale and the optimum concentration of salt in binding buffer was determined by precipitating the sample with (NH4)2SO4. The optimum salt concentration used in binding buffer is 30%(w/v) (NH4)2SO4 in 20 mM sodium phosphate buffer at pH 8.0. Phenyl (high sub) ligand showed the best result of GFP purification in terms of yield and purity. Maximum recovery (82%) of recombinant GFP was achieved with phenyl (high sub) ligand. The optimum elution buffer for scaled-up process was determined by conducting a stepwise elution with 0–15%(w/v) (NH4)2SO4 salt solution and the result showed that the GFP absorbed at the ligand was mostly eluted by using a buffer with the absence of (NH4)2SO4. The effect of GFP concentration in initial sample on the dynamic binding capacity was also studied by running a scaled-up hydrophobic interaction chromatography using HiPrep™ HIC Phenyl (high sub) 16/10 20mL packed-bed column connected to Äkta FPLC.

Effects of cold stress and heat stress on coral fluorescence in reef-building corals

Widespread temperature stress has caused catastrophic coral bleaching events that have been devastating for coral reefs. Here, we evaluate whether coral fluorescence could be utilized as a noninvasive assessment for coral health. We conducted cold and heat stress treatments on the branching coral Acropora yongei, and found that green fluorescent protein (GFP) concentration and fluorescence decreased with declining coral health, prior to initiation of bleaching. Ultimately, cold-treated corals acclimated and GFP concentration and fluorescence recovered. In contrast, heat-treated corals eventually bleached but showed strong fluorescence despite reduced GFP concentration, likely resulting from the large reduction in shading from decreased dinoflagellate density. Consequently, GFP concentration and fluorescence showed distinct correlations in non-bleached and bleached corals. Green fluorescence was positively correlated with dinoflagellate photobiology, but its closest correlation was with coral growth suggesting that green fluorescence could be used as a physiological proxy for health in some corals.

Bromomaleimide-linked bioconjugates are cleavable in mammalian cells.

Bromomaleimide-linked bioconjugates are cleavable in mammalian cells.

Simulation of dissolved CO2 gradients in a scale‐down system: A metabolic and transcriptional study of recombinant Escherichia coli

Deficient mixing in industrial-scale bioreactors is an important concern as it results in a heterogeneous environment that may affect microbial cell physiology. Dissolved carbon dioxide (dCO(2) ) fluctuations, which can occur in large-scale bioreactors, were simulated for the first time and their effects were evaluated on Escherichia coli expressing recombinant green fluorescent protein (GFP). The dCO(2) gradients were simulated by continuously circulating the medium between two vessels of a scale-down system to mimic mean circulation times (t(c) ) of 50, 170, and 375 s. Specific growth rate (μ) decreased by 11% and acetate concentration increased by 23% at the highest t(c) compared to reference cultures. An increase in the time needed for attaining maximum GFP concentration was also observed. The effect of dCO(2) fluctuations on the transcriptional levels of genes involved in the glutamate decarboxylase (gadA and gadC) and α-ketoglutarate dehydrogenase (sucA and sucB) were analyzed by quantitative real time PCR. Such genes are known to be highly over- or underexpressed at elevated constant dCO(2) . Expression levels of gadA and gadC increased up to 60% and 72%, and sucA decreased 1.8-fold in the culture performed at the highest t(c) . Only a minor decrease of sucB expression was observed at t(c) of 170 and 375 s. Although exposure to continuous high dCO(2) can affect culture performance, in this work it was shown that E. coli is able to rescue its metabolism in very short times when cells are intermittently returned to low dCO(2) conditions after being exposed to high dCO(2) .

High yield secretion of heterologous proteins in Corynebacterium glutamicum using its own Tat-type signal sequence

Efficient protein secretion, the basis of large-scale production of many compounds central to the biotechnology industry, is achieved by signal peptide and propeptide optimization in addition to optimizing host factors affecting heterologous protein production. Here, we fused green fluorescent protein (GFP) to the recently identified Tat-type secretory signal peptide of CgR0949 to demonstrate a high-yield protein secretion system of Corynebacterium glutamicum. The resultant secretion vector facilitated effective secretion of active-form GFP (20 mg l(-1)) into C. glutamicum culture medium. The expression of GFP was enhanced 2.9-fold using the Shine-Dalgarno sequence of triosephosphate isomerase in the secretion vector. Moreover, GFP drastically accumulated in the culture supernatant upon addition of calcium chloride even though Ca(2+) addition did neither enhanced the transcription of gfp nor resulted in the accumulation of cytosolic GFP. Active-form GFP concentration reached 1.8 g l(-1) after 48-h incubation in a jar fermentor. Likewise, α-amylase accumulation in C. glutamicum cultures was also enhanced by Ca(2+) addition, suggesting that Ca(2+) may affect general protein secretion in C. glutamicum.

Adaptation by stochastic switching of a monostable genetic circuit in Escherichia coli

Stochastic switching is considered as a cost-saving strategy for adaptation to environmental challenges. We show here that stochastic switching of a monostable circuit can mediate the adaptation of the engineered OSU12-hisC Escherichia coli strain to histidine starvation. In this strain, the hisC gene was deleted from the His operon and placed under the control of a monostable foreign promoter. In response to histidine depletion, the OSU12-hisC population shifted to a higher HisC expression level, which is beneficial under starving conditions but is not favoured by the monostable circuit. The population shift was accompanied by growth recovery and was reversible upon histidine addition. A weak directionality in stochastic switching of hisC was observed in growing microcolonies under histidine-free conditions. Directionality and fate decision were in part dependent on the initial cellular status. Finally, microarray analysis indicated that OSU12-hisC reorganized its transcriptome to reach the appropriate physiological state upon starvation. These findings suggest that bacteria do not necessarily need to evolve signalling mechanisms to control gene expression appropriately, even for essential genes.

Green fluorescent protein regulation in the coral Acropora yongei during photoacclimation

Reef-building corals inhabit high light environments and are dependent on photosynthetic endosymbiotic dinoflagellates for nutrition. While photoacclimation responses of the dinoflagellates to changes in illumination are well understood, host photoacclimation strategies are poorly known. This study investigated fluorescent protein expression in the shallow-water coral Acropora yongei during a 30 day laboratory photoacclimation experiment in the context of its dinoflagellate symbionts. Green fluorescent protein (GFP) concentration measured by Western blotting changed reversibly with light intensity. The first 15 days of the photoacclimation experiment led to a ∼1.6 times increase in GFP concentration for high light corals (900 μmol quanta m⁻² s⁻¹) and a ∼4 times decrease in GFP concentration for low light corals (30 μmol quanta m⁻² s⁻¹) compared with medium light corals (300 μmol quanta m⁻² s⁻¹). Green fluorescence increased ∼1.9 times in high light corals and decreased ∼1.9 times in low light corals compared with medium light corals. GFP concentration and green fluorescence intensity were significantly correlated. Typical photoacclimation responses in the dinoflagellates were observed including changes in density, photosynthetic pigment concentration and photosynthetic efficiency. Although fluorescent proteins are ubiquitous and abundant in scleractinian corals, their functions remain ambiguous. These results suggest that scleractinian corals regulate GFP to modulate the internal light environment and support the hypothesis that GFP has a photoprotective function. The success of photoprotection and photoacclimation strategies, in addition to stress responses, will be critical to the fate of scleractinian corals exposed to climate change and other stressors.

In vitro propagation of plant virus using different forms of plant tissue culture and modes of culture operation

Plant virus accumulation was investigated in vitro using three different forms of plant tissue culture. Suspended cells, hairy roots and shooty teratomas of Nicotiana benthamiana were infected with tobacco mosaic virus (TMV) using the same initial virus:biomass ratio. Viral infection did not affect tissue growth or morphology in any of the three culture systems. Average maximum virus concentrations in hairy roots and shooty teratomas were similar and about an order of magnitude higher than in suspended cells. Hairy roots were considered the preferred host because of their morphological stability in liquid medium and relative ease of culture. The average maximum virus concentration in the hairy roots was 0.82 ± 0.14 mg g −1 dry weight; viral coat protein represented a maximum of approximately 6% of total soluble protein in the biomass. Virus accumulation in hairy roots was investigated further using different modes of semi-continuous culture operation aimed at prolonging the root growth phase and providing nutrient supplementation; however, virus concentrations in the roots were not enhanced compared with simple batch culture. The relative infectivity of virus in the biomass declined by 80–90% during all the cultures tested, irrespective of the form of plant tissue used or mode of culture operation. Hairy root cultures inoculated with a transgenic TMV-based vector in batch culture accumulated green fluorescent protein (GFP); however, maximum GFP concentrations in the biomass were relatively low at 39 μg g −1 dry weight, probably due to genetic instability of the vector. This work highlights the advantages of using hairy roots for in vitro propagation of TMV compared with shooty teratomas and suspended plant cells, and demonstrates that batch root culture is more effective than semi-continuous operations for accumulation of high virus concentrations in the biomass.

Sterilization of medical devices by ethylene oxide, determination of the dissipation of residues, and use of Green Fluorescent Protein as an indicator of process control.

Ethylene oxide (EO) is used to sterilize Oxygenator and Tubing applied to heart surgery. Residual levels of EO and its derivatives, ethylene chlorohydrin (ECH) and ethylene glycol (EG), may be hazardous to the patients. Therefore, it must be removed by the aeration process. This study aimed to estimate the minimum aeration time for these devices to attain safe limits for use (avoiding excessive aeration time) and to evaluate the Green Fluorescent Protein (GFP) as a biosensor capable of best indicating the distribution and penetration of EO gas throughout the sterilization chamber. Sterilization cycles of 2, 4, and 8 h were monitored by Bacillus atrophaeus ATCC 9372 as a biological indicator (BI) and by the GFP. Residual levels of EO, ECH, and EG were determined by gas chromatography (GC), and the residual dissipation was studied. Safe limits were reached right after the sterilization process for Oxygenator and after 204 h of aeration for Tubing. In the 2 h cycle, the GFP concentration decreased from 4.8 (+/-3.2)% to 7.5 (+/-2.5)%. For the 4 h cycle, the GFP concentration decreased from 17.4 (+/-3.0)% to 21.5 (+/-6.8)%, and in the 8 h cycle, it decreased from 22.5 (+/-3.2)% to 23.9 (+/-3.9)%. This finding showed the potentiality for GFP applications as an EO biosensor.

Combination of On-Chip Field Amplification and Bovine Serum Albumin Sweeping for Ultrasensitive Detection of Green Fluorescent Protein

We report a highly effective on-chip preconcentration method by combining field-amplified sample injection (FASI) and bovine serum albumin (BSA) sweeping for ultrasensitive detection of green fluorescent protein (GFP) on a simple cross-channel microchip device. With the formation of a stagnant sample/running buffer boundary by balancing the hydrodynamic flow and the electro-osmotic flow (EOF), GFP molecules can be continuously injected into the sample loading channel and stacked. We have also demonstrated that BSA is a very effective pseudo-stationary phase for sweeping concentration of proteins in comparison to the commonly used micelles. The combination of FASI and BSA sweeping yields a concentration factor of 3570 and a limit of detection of 8.4 pM for GFP. Using this method, we have separated GFP and GFP-insulin-like growth factor-I (GFP-IGF-I) fusion protein. The entire assay (GFP concentration, matrix elimination, and electrophoretic separation) can be completed within <5 min. Furthermore, we have successfully applied this method for the detection of GFP expression of E. coli cells and the GFP content in single E. coli cells.

Metabolic and transcriptional response of recombinant Escherichia coli to elevated dissolved carbon dioxide concentrations

The effect of dissolved carbon dioxide (dCO(2)) concentration on the stoichiometric and kinetic constants and by-product accumulation was determined for Escherichia coli cells producing recombinant green fluorescent protein (GFP). Constant dCO(2), in the range of 20-300 mbar, was maintained during batch cultures by manipulating the inlet gas composition. As dCO(2) increased, specific growth rate (micro) decreased, and acetate accumulation and the time for onset of GFP production increased. Maximum biomass yield on glucose and GFP concentration were affected for dCO(2) above 70 and 150 mbar, respectively. Expression analysis of 16 representative genes showed that E. coli can respond at the transcriptional level upon exposure to increasing dCO(2), and revealed possible mechanisms responsible for the detrimental effects of high dCO(2). Genes studied included those involved in decarboxylation reactions (aceF, icdA, lpdA, sucA, sucB), genes from pathways of production and consumption of acetate (ackA, poxB, acs, aceA, fadR), genes from gluconeogenic and anaplerotic metabolism (pckA, ppc), genes from the acid resistance (AR) systems (adiA, gadA, gadC), and the heterologous gene (gfp). The transcription levels of tricarboxylic acid (TCA) cycle genes (icdA, sucA, sucB) and glyoxylate shunt (aceA) decreased as dCO(2) increased, whereas fadR (that codes for a negative regulator of the glyoxylate operon) and poxB (that codes for PoxB which is involved in acetate production from pyruvate) were up-regulated as dCO(2) increased up to 150 mbar. Furthermore, transcription levels of genes from the AR systems increased as dCO(2) increased up to 150 mbar, indicating that elevated dCO(2) triggers an acid stress response in E. coli cells. Altogether, such results suggest that the increased acetate accumulation and reduction in mu, biomass yield and maximum GFP concentration under high dCO(2) resulted from a lower carbon flux to TCA cycle, the concomitant accumulation of acetyl-CoA or pyruvate, and the acidification of the cytoplasm.

Fluorescence Correlation Spectroscopy In Live Bacillus Subtilis Cells: An In Vivo Study Of Transcriptional Regulation

Fluorescence correlation spectroscopy (FCS) is a useful technique for characterizing the mobility and concentration of fluorescent molecules both in vitro and in vivo (1). We utilize two photon FCS to characterize the concentration and mobility of fluorescent molecules within living cells of Bacillus subtilis. Autocorrelation functions were measured in bacteria expressing green fluorescent protein(GFP) under the lac promoter in both nutrient rich and nutrient poor culture medium. Although considerable heterogeneity was evident from cell to cell, on average, both intracellular concentration and mobility were found to be dependent upon culture medium and Isopropyl β-D-1-thiogalactopyranoside (IPTG) concentration. We also investigated bacteria expressing GFP under control of native promoters for transcription factors (TF) involved in the regulation of the carbon metabolic cycle in Bacillus subtilis. The GFP concentration, which should be related to TF concentration, was investigated for single cells and cell populations under different metabolic conditions. Some photobleaching was observed during the course of the measurements as a depletion in the average fluorescence intensity. This is due to the small size of the bacteria (∼10 fL) and low basal expression levels of GFP (∼100 nM) in the absence of IPTG. Methods to take this into account during data analysis are discussed.1. Schwille, P., U. Haupts, S. Maiti, and W.W. Webb. 1999. Molecular dynamics in living cells observed by fluorescence correlation spectroscopy with one- and two-photon excitation. Biophys J 77:2251-2265.

Affinity monolith preconcentrators for polymer microchip capillary electrophoresis

Developments in biology are increasing demands for rapid, inexpensive, and sensitive biomolecular analysis. In this study, polymer microdevices with monolithic columns and electrophoretic channels were used for biological separations. Glycidyl methacrylate-co-ethylene dimethacrylate monolithic columns were formed within poly(methyl methacrylate) microchannels by in situ photopolymerization. Flow experiments in these columns demonstrated retention and then elution of amino acids under conditions optimized for sample preconcentration. To enhance analyte selectivity, antibodies were immobilized on monoliths, and subsequent lysozyme treatment blocked nonspecific adsorption. The enrichment capability and selectivity of these affinity monoliths were evaluated by purifying fluorescently tagged amino acids from a mixture containing green fluorescent protein (GFP). Twenty-fold enrichment and 91% recovery were achieved for the labeled amino acids, with a >25 000-fold reduction in GFP concentration, as indicated by microchip electrophoresis analysis. These devices should provide a simple, inexpensive, and effective platform for trace analysis in complex biological samples.

Green Fluorescent Protein as a Tissue Marker in Transgenic Maize Seed

ABSTRACTSeed tissues (endosperm, embryo, and pericarp) are often separated into tissue‐enriched fractions by wet‐ or dry‐milling methods for use in food, feed, and industrial products. Seed tissue markers that are sensitive and readily quantifiable would be useful to optimize fractionation processes. To meet this need for tissue markers, we set out to produce and characterize different transgenic maize lines, each containing green fluorescent protein (GFP) in either endosperm or embryo. We examined mRNA transcripts using expressed sequence tag (EST) profiles of several major seed proteins and selected several with strong seed tissue preferences. Stably transformed maize lines were produced, and visual observation of fluorescence confirmed the presence of GFP in the desired tissues. To establish the utility of this grain for evaluating the effectiveness or separation efficiencies of fractionation processes, transgenic kernels were hand‐dissected into pericarp, endosperm, and embryo fractions and the GFP concentration in each fraction was determined. The GFP distribution between fractions of each transgenic event was calculated from GFP concentration and mass balance, which enabled the determination of GFP yield based on the hand‐dissection fractionation data and the amount of tissue contamination in each fraction. Our transgenic lines exhibited strong tissue preference for either embryo or endosperm. These lines should be useful for assessing separation efficiencies in maize fractionation processes.

Phenotypic plasticity of Escherichia coli at initial stage of symbiosis with Dictyostelium discoideum

We observed the change in the physiological state of Escherichia coli cells at the initial stage for establishing a new symbiotic relationship with Dictyostelium discoideum cells. For the physiological state, we monitored green fluorescence intensity due to a green fluorescent protein (GFP) gene integrated into the chromosome by flow cytometry (FCM). On co-cultivation of the two species, a new population of E. coli cells with increased GFP concentration appeared, and when the formation of mucoidal colonies housing the coexisting two species began, most E. coli cells were from the new population. Further experiments suggest that the physiological change is induced by interaction with D. discoideum cells and is reversible, although the processes of the changes in both directions seem to proceed gradually. The observed phenotypic plasticity, together with natural selection under a co-cultivation environment, may be important for leading to the evolution of a new symbiotic system.