Integrated bioprocessing in Saccharomyces cerevisiae using green fluorescent protein as a fusion partner

In this study, we examine the use of green fluorescent protein (GFP) for monitoring a hexokinase (HXK)-GFP fusion protein in Saccharomyces cerevisiae for various events including expression, degradation, purification, and localization. The fusion, HXK-EK-GFP-6 x His, was constructed where the histidine tag (6 x His) would allow for convenient affinity purification, and the enterokinase (EK) cleavage site would be used for separation of HXK from GFP after affinity purification. Our results showed that both HXK and GFP remained active in the fusion and, more importantly, that there was a linear correlation between HXK activity and GFP fluorescence. Enterokinase cleavage studies revealed that both GFP fluorescence intensity and HXK activity remained unchanged after separation of the fusion proteins, which indicated that fusion of GFP did not cause structural alteration of HXK and thus did not affect the enzymatic activity of HXK. We also found that degradation of the fusion protein occurred, and that degradation was limited to HXK with GFP remaining intact in the fusion. Confocal microscopy studies showed that while GFP was distributed evenly in the yeast cytosol, HXK-GFP fusion followed the correct localization of HXK, which resulted in a di-localization of both cytosol and the nucleus. GFP proved to be a useful fusion partner that may lead to the possibility of integrating the bioprocesses by quantitatively following the entire process visually.     

System for measuring oxygen consumption rates of mammalian cells in static culture under hypoxic conditions

Estimating the oxygen consumption rates (OCRs) of mammalian cells in hypoxic environments is essential for designing and developing a three‐dimensional (3‐D) cell culture system. However, OCR measurements under hypoxic conditions are infrequently reported in the literature. Here, we developed a system for measuring OCRs at low oxygen levels. The system injects nitrogen gas into the environment and measures the oxygen concentration by an optical oxygen microsensor that consumes no oxygen. The developed system was applied to HepG2 cells in static culture. Specifically, we measured the spatial profiles of the local dissolved oxygen concentration in the medium, then estimated the OCRs of the cells. The OCRs, and also the pericellular oxygen concentrations, decreased nonlinearly as the oxygen partial pressure in the environment decreased from 19% to 1%. The OCRs also depended on the culture period and the matrix used for coating the dish surface. Using this system, we can precisely estimate the OCRs of various cell types under environments that mimic 3‐D culture conditions, contributing crucial data for an efficient 3‐D culture system design. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 32:189–197, 2016     

Laser-scanning lithography (LSL) for the soft lithographic patterning of cell-adhesive self-assembled monolayers

We report the development of laser-scanning lithography (LSL), which employs a laser-scanning confocal microscope to pattern photoresists that can be utilized, for example, in the fabrication of masters for use in soft lithography. This convenient technique provides even exposure across the entire view field and facilitates accurate alignment of successive photoresist exposures. Features on the scale of 3 microm have been achieved to date with a 10x objective (NA 0.45). Virtual masks, instructions for laser irradiation, were drawn using the Region of Interest (ROI) function of a Zeiss LSM 510 microscope. These regions were then exposed to a 458 nm argon laser for 32 micros (0.9 mW/microm(2)). Differential interference contrast (DIC) imaging was utilized with a non-destructive 514 nm argon laser as an immediate quality check of each exposure, to align successive exposures, and to reduce chromatic aberration between imaging and exposure. Developed masters were replica-molded with poly(dimethylsiloxane) (PDMS); these masters were then utilized for microcontact printing of cell-adhesive self-assembled monolayers (SAMs) to demonstrate the utility of this process. Initial studies confirmed that human dermal fibroblast adhesion and spreading were limited to cell-adhesive SAM areas. LSL is a rapid, flexible, and readily available technique that will accelerate master design and preparation; moreover, it can be applied to additional forms of photolithography and photopolymerization for studies in cell biology, biomaterials design and evaluation, materials science, and surface chemistry.     

Determination of oxygen gradients in engineered tissue using a fluorescent sensor

Nutrient and oxygen supply of cells are crucial to tissue engineering in general. If a sufficient supply cannot be maintained, the development of the tissue will slow down or even fail completely. Previous studies on oxygen supply have focused on measurement of oxygen partial pressures (pO(2)) in culture media or described the use of invasive techniques with spatially limited resolution. The experimental setup described here allows for continuous, noninvasive, high-resolution pO(2) measurements over the cross-section of cultivated tissues. Applying a recently developed technique for time-resolved pO(2) sensing using optical sensor foils, containing luminescent O(2)-sensitive indicator dyes, we were able to monitor and analyze gradients in the oxygen supply in a tissue over a 3-week culture period. Cylindrical tissue samples were immobilized on top of the sensors. By measuring the luminescence decay time, two-dimensional pO(2) distributions across the tissue section in contact with the foil surface were determined. We applied this technique to cartilage explants and to tissue-engineered cartilage. For both tissue types, changes were detected in monotonously decreasing gradients of pO(2) from the surface with high pO(2) to minimum pO(2) values in the center of the samples. Nearly anoxic conditions were observed in tissue constructs ( approximately 0 Torr) but not in excised cartilage discs ( approximately 20 Torr) after 1 day. Furthermore, the oxygen supply seemed to strongly depend on cell density and cell function. Additionally, histological analysis revealed a maximum depth of approximately 1.3 mm of regular cartilage development in constructs grown under the applied culture conditions. Correlating analytical and histological analysis with the oxygen distributions, we found that pO(2) values below 11 Torr might impair proper tissue development in the center. The results illustrate that the method developed is an ideal one to precisely assess the oxygen demand of cartilage cultures.     

The use of calcium blockers to study biochemical behaviour of Saccharomyces cerevisiae cells

Altered expression of cell adhesion molecule expression has been implicated in a variety of chronic inflammatory conditions. Regulation of adhesion molecule expression by specific redox sensitive mechanisms has been reported. Grape seed proanthocyanidins have been reported to have potent antioxidant properties. We evaluated the effects of grape seed proanthocyanidin extract (GSPE) on the expression of TNF-induced ICAM-1 and VCAM-1 expression in primary human umbilical vein endothelial cells (HUVEC). GSPE at low concentrations (1-5 g/ml), down-regulated TNF-induced VCAM-1 expression but not ICAM-1 expression in HUVEC. Such regulation of inducible VCAM-1 by GSPE was also observed at the mRNA expression level. A cell-cell co-culture assay was performed to verify whether the inhibitory effect of GSPE on the expression of VCAM-1 was also effective in down-regulating actual endothelial cell/leukocyte interaction. GSPE treatment significantly decreased TNF-induced adherence of T-cells to HUVEC. Although several studies have postulated NF-B as the molecular site where redox active substances act to regulate agonist-induced ICAM-1 and VCAM-1 gene expression, inhibition of inducible VCAM-1 gene expression by GSPE was not through a NF-B-dependent pathway as detected by a NF-B reporter assay. The potent inhibitory effect of low concentrations of GSPE on agonist-induced VCAM-1 expression suggests therapeutic potential of this extract in inflammatory conditions and other pathologies involving altered expression of VCAM-1.     

Resistance to the Brownian movement of red blood cells on flat horizontal surfaces

The movements of red blood cells (RBC), suspended in plasma, on plastic, glass, rhodium metal plate, siliconized glass, and siliconized rhodium were recorded on cinéfilm and analyzed. Values for the drag coefficient were calculated, using Einstein's theory of Brownian movement, and compared with the theoretical Stokes' hydrodynamic drag. The difference between the computed and Stokes' values gave the frictional coefficient or resistance resulting from the interaction of the cells, with the test surface. Of the three uncoated test surfaces, plastic was found to have the least interaction with the RBC. The frictional coefficient for plastic was found to be 1.75×10⁻⁷ N s m⁻¹ compared with a value of 2.82×10⁻⁷ N s m⁻¹ for rhodium metal, which had the largest interaction. Upon siliconization of the test surfaces, the interaction decreased by 40%. Reduction in the pH of the suspending plasma increased the interaction between the cells and the uncoated test surfaces, but the pH effect of diminished when the surfaces were siliconized.     

Impact of operating variables on the expanded bed adsorption of Saccharomyces cerevisiae cells using a concanavalin A derivatized perfluorocarbon

The use of fluidizable affinity adsorbents for the adsorption of cells in expanded mode is investigated. Affinity adsorbents have been synthesized by immobilizing the lectin Concanavalin A onto the surface of triazine-activated perfluorocarbon-solids. The adsorbents were found to adsorb Saccharomyces cerevisiae cells from solution with adsorption capacities of up to 6.8 x 10(9) cells mL(-1). Adsorption kinetics were rapid with a time constant of 相似文献   

The challenges of using fluorescent probes to detect and quantify specific reactive oxygen species in living cells

Background

Small molecule fluorescent probes are vital tools for monitoring reactive oxygen species in cells.

Scope of review

The types of probe available, the extent to which they are specific or quantitative and complications in interpreting results are discussed.

Major conclusions

Most commonly used probes (e.g. dihydrodichlorofluorescein, dihydrorhodamine) have some value in providing information on changes to the redox environment of the cell, but they are not specific for any one oxidant and the response is affected by numerous chemical interactions and not just increased oxidant generation. These probes generate the fluorescent end product by a free radical mechanism, and to react with hydrogen peroxide they require a metal catalyst. Probe radicals can react with oxygen, superoxide, and various antioxidant molecules, all of which influence the signal. Newer generation probes such as boronates act by a different mechanism in which nucleophilic attack by the oxidant on a blocking group releases masked fluorescence. Boronates react with hydrogen peroxide, peroxynitrite, hypochlorous acid and in some cases superoxide, so are selective but not specific. They react with hydrogen peroxide very slowly, and kinetic considerations raise questions about how the reaction could occur in cells.

General significance

Data from oxidant-sensitive fluorescent probes can provide some information on cellular redox activity but is widely misinterpreted. Recently developed non-redox probes show promise but are not generally available and more information on specificity and cellular reactions is needed. We do not yet have probes that can quantify cellular production of specific oxidants. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn.     

Microscale decoupling of sediment oxygen consumption and microbial biomass in an oligotrophic lake

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A novel 1,8-naphthalimide-based fluorescent chemosensor for the detection of HSA in living cells

Human serum albumin (HSA) is an essential protein for maintaining human health. Accurate detection and quantification of HSA are of great significance for disease diagnosis and biochemical research. Here, a new HSA fluorescent probe BNPE based on the 1,8-naphthalimide fluorophore was designed and synthesized. The probe could recognize HSA through a twisted intramolecular charge transfer mechanism, effectively avoid the interference of most substances, and realize HSA fluorescence imaging in living cells.     

Detection of biomolecular interaction between biotin and streptavidin on a self-assembled monolayer using magnetic nanoparticles

For developing a magnetic bioassay system, an investigation to determine the presence of a specific biomolecular interaction between biotin and streptavidin was done using magnetic nanoparticles and a silicon substrate with a self-assembled monolayer. Streptavidin was immobilized on the magnetic particles, and biotin was attached to the monolayer-modified substrate. The reaction of streptavidin-modified magnetic particles on the biotin-modified substrate was clearly observed under an optical microscope. The magnetic signals from the particles were detected using a magnetic force microscope. The results of this study demonstrate that the combination of a monolayer-modified substrate with biomolecule-modified magnetic particles is useful for detecting biomolecular interactions in medical and diagnostic analyses.     

Enzyme induction by oxygen in the accumulation of cytochrome P-450 during batch fermentations in 20% d-glucose with Saccharomyces cerevisiae

Production of cytochrome P-450 [RH, reduced-flavoprotein:oxygen oxidoreductase (RH-hydroxylating), EC 1.14.14.1] by Saccharomyces cerevisiae NCYC 754, grown in batch culture on 20% d-glucose medium, was markedly affected by the speed of the orbital shaker. Oxygenation rather than agitation was confirmed as the likely cause of this effect using an optimized system in a microprocessor-controlled 4 litre batch fermenter. Oxygen may be acting as a substrate inducer of the cytochrome P-450 in this yeast.     

A spectrophotometric biochemical oxygen demand determination method using 2,6-dichlorophenolindophenol as the redox color indicator and the eukaryote Saccharomyces cerevisiae

A method to determine the spectrophotometric biochemical oxygen demand (BOD(sp)) was studied with high sensitivity and reproducibility by employing 2,6-dichlorophenolindophenol (DCIP) as a redox color indicator, the yeast Saccharomyces cerevisiae, and a temperature-controlling system providing a three-consecutive-stir unit. The absorbance of DCIP decreased due to the metabolism of organic substances in aqueous samples by S. cerevisiae. Under optimum conditions, a calibration curve for glucose glutamic acid concentration between 1.1 and 22mg O(2) L(-1) (r=0.988, six points, n=3) was obtained when the incubation mixture was incubated for 10min at 30 degrees C. The reproducibility of the optical responses in the calibration curve was 1.77% (average of relative standard deviations; RSD(av)). Subsequently, the characterization of this method was studied. The optical responses to pure organic substances and the influence of chloride ions, artificial seawater, and heavy metal ions on the sensor response were investigated before use with real samples. Measurements of real samples using river water were performed and compared with those obtained using the BOD(5) method. Finally, stable responses were obtained for 36 days when the yeast cell suspension was stored at 4 degrees C (response reduction, 89%; RSD(av) value for 9 testing days, 8.4%).     

Optimizing the biosynthesis of oxygenated and acetylated Taxol precursors in Saccharomyces cerevisiae using advanced bioprocessing strategies

Taxadien‐5α‐hydroxylase and taxadien‐5α‐ol O‐acetyltransferase catalyze the oxidation of taxadiene to taxadien‐5α‐ol and subsequent acetylation to taxadien‐5α‐yl‐acetate in the biosynthesis of the blockbuster anticancer drug, paclitaxel (Taxol®). Despite decades of research, the promiscuous and multispecific CYP725A4 enzyme remains a major bottleneck in microbial biosynthetic pathway development. In this study, an interdisciplinary approach was applied for the construction and optimization of the early pathway in Saccharomyces cerevisiae, across a range of bioreactor scales. High‐throughput microscale optimization enhanced total oxygenated taxane titer to 39.0 ± 5.7 mg/L and total taxane product titers were comparable at micro and minibioreactor scale at 95.4 ± 18.0 and 98.9 mg/L, respectively. The introduction of pH control successfully mitigated a reduction of oxygenated taxane production, enhancing the potential taxadien‐5α‐ol isomer titer to 19.2 mg/L, comparable with the 23.8 ± 3.7 mg/L achieved at microscale. A combination of bioprocess optimization and increased gas chromatography‐mass spectrometry resolution at 1 L bioreactor scale facilitated taxadien‐5α‐yl‐acetate detection with a final titer of 3.7 mg/L. Total oxygenated taxane titers were improved 2.7‐fold at this scale to 78 mg/L, the highest reported titer in yeast. Critical parameters affecting the productivity of the engineered strain were identified across a range of scales, providing a foundation for the development of robust integrated bioprocess control systems.     

Dual fluorescence nanoconjugates for ratiometric detection of reactive oxygen species in inflammatory cells

Reactive oxygen species (ROS) are largely produced under pathological situations. To understand the etiology of disease, it is urgent to develop efficacious probes for detecting ROS. Herein, a novel nanoconjugate detection system constructed from gold clusters (AuNCs) and quantum dots (QDs) for fluorescence ratiometric‐sensing ROS was reported. Upon interacting with ROS, the red emission fluorescence (645 nm from QDs) in the detection system gradually decreased, while the green fluorescence (480 nm from AuNCs) changed little. The fluorescence ratio at the 2 wavelengths (I_{480 nm}/I_{645 nm}) was linearly correlated with the ROS, which could be used for the real‐time ratiometric detection of ROS. The developed nanoconjugates could be applied to monitor the ROS in inflammatory cells for its ability of generating abundant ROS and uptaking ability to nanoparticles. The stimulated ROS in inflammatory cells were monitored by AuNC‐QD and the results were consistent with the traditional 2′, 7′‐dichlorofluorescin diacetate method, confirming the reliability of the developed method. Featured with the merits of higher photostability, low background, high accuracy of ratiometric detection, the AuNC‐QD conjugate demonstrated its potential to be the probe for real‐time ROS detection in inflammatory cells.      

Detecting freeze injury and seasonal cold-hardening of cells and tissues in the gall fly larvae, Eurosta solidaginis (Diptera: Tephritidae) using fluorescent vital dyes

This study identified a hierarchy in levels of cold tolerance for diverse tissues from larvae of Eurosta solidaginis. Following freezing at -80 degrees C, larval survival and the viability of specific tissues were assessed using membrane-permeant DNA stain (SYBY-14) and propidium iodide.Integumentary muscle, hemocytes, tracheae, and the crystal-containing portion of the Malpighian tubules were most susceptible to freezing injury. A second group consisting of fat body, salivary glands, and the proximal region of the Malpighian tubules were intermediate in their susceptibility, while the foregut, midgut, and hindgut were the most resistant to freezing injury. Seasonal increases in larval cold tolerance were closely matched by changes in the cold tolerance of individual tissues. Compared to larvae collected in September, the survival rates for each of the six tissues tested from October-collected larvae increased by 20-30%. The survival rate in all tissues was notably higher than that of whole animals, indicating that larval death could not be explained by the mortality in any of the tissues we tested. This method will be useful for assessing the nature of chilling/freezing injury, the role cryoprotectants, and cellular changes promoting cold tolerance.