共查询到20条相似文献,搜索用时 31 毫秒
1.
Najaf A Shah Richard J Laws Bradley Wardman Lue Ping Zhao John L Hartman IV 《BMC systems biology》2007,1(1):3-14
Background
Genome-wide mutant strain collections have increased demand for high throughput cellular phenotyping (HTCP). For example, investigators use HTCP to investigate interactions between gene deletion mutations and additional chemical or genetic perturbations by assessing differences in cell proliferation among the collection of 5000 S. cerevisiae gene deletion strains. Such studies have thus far been predominantly qualitative, using agar cell arrays to subjectively score growth differences. Quantitative systems level analysis of gene interactions would be enabled by more precise HTCP methods, such as kinetic analysis of cell proliferation in liquid culture by optical density. However, requirements for processing liquid cultures make them relatively cumbersome and low throughput compared to agar. To improve HTCP performance and advance capabilities for quantifying interactions, YeastXtract software was developed for automated analysis of cell array images. 相似文献2.
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Johanna Panula-Perälä Juozas Šiurkus Antti Vasala Robert Wilmanowski Marco G Casteleijn Peter Neubauer 《Microbial cell factories》2008,7(1):1-12
Background
Here we describe a novel cultivation method, called EnBase?, or enzyme-based-substrate-delivery, for the growth of microorganisms in millilitre and sub-millilitre scale which yields 5 to 20 times higher cell densities compared to standard methods. The novel method can be directly applied in microwell plates and shake flasks without any requirements for additional sensors or liquid supply systems. EnBase is therefore readily applicable for many high throughput applications, such as DNA production for genome sequencing, optimisation of protein expression, production of proteins for structural genomics, bioprocess development, and screening of enzyme and metagenomic libraries.Results
High cell densities with EnBase are obtained by applying the concept of glucose-limited fed-batch cultivation which is commonly used in industrial processes. The major difference of the novel method is that no external glucose feed is required, but glucose is released into the growth medium by enzymatic degradation of starch. To cope with the high levels of starch necessary for high cell density cultivation, starch is supplied to the growing culture suspension by continuous diffusion from a storage gel. Our results show that the controlled enzyme-based supply of glucose allows a glucose-limited growth to high cell densities of OD600 = 20 to 30 (corresponding to 6 to 9 g l-1 cell dry weight) without the external feed of additional compounds in shake flasks and 96-well plates. The final cell density can be further increased by addition of extra nitrogen during the cultivation. Production of a heterologous triosphosphate isomerase in E. coli BL21(DE3) resulted in 10 times higher volumetric product yield and a higher ratio of soluble to insoluble product when compared to the conventional production method.Conclusion
The novel EnBase method is robust and simple-to-apply for high cell density cultivation in shake flasks and microwell plates. The potential of the system is that the microbial growth rate and oxygen consumption can be simply controlled by the amount (and principally also by the activity) of the starch-degrading enzyme. This solves the problems of uncontrolled growth, oxygen limitation, and severe pH drop in shaken cultures. In parallel the method provides the basis for enhanced cell densities. The feasibility of the new method has been shown for 96-well plates and shake flasks and we believe that it can easily be adapted to different microwell and deepwell plate formats and shake flasks. Therefore EnBase will be a helpful tool especially in high throughput applications. 相似文献5.
Mirja Krause Kaisa Ukkonen Tatu Haataja Maria Ruottinen Tuomo Glumoff Antje Neubauer Peter Neubauer Antti Vasala 《Microbial cell factories》2010,9(1):11
Background
Cultivations for recombinant protein production in shake flasks should provide high cell densities, high protein productivity per cell and good protein quality. The methods described in laboratory handbooks often fail to reach these goals due to oxygen depletion, lack of pH control and the necessity to use low induction cell densities. In this article we describe the impact of a novel enzymatically controlled fed-batch cultivation technology on recombinant protein production in Escherichia coli in simple shaken cultures. 相似文献6.
Background
Microbial biofilms exist all over the natural world, a distribution that is paralleled by metal cations and oxyanions. Despite this reality, very few studies have examined how biofilms withstand exposure to these toxic compounds. This article describes a batch culture technique for biofilm and planktonic cell metal susceptibility testing using the MBEC assay. This device is compatible with standard 96-well microtiter plate technology. As part of this method, a two part, metal specific neutralization protocol is summarized. This procedure minimizes residual biological toxiCity arising from the carry-over of metals from challenge to recovery media. Neutralization consists of treating cultures with a chemical compound known to react with or to chelate the metal. Treated cultures are plated onto rich agar to allow metal complexes to diffuse into the recovery medium while bacteria remain on top to recover. Two difficulties associated with metal susceptibility testing were the focus of two applications of this technique. First, assays were calibrated to allow comparisons of the susceptibility of different organisms to metals. Second, the effects of exposure time and growth medium composition on the susceptibility of E. coli JM109 biofilms to metals were investigated. 相似文献7.
Aims
Antibiotics can act as signal molecules and affect bacterial gene expression, physiology and virulence. The purpose of this study was to determine whether subinhibitory antibiotic concentrations alter gene expression and physiology of Listeria monocytogenes.Methods and Results
Using an agar‐based screening assay with promoter fusions, 14 of 16 antibiotics induced or repressed expression of one or more stress and/or virulence genes. Despite ampicillin‐induced up‐regulation of PinlA‐lacZ expression, Caco‐2 cell invasion was not affected. Subinhibitory concentrations of ampicillin and tetracycline caused up‐ and down‐regulation of stress response genes, respectively, but both antibiotics caused increased sensitivity to acid stress. Six combinations of gene‐antibiotic were quantified in broth cultures and five of the six resulted in the same expression pattern as the agar‐based assay.Conclusions
Antibiotics affect virulence and/or stress gene expression; however, altered expression could not predict changes in phenotypic behaviour. Subinhibitory concentrations of antibiotics led to increased acid sensitivity, and we speculate that this is attributed to changes in cell envelope or reduced σB‐dependent gene expression.Significance and Impact of the Study
Although subinhibitory concentrations of antibiotics affect gene expression in L. monocytogenes, the changes did not increase virulence but did enhance the acid sensitivity. 相似文献8.
During batch growth on mixtures of two growth-limiting substrates, microbes consume the substrates either sequentially (diauxie)
or simultaneously. The ubiquity of these growth patterns suggests that they may be driven by a universal mechanism common
to all microbial species. Recently, we showed that a minimal model accounting only for enzyme induction and dilution, the
two processes that occur in all microbes, explains the phenotypes observed in batch cultures of various wild-type and mutant/recombinant
cells (Narang and Pilyugin in J. Theor. Biol. 244:326–348, 2007). Here, we examine the extension of the minimal model to continuous cultures. We show that: (1) Several enzymatic trends,
attributed entirely to cross-regulatory mechanisms, such as catabolite repression and inducer exclusion, can be quantitatively
explained by enzyme dilution. (2) The bifurcation diagram of the minimal model for continuous cultures, which classifies the
substrate consumption pattern at any given dilution rate and feed concentrations, provides a precise explanation for the empirically
observed correlations between the growth patterns in batch and continuous cultures. (3) Numerical simulations of the model
are in excellent agreement with the data. The model captures the variation of the steady state substrate concentrations, cell
densities, and enzyme levels during the single- and mixed-substrate growth of bacteria and yeasts at various dilution rates
and feed concentrations. This variation is well approximated by simple analytical expressions that furnish deep physical insights.
(4) Since the minimal model describes the behavior of the cells in the absence of cross-regulatory mechanisms, it provides
a rigorous framework for quantifying the effect of these mechanisms. We illustrate this by analyzing several data sets from
the literature. 相似文献
9.
Aims
This study aimed to examine the effects of seven different isothiocyanates against the growth and development of three important soil borne potato pathogens, (Colletotrichum coccodes, Rhizoctonia solani and Helminthosporium solani).Methods
The study was carried out using an agar diffusion assay. The radial growth of fungal pathogens grown on agar containing different ITCs at a range of concentrations was compared to that of growth on control agar plates that did not contain ITCs.Results
Results varied depending on the specific isothiocyanate incorporated into the agar. They ranged from those which showed a significant effect on fungal growth to those which appeared to have little or no effect. Where a suppressive effect was observed, due to the presence of the isothiocyanate, fungal colony growth decreased as the concentration of the incorporated isothiocyanate increased.Conclusions
Results from this study indicate that fungal growth can be inhibited by exposure to ITCs. However the results observed are specific to the ITC structure and exposure concentration. 相似文献10.
Tuty Asmawaty Abdul Kadir Ahmad A Mannan Andrzej M Kierzek Johnjoe McFadden Kazuyuki Shimizu 《Microbial cell factories》2010,9(1):88
Background
It is quite important to simulate the metabolic changes of a cell in response to the change in culture environment and/or specific gene knockouts particularly for the purpose of application in industry. If this could be done, the cell design can be made without conducting exhaustive experiments, and one can screen out the promising candidates, proceeded by experimental verification of a select few of particular interest. Although several models have so far been proposed, most of them focus on the specific metabolic pathways. It is preferred to model the whole of the main metabolic pathways in Escherichia coli, allowing for the estimation of energy generation and cell synthesis, based on intracellular fluxes and that may be used to characterize phenotypic growth. 相似文献11.
Aims
To investigate the effects of temperature and medium composition on growth/aflatoxin inhibitory activities of terpenoids gossypol, gossypolone and apogossypolone against Aspergillus flavus and A. parasiticus.Methods and Results
The compounds were tested at a concentration of 100 μg ml?1 in a Czapek Dox (Czapek) agar medium at 25, 31 and 37°C. Increased incubation temperature marginally increased growth inhibition caused by these compounds, but reduced the aflatoxin inhibition effected by gossypol. Gossypolone and apogossypolone retained good aflatoxin inhibitory activity against A. flavus and A. parasiticus at higher incubation temperatures. However, increased temperature also significantly reduced aflatoxin production in control cultures. The effects of the terpenoids on fungal growth and aflatoxin production against the same fungi were also determined in Czapek, Czapek with a protein/amino acid addendum and yeast extract sucrose (YES) media. Growth of these fungi in the protein‐supplemented Czapek medium or in the YES medium greatly reduced the growth inhibition effects of the terpenoids. Apogossypolone displayed strong anti‐aflatoxigenic activity in the Czapek medium, but this activity was significantly reduced in the protein‐amended Czapek and YES media. Gossypol, which displayed little to no aflatoxin inhibitory activity in the Czapek medium, did yield significant anti‐aflatoxigenic activity in the YES medium.Conclusions
Incubation temperature and media composition are important parameters involved in the regulation of aflatoxin production in A. flavus and A. parasiticus. These parameters also affect the potency of growth and aflatoxin inhibitory activities of these gossypol‐related compounds against aflatoxigenic fungi.Significance and Impact of the Study
Studies utilizing gossypol‐related compounds as inhibitory agents of biological activities should be interpreted with caution due to compound interaction with multiple components of the test system, especially serum proteins. 相似文献12.
Nipaporn Kanthong Nuanpan Khemnu Sa-Nga Pattanakitsakul Prida Malasit Timothy W Flegel 《BMC microbiology》2010,10(1):14
Background
It is known that insects and crustaceans can carry simultaneous, active infections of two or more viruses without showing signs of disease, but it was not clear whether co-infecting viruses occupied the same cells or different cells in common target tissues. Our previous work showed that successive challenge of mosquito cell cultures followed by serial, split-passage resulted in stabilized cultures with 100% of the cells co-infected with Dengue virus (DEN) and an insect parvovirus (densovirus) (DNV). By addition of Japanese encephalitis virus (JE), we tested our hypothesis that stable, persistent, triple-virus co-infections could be obtained by the same process. 相似文献13.
Niraj Kumar Patrick Gammell Paula Meleady Michael Henry Martin Clynes 《BMC biotechnology》2008,8(1):42
Background
To ensure maximal productivity of recombinant proteins (rP) during production culture it is typical to encourage an initial phase of rapid cell proliferation to achieve high biomass followed by a stationary phase where cellular energies are directed towards production of rP. During many such biphasic cultures, the initial phase of rapid cell growth at 37°C is followed by a growth arrest phase induced through reduction of the culture temperature. Low temperature induced growth arrest is associated with many positive phenotypes including increased productivity, sustained viability and an extended production phase, although the mechanisms regulating these phenotypes during mild hypothermia are poorly understood. 相似文献14.
Boris Wilmes Angelika Hartung Michael Lalk Manuel Liebeke Thomas Schweder Peter Neubauer 《Microbial cell factories》2010,9(1):72
Background
Pseudoalteromonas haloplanktis is a cold-adapted γ-proteobacterium isolated from Antarctic sea ice. It is characterized by remarkably high growth rates at low temperatures. P. haloplanktis is one of the model organisms of cold-adapted bacteria and has been suggested as an alternative host for the soluble overproduction of heterologous proteins which tend to form inclusion bodies in established expression hosts. Despite the progress in establishing P. haloplanktis as an alternative expression host the cell densities obtained with this organism, which is unable to use glucose as a carbon source, are still low. Here we present the first fed-batch cultivation strategy for this auspicious alternative expression host. 相似文献15.
16.
Carmen Di Franco Elena Beccari Tiziana Santini Giuseppe Pisaneschi Giorgio Tecce 《BMC microbiology》2002,2(1):33-15
Background
Bacillus mycoides Flügge, a Gram-positive, non-motile soil bacterium assigned to Bacillus cereus group, grows on agar as chains of cells linked end to end, forming radial filaments curving clock- or counter-clockwise (SIN or DX morphotypes). The molecular mechanism causing asymmetric curving is not known: our working hypothesis considers regulation of filamentous growth as the prerequisite for these morphotypes. 相似文献17.
J. Rivadeneira M. Carina Audisio A.R. Boccaccini A.A. Gorustovich 《Journal of applied microbiology》2013,115(2):604-612
Aims
To assess the antibacterial efficacy of new composite materials developed from microparticles of 45S5 bioactive glass (BG) and agar–gelatin films.Methods and Results
In vitro antibacterial activity was evaluated against Staphylococcus spp. because of the importance of this pathogen in damaged tissues and in failures associated with biomaterial implants. To our knowledge, this is the first paper reporting on the suitable combination of BG and agar–gelatin for bioactive and antibacterial films. Bacterial suspensions up or below 105 CFU ml?1 reflecting situations of wound infection and of noninfection, respectively, were prepared and then put in contact with the biomaterials at 37°C. After 24 and 48 h of incubation, the pH value was measured and the staphylococci strains viability was determined by counting in Mueller–Hinton agar plates. Moreover, the biomaterials were prepared for observation under scanning electron microscopy (SEM). Biocomposites (BCs) showed a strong antibacterial effect against all staphylococci strains tested. Some differences were found depending on the strain, the inoculum size and the contact time. This effect was correlated with an alkalinization of the media. By SEM analyses, no bacterial presence was observed on the surface of BCs in any of the cell concentrations tested at any time.Conclusions
Overall, the coating of 45S5 BG on agar–gelatin films promoted BCs with strong antistaphylococcal activity. The effect was efficient under bacterial concentration up or below 105 CFU ml?1. Additionally, none of the strains were found on BCs surfaces.Significance and Impact of Study
45S5 bioglass/agar–gelatin biocomposite films are reported for the first time. The results suggest a potential application as wound dressing. 相似文献18.
Background
High resolution ultrasonography (HR-US) can monitor the molecular changes and biochemical interactions between proteins in real-time. The aim of this study was to use HR-US to characterize the real-time interactions between plasminogen coated beads and PrPSc and to determine if this approach could be applied to the identification of animals affected by prion diseases. Plasminogen, immobilized to beads, was used as a capturing tool for PrPSc in brain homogenates from scrapie affected sheep and the binding reaction was monitored in real-time in an ultrasonic cell. 相似文献19.
Magnóliade A Campos Marilia S Silva Cláudio P Magalhães Simone G Ribeiro Rafael PD Sarto Eduardo A Vieira Maria F Grossi de Sá 《Microbial cell factories》2008,7(1):1-10
Background
The large-scale production of G-protein coupled receptors (GPCRs) for functional and structural studies remains a challenge. Recent successes have been made in the expression of a range of GPCRs using Pichia pastoris as an expression host. P. pastoris has a number of advantages over other expression systems including ability to post-translationally modify expressed proteins, relative low cost for production and ability to grow to very high cell densities. Several previous studies have described the expression of GPCRs in P. pastoris using shaker flasks, which allow culturing of small volumes (500 ml) with moderate cell densities (OD600 ~15). The use of bioreactors, which allow straightforward culturing of large volumes, together with optimal control of growth parameters including pH and dissolved oxygen to maximise cell densities and expression of the target receptors, are an attractive alternative. The aim of this study was to compare the levels of expression of the human Adenosine 2A receptor (A2AR) in P. pastoris under control of a methanol-inducible promoter in both flask and bioreactor cultures.Results
Bioreactor cultures yielded an approximately five times increase in cell density (OD600 ~75) compared to flask cultures prior to induction and a doubling in functional expression level per mg of membrane protein, representing a significant optimisation. Furthermore, analysis of a C-terminally truncated A2AR, terminating at residue V334 yielded the highest levels (200 pmol/mg) so far reported for expression of this receptor in P. pastoris. This truncated form of the receptor was also revealed to be resistant to C-terminal degradation in contrast to the WT A2AR, and therefore more suitable for further functional and structural studies.Conclusion
Large-scale expression of the A2AR in P. pastoris bioreactor cultures results in significant increases in functional expression compared to traditional flask cultures. 相似文献20.