New Device for High-Throughput Viability Screening of Flow Biofilms

ABSTRACT Control of biofilms requires rapid methods to identify compounds effective against them and to isolate resistance-compromised mutants for identifying genes involved in enhanced biofilm resistance. While rapid screening methods for microtiter plate well (“static”) biofilms are available, there are no methods for such screening of continuous flow biofilms (“flow biofilms”). Since the latter biofilms more closely approximate natural biofilms, development of a high-throughput (HTP) method for screening them is desirable. We describe here a new method using a device comprised of microfluidic channels and a distributed pneumatic pump (BioFlux) that provides fluid flow to 96 individual biofilms. This device allows fine control of continuous or intermittent fluid flow over a broad range of flow rates, and the use of a standard well plate format provides compatibility with plate readers. We show that use of green fluorescent protein (GFP)-expressing bacteria, staining with propidium iodide, and measurement of fluorescence with a plate reader permit rapid and accurate determination of biofilm viability. The biofilm viability measured with the plate reader agreed with that determined using plate counts, as well as with the results of fluorescence microscope image analysis. Using BioFlux and the plate reader, we were able to rapidly screen the effects of several antimicrobials on the viability of Pseudomonas aeruginosa PAO1 flow biofilms.

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High-Throughput Chemometric Quality Assessment of Extra Virgin Olive Oils Using a Microtiter Plate Reader

Sensors ◽

10.3390/s19194169 ◽

2019 ◽

Vol 19 (19) ◽

pp. 4169

Author(s):

Huihui He ◽

Weiying Lu

Keyword(s):

Quality Assessment ◽

High Throughput ◽

Latent Variables ◽

Scatter Correction ◽

Principal Component ◽

Microtiter Plate ◽

Food Ingredients ◽

Virgin Olive Oils ◽

Olive Oils ◽

Plate Reader

A commercially available microtiter plate reader was applied as a high-throughput counterpart of ultraviolet-visible (UV–Vis) spectrophotometer to identify the producing location of extra virgin olive oils (EVOOs). Multiplicative scatter correction and the first derivative was used to denoise the UV–Vis spectra and eliminate the effects of background drift. The spectra were analyzed using chemometrics methods including the principal component analysis (PCA) and the partial least squares-discriminant analysis (PLS-DA). The PLS-DA model on full spectra using 5 latent variables showed a classification accuracy of 97.92% by cross-validation. The overall results demonstrated that the use of a UV–Vis spectrophotometer based on the microtiter plate reader combined with chemometrics can be applied to the quality assessment of EVOOs. It is demonstrated that the microtiter plate reader can be a high-throughput tool in the quality assessment of food ingredients.

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High-Throughput Growth Assay for Toxoplasma gondii Using Yellow Fluorescent Protein

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.47.1.309-316.2003 ◽

2003 ◽

Vol 47 (1) ◽

pp. 309-316 ◽

Cited By ~ 146

Author(s):

Marc-Jan Gubbels ◽

Catherine Li ◽

Boris Striepen

Keyword(s):

Toxoplasma Gondii ◽

High Throughput ◽

Fluorescent Protein ◽

Yellow Fluorescent Protein ◽

Host Cells ◽

Rapid Screening ◽

Published Data ◽

Parasite Line ◽

Wild Type ◽

Growth Assay

ABSTRACT A high-throughput growth assay for the protozoan parasite Toxoplasma gondii was developed based on a highly fluorescent transgenic parasite line. These parasites are stably transfected with a tandem yellow fluorescent protein (YFP) and are 1,000 times more fluorescent than the wild type. Parasites were inoculated in optical-bottom 384-well culture plates containing a confluent monolayer of host cells, and growth was monitored by using a fluorescence plate reader. The signal was linearly correlated with parasite numbers over a wide array. Direct comparison of the YFP growth assay with the β-galactosidase growth assay by using parasites expressing both reporters demonstrated that the assays' sensitivities were comparable but that the accuracy of the YFP assay was higher, especially at higher numbers of parasites per well. Determination of the 50%-inhibitory concentrations of three known growth-inhibiting drugs (cytochalasin D, pyrimethamine, and clindamycin) resulted in values comparable to published data. The delayed parasite death kinetics of clindamycin could be measured without modification of the assay, making this assay very versatile. Additionally, the temperature-dependent effect of pyrimethamine was assayed in both wild-type and engineered drug-resistant parasites. Lastly, the development of mycophenolic acid resistance after transfection of a resistance gene in T. gondii was followed. In conclusion, the YFP growth assay limits pipetting steps to a minimum, is highly versatile and amendable to automation, and should enable rapid screening of compounds to fulfill the need for more efficient and less toxic antiparasitic drugs.

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Biosensor-based multi-gene pathway optimization for enhancing the production of glycolate

Applied and Environmental Microbiology ◽

10.1128/aem.00113-21 ◽

2021 ◽

Author(s):

Shumin Xu ◽

Linpei Zhang ◽

Shenghu Zhou ◽

Yu Deng

Keyword(s):

High Throughput ◽

High Throughput Screening ◽

Metabolic Flux ◽

Agar Plate ◽

Expression Level ◽

Rapid Screening ◽

Screening Methods ◽

Synthetic Pathway ◽

Gene Pathway ◽

Pathway Optimization

Glycolate is widely used in industry, especially in the fields of chemical cleaning, cosmetics, and medical materials, and has broad market prospects for the future. Recent advances in metabolic engineering and synthetic biology have significantly improved the titer and yield of glycolate. However, an expensive inducer was used in previous studies that is not feasible for use in large-scale industrial fermentations. To constitutively biosynthesize glycolate, the expression level of each gene of the glycolate synthetic pathway needs to be systemically optimized. The main challenge of multi-gene pathway optimization is being able to select or screen the optimum strain from the randomly assembled library by an efficient high-throughput method within a short period of time. To overcome these challenges, we firstly established a glycolate-responsive biosensor and developed agar plate- and 48-well deep well plate-scale high-throughput screening methods for rapid screening of superior glycolate producers from a large library. A total of 22 gradient strength promoter-5′-UTR complexes were randomly cloned upstream of the genes of the glycolate synthetic pathway, generating a large random assembled library. After rounds of screening, the optimum strain was obtained from 6×105 transformants in a week, and it achieved a titer of 40.9 ± 3.7 g/L glycolate in a 5-L bioreactor. Furthermore, high expression levels of the enzymes YcdW and GltA were found to promote glycolate production, whereas AceA has no obvious impact on glycolate production. Overall, the glycolate biosensor-based pathway optimization strategy presented in this work provides a paradigm for other multi-gene pathway optimizations. Importance The use of strong promoters, such as pTrc and T7, to control gene expression not only need adding expensive inducers but also results in excessive protein expression that may be resulting in unbalanced metabolic flux and the waste of cellular building blocks and energy. To balance the metabolic flux of glycolate biosynthesis, the expression level of each gene needs to be systemically optimized in a constitutive manner. However, the lack of a high-throughput screening methods restricted the glycolate synthetic pathway optimization. Our work firstly established a glycolate-response biosensor, then agar plate and 48-well plate scale high-throughput screening methods were developed for rapid screening of optimum pathways from a large library. Finally, we obtained a glycolate producing strain with good biosynthetic performance, and the use of the expensive inducer IPTG was avoided, which broadens our understanding about the mechanism of glycolate synthesis.

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Spectral Unmixing Plate Reader: High-Throughput, High-Precision FRET Assays in Living Cells

SLAS DISCOVERY Advancing Life Sciences ◽

10.1177/1087057116679637 ◽

2016 ◽

Vol 22 (3) ◽

pp. 250-261 ◽

Cited By ~ 5

Author(s):

Tory M. Schaaf ◽

Kurt C. Peterson ◽

Benjamin D. Grant ◽

David D. Thomas ◽

Gregory D. Gillispie

Keyword(s):

High Throughput ◽

High Throughput Screening ◽

Structural Changes ◽

Fluorescent Protein ◽

Fluorescence Emission ◽

Resonance Energy ◽

Living Cells ◽

Spectral Unmixing ◽

Fluorescence Emission Spectrum ◽

Plate Reader

We have developed a microplate reader that records a complete high-quality fluorescence emission spectrum on a well-by-well basis under true high-throughput screening (HTS) conditions. The read time for an entire 384-well plate is less than 3 min. This instrument is particularly well suited for assays based on fluorescence resonance energy transfer (FRET). Intramolecular protein biosensors with genetically encoded green fluorescent protein (GFP) donor and red fluorescent protein (RFP) acceptor tags at positions sensitive to structural changes were stably expressed and studied in living HEK cells. Accurate quantitation of FRET was achieved by decomposing each observed spectrum into a linear combination of four component (basis) spectra (GFP emission, RFP emission, water Raman, and cell autofluorescence). Excitation and detection are both conducted from the top, allowing for thermoelectric control of the sample temperature from below. This spectral unmixing plate reader (SUPR) delivers an unprecedented combination of speed, precision, and accuracy for studying ensemble-averaged FRET in living cells. It complements our previously reported fluorescence lifetime plate reader, which offers the feature of resolving multiple FRET populations within the ensemble. The combination of these two direct waveform-recording technologies greatly enhances the precision and information content for HTS in drug discovery.

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A High-Throughput Mechanical Activator for Cartilage Engineering Enables Rapid Screening of in vitro Response of Tissue Models to Physiological and Supra-Physiological Loads

Cells Tissues Organs ◽

10.1159/000514985 ◽

2021 ◽

pp. 1-19

Author(s):

Elisa Capuana ◽

Davide Marino ◽

Roberto Di Gesù ◽

Vincenzo La Carrubba ◽

Valerio Brucato ◽

...

Keyword(s):

High Throughput ◽

Compressive Loading ◽

Cartilage Degeneration ◽

Early Response ◽

Rapid Screening ◽

New Device ◽

Cartilage Engineering ◽

Physiological Loading ◽

Tgf Β1

Articular cartilage is crucially influenced by loading during development, health, and disease. However, our knowledge of the mechanical conditions that promote engineered cartilage maturation or tissue repair is still incomplete. Current in vitro models that allow precise control of the local mechanical environment have been dramatically limited by very low throughput, usually just a few specimens per experiment. To overcome this constraint, we have developed a new device for the high throughput compressive loading of tissue constructs: the High Throughput Mechanical Activator for Cartilage Engineering (HiT-MACE), which allows the mechanoactivation of 6 times more samples than current technologies. With HiT-MACE we were able to apply cyclic loads in the physiological (e.g., equivalent to walking and normal daily activity) and supra-physiological range (e.g., injurious impacts or extensive overloading) to up to 24 samples in one single run. In this report, we compared the early response of cartilage to physiological and supra-physiological mechanical loading to the response to IL-1β exposure, a common but rudimentary in vitro model of cartilage osteoarthritis. Physiological loading rapidly upregulated gene expression of anabolic markers along the TGF-β1 pathway. Notably, TGF-β1 or serum was not included in the medium. Supra-physiological loading caused a mild catabolic response while IL-1β exposure drove a rapid anabolic shift. This aligns well with recent findings suggesting that overloading is a more realistic and biomimetic model of cartilage degeneration. Taken together, these findings showed that the application of HiT-MACE allowed the use of larger number of samples to generate higher volume of data to effectively explore cartilage mechanobiology, which will enable the design of more effective repair and rehabilitation strategies for degenerative cartilage pathologies.

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Beeporter: tools for high-throughput analyses of pollinator-virus infections

10.22541/au.161884293.36210275/v1 ◽

2021 ◽

Author(s):

Jay Evans ◽

Olubukola Banmeke ◽

Evan Palmer-Young ◽

Yanping Chen ◽

Eugene Ryabov

Keyword(s):

Virus Infection ◽

High Throughput ◽

Honey Bees ◽

High Throughput Screening ◽

Fluorescent Protein ◽

Imaging System ◽

Screening Methods ◽

Cdna Clones ◽

Virus Infections ◽

Deformed Wing Virus

Pollinators are in decline thanks to the combined stresses of disease, pesticides, habitat loss, and climate. Honey bees face numerous pests and pathogens but arguably none are as devastating as Deformed wing virus (DWV). Understanding host-pathogen interactions and virulence of DWV in honey bees is slowed by the lack of cost-effective high-throughput screening methods for viral infection. Currently, analysis of virus infection in bees and their colonies is tedious, requiring a well-equipped molecular biology laboratory and the use of hazardous chemicals. Here we describe cDNA clones of DWV tagged with green fluorescent protein (GFP) or nanoluciferase (nLuc), providing high-throughput detection and quantification of virus infections. GFP fluorescence is recorded non-invasively in living bees via commonly available long-wave UV light sources and a smartphone camera or a standard ultraviolet transilluminator gel imaging system. Nonlethal monitoring with GFP allows high-throughput screening and serves as a direct breeding tool for identifying honey bee parents with increased antivirus resistance. Expression using the nLuc reporter strongly correlates with virus infection levels and is especially sensitive. Using multiple reporters, it is also possible to visualize competition, differential virulence, and host tissue targeting by co-occuring pathogens. Finally, it is possible to directly assess the risk of cross-species ‘spillover’ from honey bees to other pollinators and vice versa.

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High Throughput Screening Methods

10.1039/9781782626770 ◽

2016 ◽

Cited By ~ 4

Keyword(s):

High Throughput ◽

High Throughput Screening ◽

Screening Methods

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Rapid Screening Methods for Bleeding Disorders. A Three Year Survey

Thrombosis and Haemostasis ◽

10.1055/s-0038-1654846 ◽

1964 ◽

Vol 11 (02) ◽

pp. 506-512 ◽

Cited By ~ 1

Author(s):

V. A Lovric ◽

J Margolis

Keyword(s):

Laboratory Tests ◽

Capillary Blood ◽

Screening Tests ◽

Rapid Screening ◽

Screening Methods ◽

Bleeding Disorders ◽

Routine Laboratory ◽

Clotting Time ◽

Kaolin Clotting Time ◽

In Infants And Children

SummaryAn adaptation of “kaolin clotting time” and prothrombin time for use on haemolysed capillary blood provided simple and sensitive screening tests suitable for use in infants and children. A survey of three year’s experience shows that these are reliable routine laboratory tests for detection of latent coagulation disorders.

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