scholarly journals Fluorescence-Activated Droplet Sorting of Polyethylene terephthalate Degrading Enzymes

2021 ◽  
Author(s):  
Yuxin Qiao ◽  
Ran Hu ◽  
Dongwei Chen ◽  
Li Wang ◽  
Ye Fu ◽  
...  
Keyword(s):  
Polyethylene Terephthalate ◽  
Staphylococcus Epidermidis ◽  
High Throughput Screening ◽  
Single Cells ◽  
Critical Factors ◽  
Textile Mill ◽  
Factors Associated ◽  
Specificity And Sensitivity ◽  
Fluorogenic Probe ◽  
Droplet Sorting

It is a great challenge to expand the spectrum of enzymes that can decompose synthetic plastics such as Polyethylene terephthalate (PET). However, a bottleneck remains due to the lack of techniques for detecting and sorting environmental microorganisms with vast diversity and abundance. Here, we developed a fluorescence-activated droplet sorting (FADS) pipeline for high-throughput screening of PET-degrading microorganisms or enzymes (PETases). The pipeline comprises three steps: generation of droplets encapsulating single cells, picoinjection of Fluorescein dibenzoate (FDBz) as the fluorogenic probe, and screening of droplets to obtain PET-degrading cells. We characterized critical factors associated with the method, including specificity and sensitivity for discriminating PETase from other enzymes, and optimized its performance and compatibility with environmental samples. The system was used to screen an environmental sample from a PET textile mill, successfully obtained PET-degrading species belonging to 9 different genera. Moreover, two putative PETases from isolates Kineococcus endophyticus Un-5 and Staphylococcus epidermidis Un-C2-8 were genetically derived, heterologously expressed, and preliminarily validated for PET-degrading activities. We speculate the FADS pipeline can be widely adopted to discover new PET-degrading microorganisms and enzymes from various environments, as well as directed evolution of PETases using synthetic biology.

scholarly journals Raman-Activated Droplet Sorting (RADS) for Label-Free High-Throughput Screening of Microalgal Single-Cells

2017 ◽  
Vol 89 (22) ◽  
pp. 12569-12577 ◽  
Author(s):  
Xixian Wang ◽  
Lihui Ren ◽  
Yetian Su ◽  
Yuetong Ji ◽  
Yaoping Liu ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Single Cells ◽  
Label Free ◽  
Droplet Sorting

scholarly journals High-Throughput Screening of Higher Α-Amylase Producing Bacillus Licheniformis by Fluorescence-Activated Droplet Sorting

2021 ◽  
Author(s):  
Huiling Yuan ◽  
Ran Tu ◽  
Xinwei Tong ◽  
Yuping Lin ◽  
Qinhong Wang
Keyword(s):  
Bacillus Licheniformis ◽  
High Throughput ◽  
High Throughput Screening ◽  
Single Cells ◽  
Random Mutagenesis ◽  
Strain Improvement ◽  
Industrial Applications ◽  
Cell Factory ◽  
Temperature Plasma ◽  
Droplet Sorting

Abstract Backgroundα-Amylases is one of the most important starch degrading enzymes and has the widest range of industrial applications. Bacillus licheniformis has been widely used as a cell factory for industrial production of amylase. However, difficulties in genetic modification of B. licheniformis have limited its widespread use. Directed evolution, based on the combination of random mutagenesis and high throughput screening (HTS), has been proven an effective strategy in strain improvement for increasing the productivity, but it requires a suitable HTS system to screen the desired mutants. Droplet-based microfluidics has emerged as a powerful tool for single-cell screening with ultra-high throughput, however, the accessibility of a droplet microfluidic HTS platform to users having no background in microfluidics is still an issue. ResultsHere, we first developed a microfluidic HTS platform based on fluorescence-activated droplet sorting (FADS) technology. This platform allowed (i) encapsulation of single cells in monodisperse water-in-oil droplets; (ii) cell growth and protein production in droplets; (iii) sorting of droplets based on their fluorescence intensities. To validate the platform, a model selection experiment of a binary mixture of Bacillus strains was performed and a 45.6-fold enrichment was achieved at a sorting rate of 300 droplets per second. Furthermore, we used the platform for the selection of higher α-amylase-producing strains from a library of B. licheniformis strains (a strain already used at industrial-scale). The B. licheniformis mutant library was generated by atmospheric and room temperature plasma (ARTP) mutagenesis. The clones displaying over 50% improvement in α-amylases productivity compared to the wild-type were isolated.ConclusionsWe established an efficient droplet microfluidic platform which consisted of droplet generation, droplet incubation, and sorting of droplets with a throughput of up to 1 × 106 droplets per h. The screening platform was demonstrated by successfully identifying B. licheniformis clones with improved α-amylase production. We believe that the droplet platform could be extended to the development of other industrially valuable strains.

Risk Score Based on Critical Factors Associated with COVID-19

2021 ◽  
Vol 19 (6) ◽  
pp. 1090-1098
Author(s):  
Rafael Martinez Pelaez ◽  
Josue Roberto Bermeo Escalona ◽  
Jose Luis Ayala Herrera ◽  
Solange Ivette Rivera Manrique ◽  
Abraham Sanchez Ruiz ◽  
...  
Keyword(s):  
Risk Score ◽  
Critical Factors ◽  
Factors Associated

Encapsulation of single cells on a microfluidic device integrating droplet generation with fluorescence-activated droplet sorting

2013 ◽  
Vol 15 (3) ◽  
pp. 553-560 ◽  
Author(s):  
Liang Wu ◽  
Pu Chen ◽  
Yingsong Dong ◽  
Xiaojun Feng ◽  
Bi-Feng Liu
Keyword(s):  
Microfluidic Device ◽  
Single Cells ◽  
Droplet Generation ◽  
Droplet Sorting

scholarly journals Plasma Biomarkers: Potent Screeners of Alzheimer’s Disease

2019 ◽  
Vol 34 (5) ◽  
pp. 290-301 ◽  
Author(s):  
Muhammad Naveed ◽  
Shamsa Mubeen ◽  
Abeer Khan ◽  
Sehrish Ibrahim ◽  
Bisma Meer
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Plasma Proteins ◽  
Great Accuracy ◽  
Vital Role ◽  
Brain Cell ◽  
Plasma Biomarkers ◽  
Factors Associated ◽  
Specificity And Sensitivity ◽  
Complex Chronic Disease

Alzheimer’s disease (AD), a neurological disorder, is as a complex chronic disease of brain cell death that usher to cognitive decline and loss of memory. Its prevalence differs according to risk factors associated with it and necropsy performs vital role in its definite diagnosis. The stages of AD vary from preclinical to severe that proceeds to death of patient with no availability of treatment. Biomarker may be a biochemical change that can be recognized by different emerging technologies such as proteomics and metabolomics. Plasma biomarkers, 5-protein classifiers, are readily being used for the diagnosis of AD and can also predict its progression with a great accuracy, specificity, and sensitivity. In this review, upregulation or downregulation of few plasma proteins in patients with AD has also been discussed, when juxtaposed with control, and thus serves as potent biomarker in the diagnosis of AD.

scholarly journals Mitochondrial Correlation Microscopy and Nanolaser Spectroscopy — New Tools for Biophotonic Detection of Cancer in Single Cells

2005 ◽  
Vol 4 (6) ◽  
pp. 585-592 ◽  
Author(s):  
Paul L. Gourley ◽  
Judy K. Hendricks ◽  
Anthony E. McDonald ◽  
R. Guild Copeland ◽  
Keith E. Barrett ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Tumor Cells ◽  
High Throughput Screening ◽  
Laser Scanning ◽  
Spatial Organization ◽  
Early Stage ◽  
Single Cells ◽  
Normal Liver ◽  
Diagnosis And Treatment ◽  
Optical Methods

Currently, pathologists rely on labor-intensive microscopic examination of tumor cells using century-old staining methods that can give false readings. Emerging BioMicroNano-technologies have the potential to provide accurate, realtime, high-throughput screening of tumor cells without the need for time-consuming sample preparation. These rapid, nanooptical techniques may play an important role in advancing early detection, diagnosis, and treatment of disease. In this report, we show that laser scanning confocal microscopy can be used to identify a previously unknown property of certain cancer cells that distinguishes them, with single-cell resolution, from closely related normal cells. This property is the correlation of light scattering and the spatial organization of mitochondria. In normal liver cells, mitochondria are highly organized within the cytoplasm and highly scattering, yielding a highly correlated signal. In cancer cells, mitochondria are more chaotically organized and poorly scattering. These differences correlate with important bioenergetic disturbances that are hallmarks of many types of cancer. In addition, we review recent work that exploits the new technology of nanolaser spectroscopy using the biocavity laser to characterize the unique spectral signatures of normal and transformed cells. These optical methods represent powerful new tools that hold promise for detecting cancer at an early stage and may help to limit delays in diagnosis and treatment.

scholarly journals Assay Establishment and Validation of a High-Throughput Screening Platform for Three-Dimensional Patient-Derived Colon Cancer Organoid Cultures

2016 ◽  
Vol 21 (9) ◽  
pp. 931-941 ◽  
Author(s):  
Karsten Boehnke ◽  
Philip W. Iversen ◽  
Dirk Schumacher ◽  
María José Lallena ◽  
Rubén Haro ◽  
...  
Keyword(s):  
Colon Cancer ◽  
Drug Discovery ◽  
High Throughput ◽  
High Throughput Screening ◽  
Single Cells ◽  
Three Dimensional ◽  
Compound Screening ◽  
Organoid Cultures ◽  
Screening Platform ◽  
Disease Specific

The application of patient-derived three-dimensional culture systems as disease-specific drug sensitivity models has enormous potential to connect compound screening and clinical trials. However, the implementation of complex cell-based assay systems in drug discovery requires reliable and robust screening platforms. Here we describe the establishment of an automated platform in 384-well format for three-dimensional organoid cultures derived from colon cancer patients. Single cells were embedded in an extracellular matrix by an automated workflow and subsequently self-organized into organoid structures within 4 days of culture before being exposed to compound treatment. We performed validation of assay robustness and reproducibility via plate uniformity and replicate-experiment studies. After assay optimization, the patient-derived organoid platform passed all relevant validation criteria. In addition, we introduced a streamlined plate uniformity study to evaluate patient-derived colon cancer samples from different donors. Our results demonstrate the feasibility of using patient-derived tumor samples for high-throughput assays and their integration as disease-specific models in drug discovery.

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