scholarly journals High-throughput screening of high lactic acid-producing Bacillus coagulans by droplet microfluidic based flow cytometry with fluorescence activated cell sorting

RSC Advances ◽  
2019 ◽  
Vol 9 (8) ◽  
pp. 4507-4513 ◽  
Author(s):  
Xu-Dong Zhu ◽  
Xiang Shi ◽  
Shu-Wen Wang ◽  
Ju Chu ◽  
Wei-Hong Zhu ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Lactic Acid ◽  
High Throughput ◽  
Cell Sorting ◽  
High Throughput Screening ◽  
Bacillus Coagulans ◽  
Screening System ◽  
Fluorescence Activated Cell Sorting ◽  
Microfluidic Sorting

A high-throughput screening system based on droplet microfluidic sorting was developed and employed for screening of high lactic acid-producing Bacillus coagulans.

scholarly journals A High-Throughput Screening System Based on Fluorescence-Activated Cell Sorting for the Directed Evolution of Chitinase A

2021 ◽  
Vol 22 (6) ◽  
pp. 3041
Author(s):  
Gheorghita Menghiu ◽  
Vasile Ostafe ◽  
Radivoje Prodanović ◽  
Rainer Fischer ◽  
Raluca Ostafe
Keyword(s):  
High Throughput ◽  
Cell Sorting ◽  
High Throughput Screening ◽  
Screening Method ◽  
Enzymatic Reaction ◽  
Hydrolytic Enzymes ◽  
Screening System ◽  
Fluorescence Activated Cell Sorting ◽  
Fungal Cell ◽  
Chitinase A

Chitinases catalyze the degradation of chitin, a polymer of N-acetylglucosamine found in crustacean shells, insect cuticles, and fungal cell walls. There is great interest in the development of improved chitinases to address the environmental burden of chitin waste from the food processing industry as well as the potential medical, agricultural, and industrial uses of partially deacetylated chitin (chitosan) and its products (chito-oligosaccharides). The depolymerization of chitin can be achieved using chemical and physical treatments, but an enzymatic process would be more environmentally friendly and more sustainable. However, chitinases are slow-acting enzymes, limiting their biotechnological exploitation, although this can be overcome by molecular evolution approaches to enhance the features required for specific applications. The two main goals of this study were the development of a high-throughput screening system for chitinase activity (which could be extrapolated to other hydrolytic enzymes), and the deployment of this new method to select improved chitinase variants. We therefore cloned and expressed the Bacillus licheniformis DSM8785 chitinase A (chiA) gene in Escherichia coli BL21 (DE3) cells and generated a mutant library by error-prone PCR. We then developed a screening method based on fluorescence-activated cell sorting (FACS) using the model substrate 4-methylumbelliferyl β-d-N,N′,N″-triacetyl chitotrioside to identify improved enzymes. We prevented cross-talk between emulsion compartments caused by the hydrophobicity of 4-methylumbelliferone, the fluorescent product of the enzymatic reaction, by incorporating cyclodextrins into the aqueous phases. We also addressed the toxicity of long-term chiA expression in E. coli by limiting the reaction time. We identified 12 mutants containing 2–8 mutations per gene resulting in up to twofold higher activity than wild-type ChiA.

Fluorescence Activated Cell Sorting as a General Ultra-High-Throughput Screening Method for Directed Evolution of Glycosyltransferases

2010 ◽  
Vol 132 (30) ◽  
pp. 10570-10577 ◽  
Author(s):  
Guangyu Yang ◽  
Jamie R. Rich ◽  
Michel Gilbert ◽  
Warren W. Wakarchuk ◽  
Yan Feng ◽  
...  
Keyword(s):  
Directed Evolution ◽  
High Throughput ◽  
Cell Sorting ◽  
High Throughput Screening ◽  
Screening Method ◽  
Fluorescence Activated Cell Sorting

scholarly journals A Fully Automated High-Throughput Flow Cytometry Screening System Enabling Phenotypic Drug Discovery

2018 ◽  
Vol 23 (7) ◽  
pp. 697-707 ◽  
Author(s):  
John Joslin ◽  
James Gilligan ◽  
Paul Anderson ◽  
Catherine Garcia ◽  
Orzala Sharif ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Drug Discovery ◽  
High Throughput ◽  
High Throughput Screening ◽  
Screening System ◽  
Preclinical Development ◽  
Drug Identification ◽  
Therapeutic Setting ◽  
Compound Libraries ◽  
Automated Platform

The goal of high-throughput screening is to enable screening of compound libraries in an automated manner to identify quality starting points for optimization. This often involves screening a large diversity of compounds in an assay that preserves a connection to the disease pathology. Phenotypic screening is a powerful tool for drug identification, in that assays can be run without prior understanding of the target and with primary cells that closely mimic the therapeutic setting. Advanced automation and high-content imaging have enabled many complex assays, but these are still relatively slow and low throughput. To address this limitation, we have developed an automated workflow that is dedicated to processing complex phenotypic assays for flow cytometry. The system can achieve a throughput of 50,000 wells per day, resulting in a fully automated platform that enables robust phenotypic drug discovery. Over the past 5 years, this screening system has been used for a variety of drug discovery programs, across many disease areas, with many molecules advancing quickly into preclinical development and into the clinic. This report will highlight a diversity of approaches that automated flow cytometry has enabled for phenotypic drug discovery.

Improvement in oxidative stability of versatile peroxidase by flow cytometry-based high-throughput screening system

2020 ◽  
Vol 157 ◽  
pp. 107555
Author(s):  
Karla Ilić Đurđić ◽  
Selin Ece ◽  
Raluca Ostafe ◽  
Simon Vogel ◽  
Stefan Schillberg ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Oxidative Stability ◽  
High Throughput ◽  
High Throughput Screening ◽  
Versatile Peroxidase ◽  
Screening System

scholarly journals Flow cytometry based high throughput screening system for screening and improving industrially important enzymes

2009 ◽  
Vol 25 ◽  
pp. S144
Author(s):  
R. Tu ◽  
R. Prodanovic ◽  
M. Blanusa ◽  
R. Ostafe ◽  
F. Niehaus ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
High Throughput ◽  
High Throughput Screening ◽  
Screening System

scholarly journals Evaluation of Mosquito Attractant Candidates Using a High-Throughput Screening System for Aedes aegypti (L.), Culex quinquefasciatus Say. and Anopheles minimus Theobald (Diptera: Culicidae)

Insects ◽  
2021 ◽  
Vol 12 (6) ◽  
pp. 528
Author(s):  
Dae-Yun Kim ◽  
Theerachart Leepasert ◽  
Michael J. Bangs ◽  
Theeraphap Chareonviriyaphap
Keyword(s):  
Lactic Acid ◽  
Aedes Aegypti ◽  
High Throughput ◽  
Culex Quinquefasciatus ◽  
High Throughput Screening ◽  
Mosquito Species ◽  
Laboratory Strain ◽  
Screening System ◽  
Anopheles Minimus ◽  
Significant Difference

Several types of olfactometers have been used to evaluate mosquito responses to agents that mimic natural volatiles that repel or attract. The Y-tube olfactometer has been widely used to study repellents and attractants, while the high-throughput screening system assay has only been used to study repellents. Whether the high-throughput screening system assay is suitable for evaluating attractants is unknown. We evaluated the responses to four lactic-acid-based mixtures and two non-lactic-acid-based chemical lure candidates using the high-throughput screening system (HITSS) for three mosquito species (laboratory strains and field populations of both Aedes aegypti (L.) and Culex quinquefasciatus Say.; laboratory strain of Anopheles minimus Theobald) under laboratory-controlled conditions. HITSS assay results showed that KU-lure #1 elicited the greatest percent attraction for pyrethroid-resistant and -susceptible Ae. aegypti. KU-lure #6 elicited the strongest attractive response for pyrethroid-susceptible and -resistant Cx. quinquefasciatus and pyrethroid-susceptible An. minimus. The response to the lures from each species was independent of the pyrethroid susceptibility status (Ae. aegypti, p = 0.825; Cx. quinquefasciatus, p = 0.056). However, a significant difference in attraction to KU-lure #6 was observed between diurnal and nocturnal mosquitoes (Cx. quinquefasciatus vs. Ae. aegypti, p = 0.014; An. minimus vs. Ae. aegypti, p = 0.001). The laboratory-level HITSS assay effectively selects potential lure candidates. Because the host-seeking behavior differs between mosquito species, further studies are needed to develop species-specific attractants. Additional studies in semi-field screen houses using commercial traps are necessary to evaluate the accuracy of these laboratory assay results.

scholarly journals Creation of Novel Protein Transduction Domain (PTD) Mutants by a Phage Display-Based High-Throughput Screening System

2006 ◽  
Vol 29 (8) ◽  
pp. 1570-1574 ◽  
Author(s):  
Yohei Mukai ◽  
Toshiki Sugita ◽  
Tomoko Yamato ◽  
Natsue Yamanada ◽  
Hiroko Shibata ◽  
...  
Keyword(s):  
Phage Display ◽  
High Throughput ◽  
High Throughput Screening ◽  
Protein Transduction ◽  
Protein Transduction Domain ◽  
Screening System ◽  
Novel Protein
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