Enhanced avermectin production by Streptomyces avermitilis ATCC 31267 using high-throughput screening aided by fluorescence-activated cell sorting

2017 ◽  
Vol 102 (2) ◽  
pp. 703-712 ◽  
Author(s):  
Xiaomei Cao ◽  
Zhengshan Luo ◽  
Weizhu Zeng ◽  
Sha Xu ◽  
Liqing Zhao ◽  
...  
Keyword(s):  
High Throughput ◽  
Cell Sorting ◽  
High Throughput Screening ◽  
Streptomyces Avermitilis ◽  
Fluorescence Activated Cell Sorting

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 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.

An integrated high-throughput microfluidic circulatory fluorescence-activated cell sorting system (μ-CFACS) for the enrichment of rare cells

Lab on a Chip ◽  
2021 ◽  
Author(s):  
Kunpeng Cai ◽  
Shruti Mankar ◽  
Taiga Ajiri ◽  
Kentaro Shirai ◽  
Tasuku Yotoriyama
Keyword(s):  
Personalized Medicine ◽  
Regenerative Medicine ◽  
Precision Medicine ◽  
High Throughput ◽  
Cell Sorting ◽  
Next Generation ◽  
Fluorescence Activated Cell Sorting ◽  
Clinical Environments ◽  
Rare Cells ◽  
Sorting System

There is an increasing need for the enrichment of rare cells in the clinical environments of precision medicine, personalized medicine, and regenerative medicine. With the possibility of becoming the next-generation...

scholarly journals Ultra-high throughput functional enrichment of large monoamine oxidase (MAO-N) libraries by fluorescence activated cell sorting

The Analyst ◽  
2018 ◽  
Vol 143 (19) ◽  
pp. 4747-4755 ◽  
Author(s):  
Joanna C. Sadler ◽  
Andrew Currin ◽  
Douglas B. Kell
Keyword(s):  
Monoamine Oxidase ◽  
Directed Evolution ◽  
High Throughput ◽  
Cell Sorting ◽  
Oxidase Activity ◽  
Functional Enrichment ◽  
High Throughput Screen ◽  
Fluorescence Activated Cell Sorting ◽  
E Coli

A novel ultra-high throughput screen forin vivodetection of oxidase activity inE. colicells and its application to directed evolution.

scholarly journals High-throughput fluorescence-activated cell sorting for lipid hyperaccumulatingChlamydomonas reinhardtiimutants

2014 ◽  
Vol 12 (7) ◽  
pp. 872-882 ◽  
Author(s):  
Bo Xie ◽  
Dan Stessman ◽  
Jason H. Hart ◽  
Haili Dong ◽  
Yingjun Wang ◽  
...  
Keyword(s):  
High Throughput ◽  
Cell Sorting ◽  
Fluorescence Activated Cell Sorting

scholarly journals Cyto-Mine: An Integrated, Picodroplet System for High-Throughput Single-Cell Analysis, Sorting, Dispensing, and Monoclonality Assurance

2020 ◽  
Vol 25 (2) ◽  
pp. 177-189 ◽  
Author(s):  
Dimitris Josephides ◽  
Serena Davoli ◽  
William Whitley ◽  
Raphael Ruis ◽  
Robert Salter ◽  
...  
Keyword(s):  
Single Cell ◽  
Cell Line ◽  
High Throughput ◽  
Cell Sorting ◽  
High Throughput Screening ◽  
Single Cell Analysis ◽  
Microtiter Plate ◽  
Cell Analysis ◽  
Fully Integrated ◽  
Clonal Cell Lines

The primary goal of bioprocess cell line development is to obtain high product yields from robustly growing and well-defined clonal cell lines in timelines measured in weeks rather than months. Likewise, high-throughput screening of B cells and hybridomas is required for most cell line engineering workflows. A substantial bottleneck in these processes is detecting and isolating rare clonal cells with the required characteristics. Traditionally, this was achieved by the resource-intensive method of limiting dilution cloning, and more recently aided by semiautomated technologies such as cell sorting (e.g., fluorescence-activated cell sorting) and colony picking. In this paper we report on our novel Cyto-Mine Single Cell Analysis and Monoclonality Assurance System, which overcomes the limitations of current technologies by screening hundreds of thousands of individual cells for secreted target proteins, and then isolating and dispensing the highest producers into microtiter plate wells (MTP). The Cyto-Mine system performs this workflow using a fully integrated, microfluidic Cyto-Cartridge. Critically, all reagents and Cyto-Cartridges used are animal component-free (ACF) and sterile, thus allowing fast, robust, and safe isolation of desired cells.

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