scholarly journals Enzyme‐mediated hydrogel encapsulation of single cells for high‐throughput screening and directed evolution of oxidoreductases

2019 ◽  
Vol 116 (8) ◽  
pp. 1878-1886 ◽  
Author(s):  
Rosario Vanella ◽  
Duy Tien Ta ◽  
Michael A. Nash
Keyword(s):  
Directed Evolution ◽  
High Throughput ◽  
High Throughput Screening ◽  
Single Cells ◽  
Hydrogel Encapsulation

scholarly journals In vitro optoacoustic flow cytometry with light scattering referencing

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Markus Seeger ◽  
Andre C. Stiel ◽  
Vasilis Ntziachristos
Keyword(s):  
Light Scattering ◽  
Directed Evolution ◽  
High Throughput ◽  
High Throughput Screening ◽  
Single Cells ◽  
Light Flux ◽  
Focal Volume ◽  
Optoacoustic Imaging ◽  
Excitation Light

AbstractMorphological and functional optoacoustic imaging is enhanced by dedicated transgene reporters, in analogy to fluorescence methods. The development of optoacoustic reporters using protein engineering and directed evolution would be accelerated by high-throughput in-flow screening for intracellular, genetically encoded, optoacoustic contrast. However, accurate characterization of such contrast is impeded because the optoacoustic signals depend on the cell’s size and position in the flow chamber. We report herein an optoacoustic flow cytometer (OA-FCM) capable of precise measurement of intracellular optoacoustic signals of genetically-encoded chromoproteins in flow. The novel system records light-scattering as a reference for the detected optoacoustic signals in order to account for cell size and position, as well as excitation light flux in the focal volume, which we use to reference the detected optoacoustic signals to enhance the system’s precision. The OA-FCM was calibrated using micrometer-sized particles to showcase the ability to assess in-flow objects in the size range of single-cells. We demonstrate the capabilities of our OA-FCM to identify sub-populations in a mixture of two E. coli stocks expressing different reporter-proteins with a precision of over 90%. High-throughput screening of optoacoustic labels could pave the way for identifying genetically encoded optoacoustic reporters by transferring working concepts of the fluorescence field such as directed evolution and activated cell sorting.

Directed Evolution of P450 Fatty Acid Decarboxylases via High-Throughput Screening Towards Improved Catalytic Activity

2019 ◽  
Author(s):  
Huifang Xu ◽  
Weinan Liang ◽  
Linlin Ning ◽  
Yuanyuan Jiang ◽  
Wenxia Yang ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Fatty Acid ◽  
Directed Evolution ◽  
High Throughput ◽  
High Throughput Screening ◽  
Colorimetric Detection ◽  
Screening Method ◽  
Random Mutagenesis ◽  
Direct Production ◽  
Consumption Activity

P450 fatty acid decarboxylases (FADCs) have recently been attracting considerable attention owing to their one-step direct production of industrially important 1-alkenes from biologically abundant feedstock free fatty acids under mild conditions. However, attempts to improve the catalytic activity of FADCs have met with little success. Protein engineering has been limited to selected residues and small mutant libraries due to lack of an effective high-throughput screening (HTS) method. Here, we devise a catalase-deficient <i>Escherichia coli</i> host strain and report an HTS approach based on colorimetric detection of H<sub>2</sub>O<sub>2</sub>-consumption activity of FADCs. Directed evolution enabled by this method has led to effective identification for the first time of improved FADC variants for medium-chain 1-alkene production from both DNA shuffling and random mutagenesis libraries. Advantageously, this screening method can be extended to other enzymes that stoichiometrically utilize H<sub>2</sub>O<sub>2</sub> as co-substrate.

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 Photoclick‐Based High‐Throughput Screening for the Directed Evolution of Decarboxylase OleT

2020 ◽  
Author(s):  
Ulrich Markel ◽  
Pia Lanvers ◽  
Daniel F. Sauer ◽  
Malte Wittwer ◽  
Gaurao V. Dhoke ◽  
...  
Keyword(s):  
Directed Evolution ◽  
High Throughput ◽  
High Throughput Screening

scholarly journals High-Throughput Screening for Terpene-Synthase-Cyclization Activity and Directed Evolution of a Terpene Synthase

2013 ◽  
Vol 52 (21) ◽  
pp. 5571-5574 ◽  
Author(s):  
Ryan Lauchli ◽  
Kersten S. Rabe ◽  
Karolina Z. Kalbarczyk ◽  
Amulya Tata ◽  
Thomas Heel ◽  
...  
Keyword(s):  
Directed Evolution ◽  
High Throughput ◽  
High Throughput Screening ◽  
Terpene Synthase

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

Flow Cytometer-Based High-Throughput Screening System for Accelerated Directed Evolution of P450 Monooxygenases

ACS Catalysis ◽  
2012 ◽  
Vol 2 (12) ◽  
pp. 2724-2728 ◽  
Author(s):  
Anna Joëlle Ruff ◽  
Alexander Dennig ◽  
Georgette Wirtz ◽  
Milan Blanusa ◽  
Ulrich Schwaneberg
Keyword(s):  
Directed Evolution ◽  
High Throughput ◽  
High Throughput Screening ◽  
Flow Cytometer ◽  
Screening System ◽  
P450 Monooxygenases

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.

High-throughput screening methodology for the directed evolution of glycosyltransferases

2006 ◽  
Vol 3 (8) ◽  
pp. 609-614 ◽  
Author(s):  
Amir Aharoni ◽  
Karena Thieme ◽  
Cecilia P C Chiu ◽  
Sabrina Buchini ◽  
Luke L Lairson ◽  
...  
Keyword(s):  
Directed Evolution ◽  
High Throughput ◽  
High Throughput Screening
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