Multi-enzyme screening using a high-throughput genetic enzyme screening system

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Title
Multi-enzyme screening using a high-throughput genetic enzyme screening system
Author(s)
Haseong KimKil Koang Kwon; Wonjae Seong; Seung Goo Lee
Bibliographic Citation
Journal of Visualized Experiments, vol. 2016, no. 114, pp. e54059-e54059
Publication Year
2016
Abstract
The recent development of a high-throughput single-cell assay technique enables the screening of novel enzymes based on functional activities from a large-scale metagenomic library1. We previously proposed a genetic enzyme screening system (GESS) that uses dimethylphenol regulator activated by phenol or p-nitrophenol. Since a vast amount of natural enzymatic reactions produce these phenolic compounds from phenol deriving substrates, this single genetic screening system can be theoretically applied to screen over 200 different enzymes in the BRENDA database. Despite the general applicability of GESS, applying the screening process requires a specific procedure to reach the maximum flow cytometry signals. Here, we detail the developed screening process, which includes metagenome preprocessing with GESS and the operation of a flow cytometry sorter. Three different phenolic substrates (p-nitrophenyl acetate, p-nitrophenyl-β-D-cellobioside, and phenyl phosphate) with GESS were used to screen and to identify three different enzymes (lipase, cellulase, and alkaline phosphatase), respectively. The selected metagenomic enzyme activities were confirmed only with the flow cytometry but DNA sequencing and diverse in vitro analysis can be used for further gene identification
Keyword
DmpREnzyme screeningGenetic circuitsHigh-throughput screeningIssue 114MetagenomeMolecular biologyPhenolic compound
ISSN
1940-087X
Publisher
Journal of Visualized Experiments
DOI
http://dx.doi.org/10.3791/54059
Type
Article
Appears in Collections:
Division of Biomaterials Research > Synthetic Biology and Bioengineering Research Center > 1. Journal Articles
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