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

Cited 4 time in scopus
Metadata Downloads
Multi-enzyme screening using a high-throughput genetic enzyme screening system
Haseong KimKil Koang Kwon; Wonjae Seong; Seung Goo Lee
Bibliographic Citation
Journal of Visualized Experiments, vol. 2016, no. 114, pp. e54059-e54059
Publication Year
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
DmpREnzyme screeningGenetic circuitsHigh-throughput screeningIssue 114MetagenomeMolecular biologyPhenolic compound
Journal of Visualized Experiments
Appears in Collections:
Synthetic Biology and Bioengineering Research Institute > Synthetic Biology Research Center > 1. Journal Articles
Synthetic Biology and Bioengineering Research Institute > 1. Journal Articles
Files in This Item:
  • There are no files associated with this item.

Items in OpenAccess@KRIBB are protected by copyright, with all rights reserved, unless otherwise indicated.