Kil Koang KwonView all speakers
Kil Koang Kwon received his BS/MS at the Department of Microbial Engineering at Konkuk University in South Korea. He is currently completing his PhD in the Department of Chemical & Biomolecular Engineering at Korea Advanced Institute of Science and Technology (KAIST). He is also working at the Biochemicals & Synthetic Biology Research Center in Korea Research Institute of Bioscience & Biotechnology (KRIBB).
Enzyme engineering and synthetic biology are his main research interests, so he is currently working on the applications of genetic circuits along with protein engineering.
Large-scale screening of enzyme libraries is essential for the developments of cost-effective biological processes, which will be indispensable for the sustainable green chemistry. Here, we report a single cell based screening using a genetic circuit that enables the cell to respond quantitatively to aromatic molecules, which is released from substrates by the activity of target enzymes. The genetic circuit consists of two AND gates where the first one is turned on by a target enzyme and a substrate with an aromatic side chain. The aromatic molecule, in turn, could be an input signal of the second AND gate and it is turned ON by a sensor protein which initiates the transcription of a GFP fluorescent reporter gene after the recognition of aromatic molecules. We used a bacterial regulator, dmpR, as the phenol sensor protein which could be replaced by other effector binding genes such as tbuT if the effector is toluene. In cells harboring this genetic circuit, diverse enzyme activities, such as those of tyrosine phenol-lyase, lipase, cellulase, and methyl parathion hydrolase, were detected by the fluorescence emission when phenol- or nitrophenol-derivatized substrates of these enzymes were supplied. Along with these cells, a high throughput flow cytometry was successfully used to isolate a novel phosphatase from a metagenome library derived from tidal flat sediment using phenyl phosphate as a substrate. Our result shows that the genetic circuit provides a widely applicable tool for high throughput and quantitative screening of diverse activities from large-scale enzyme libraries. In addition, quantitative part characterization of the circuit by analyzing their fluorescence changes would provide useful information for further plug and play circuit design.