Construction of a Compact Array of Microplasma Jet Devices and Its Application for Random Mutagenesis of Rhodosporidium toruloides

Themes: Conversion

Keywords: Genome Engineering

Citation

Koh, H.G., Kim, J., Rao, C.V., Park, S.J., Jin, Y.S. Oct. 21, 2023. “Construction of a Compact Array of Microplasma Jet Devices and Its Application for Random Mutagenesis of Rhodosporidium toruloides.” ACS Synthetic Biology. DOI: 10.1021/acssynbio.3c00443.

Overview

Diagrams of the arrays of microplasma jet device and enclosure. (a) Diagrams of the arrays of microplasma jets with different channel lengths (M8: 8 cm, M5: 5 cm, M2: 2 cm). (b) Cutaway view of the array structure, illustrating the fabrication of the rectangular shape microchannels in quartz and the array pitch of 2 mm. (c) Photographs of the 7 × 1 array of microplasma jets with plasma ignition. A glow plasma is established when a sinusoidal voltage having a frequency of 35 kHz is delivered to the device and He is the feedstock gas. (d) Experimental arrangement of microplasma device in operation with agar plate.

A small and efficient DNA mutation-inducing machine was constructed with an array of microplasma jet devices (7 × 1) that can be operated at atmospheric pressure for microbial mutagenesis. Using this machine, we report disruption of a plasmid DNA and generation of mutants of an oleaginous yeast Rhodosporidium toruloides. Specifically, a compact-sized microplasma channel (25 × 20 × 2 mm3) capable of generating an electron density of greater than 1013 cm–3 was constructed to produce reactive species (N2*, N2+, O, OH, and Hα) under helium atmospheric conditions to induce DNA mutagenesis. The length of microplasma channels in the device played a critical role in augmenting both the volume of plasma and the concentration of reactive species. First, we confirmed that microplasma treatment can linearize a plasmid by creating nicks in vitro. Second, we treated R. toruloides cells with a jet device containing 7 microchannels for 5 min; 94.8% of the treated cells were killed, and 0.44% of surviving cells showed different colony colors as compared to their parental colony. Microplasma-based DNA mutation is energy-efficient and can be a safe alternative for inducing mutations compared to conventional methods using toxic mutagens. This compact and scalable device is amenable for industrial strain improvement involving large-scale mutagenesis.

Data

Download (1.8KB) includes:

  • Carotenoid contents
  • Plasma jet relative intensity