期刊
ENGINEERING IN LIFE SCIENCES
卷 11, 期 2, 页码 157-164出版社
WILEY
DOI: 10.1002/elsc.201000043
关键词
Droplet-based bioreactors; Emulsion; High-throughput; Microfluidics; OpdA expression
资金
- Australian National University
- CSIRO Synthetic Enzymes Emerging Science Initiative
- RCUK
- China Scholarship Council through a Chinese Government
- EPSRC [EP/D048664/1, EP/C013174/1] Funding Source: UKRI
- Engineering and Physical Sciences Research Council [EP/D048664/1, EP/C013174/1] Funding Source: researchfish
There is growing demand for high-throughput measurement of biochemical reactions in drug discovery and directed evolution programs. To meet this need, a powerful platform based on droplet-based bioreactors manipulated by microfluidic systems is being developed, which can overcome the limitations of scale and power encountered in conventional screening methods. This paper reports our progress in the synthesis of enzymes and assay of their activity within a microfluidic droplet system. The model system we use involves the organophosphorus hydrolase enzyme OpdA from Agrobacterium radiobacter and a robust microchip made from polymethyl methacrylate (PMMA). Synthesis of OpdA from cognate DNA within water-in-oil droplets was tested using both in-house and commercial in vitro transcription and translation (IVTT) kits. OpdA activity was measured using coumaphos as substrate and by monitoring the fluorescence released by its product, chlorferone. OpdA was demonstrated to be synthesized and assayed within the droplets using the commercial in vitro transcription and translation kit, although the activity measured within the droplets diminished over time, apparently due to leakage of chlorferone out of the droplets.
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