DNA origami requires long scaffold DNA to be aligned with the guidance of short staple DNA strands. Scaffold DNA is produced in Escherichia coli as a form of the M13 bacteriophage by rolling circle amplification (RCA). This study shows that RCA can be reconfigured by reducing phage protein V (pV) expression, improving the production throughput of scaffold DNA by at least 5.66-fold. The change in pV expression was executed by modifying the untranslated region sequence and monitored using a reporter green fluorescence protein fused to pV. In a separate experiment, pV expression was controlled by an inducer. In both experiments, reduced pV expression was correlated with improved M13 bacteriophage production. High-cell-density cultivation was attempted for mass scaffold DNA production, and the produced scaffold DNA was successfully folded into a barrel shape without compromising structural quality. This result suggested that scaffold DNA production throughput can be significantly improved by reprogramming the RCA in E. coli.
Bibliographical noteFunding Information:
Discovery Program through the National Research Foundation of Korea (NRF) funded by Ministry of Science and ICT (NRF-2017M3D1A1039421 and NRF- 2017M3D1A1039423). Funding for open access charge: Future Materials Discovery Program through the National Research Foundation of Korea (NRF) funded by Ministry of Science and ICT (NRF-2017M3D1A1039421 andNRF- 2017M3D1A1039423).
© 2021 The Author(s).
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