Optimizing protein V untranslated region sequence in M13 phage for increased production of single-stranded DNA for origami |
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| Authors: | Bo-Young Lee Jaewon Lee Dong June Ahn Seungwoo Lee Min-Kyu Oh |
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| Institution: | Department of Chemical & Biological Engineering, Korea University, Seoul 02841, Republic of Korea;KU-KIST Graduate School of Converging Science and Technology, Korea University, Seoul 02841, Republic of Korea;Department of Integrative Energy Engineering, Korea University, Seoul 02841, Republic of Korea;The w:i Interface Augmentation Center, Korea University, Seoul, 02841,Republic of Korea |
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| Abstract: | 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. |
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