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Optimizing protein V untranslated region sequence in M13 phage for increased production of single-stranded DNA for origami

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) ex...

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Autores principales: Lee, Bo-Young, Lee, Jaewon, Ahn, Dong June, Lee, Seungwoo, Oh, Min-Kyu
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8216270/
https://www.ncbi.nlm.nih.gov/pubmed/34110422
http://dx.doi.org/10.1093/nar/gkab455
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author Lee, Bo-Young
Lee, Jaewon
Ahn, Dong June
Lee, Seungwoo
Oh, Min-Kyu
author_facet Lee, Bo-Young
Lee, Jaewon
Ahn, Dong June
Lee, Seungwoo
Oh, Min-Kyu
author_sort Lee, Bo-Young
collection PubMed
description 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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spelling pubmed-82162702021-06-22 Optimizing protein V untranslated region sequence in M13 phage for increased production of single-stranded DNA for origami Lee, Bo-Young Lee, Jaewon Ahn, Dong June Lee, Seungwoo Oh, Min-Kyu Nucleic Acids Res Synthetic Biology and Bioengineering 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. Oxford University Press 2021-06-10 /pmc/articles/PMC8216270/ /pubmed/34110422 http://dx.doi.org/10.1093/nar/gkab455 Text en © The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research. https://creativecommons.org/licenses/by-nc/4.0/This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (http://creativecommons.org/licenses/by-nc/4.0/ (https://creativecommons.org/licenses/by-nc/4.0/) ), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com
spellingShingle Synthetic Biology and Bioengineering
Lee, Bo-Young
Lee, Jaewon
Ahn, Dong June
Lee, Seungwoo
Oh, Min-Kyu
Optimizing protein V untranslated region sequence in M13 phage for increased production of single-stranded DNA for origami
title Optimizing protein V untranslated region sequence in M13 phage for increased production of single-stranded DNA for origami
title_full Optimizing protein V untranslated region sequence in M13 phage for increased production of single-stranded DNA for origami
title_fullStr Optimizing protein V untranslated region sequence in M13 phage for increased production of single-stranded DNA for origami
title_full_unstemmed Optimizing protein V untranslated region sequence in M13 phage for increased production of single-stranded DNA for origami
title_short Optimizing protein V untranslated region sequence in M13 phage for increased production of single-stranded DNA for origami
title_sort optimizing protein v untranslated region sequence in m13 phage for increased production of single-stranded dna for origami
topic Synthetic Biology and Bioengineering
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8216270/
https://www.ncbi.nlm.nih.gov/pubmed/34110422
http://dx.doi.org/10.1093/nar/gkab455
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