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Seamless Insert-Plasmid Assembly at High Efficiency and Low Cost
Seamless cloning methods, such as co-transformation cloning, sequence- and ligation-independent cloning (SLIC) or the Gibson assembly, are essential tools for the precise construction of plasmids. The efficiency of co-transformation cloning is however low and the Gibson assembly reagents are expensi...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2016
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4830597/ https://www.ncbi.nlm.nih.gov/pubmed/27073895 http://dx.doi.org/10.1371/journal.pone.0153158 |
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author | Benoit, Roger M. Ostermeier, Christian Geiser, Martin Li, Julia Su Zhou Widmer, Hans Auer, Manfred |
author_facet | Benoit, Roger M. Ostermeier, Christian Geiser, Martin Li, Julia Su Zhou Widmer, Hans Auer, Manfred |
author_sort | Benoit, Roger M. |
collection | PubMed |
description | Seamless cloning methods, such as co-transformation cloning, sequence- and ligation-independent cloning (SLIC) or the Gibson assembly, are essential tools for the precise construction of plasmids. The efficiency of co-transformation cloning is however low and the Gibson assembly reagents are expensive. With the aim to improve the robustness of seamless cloning experiments while keeping costs low, we examined the importance of complementary single-stranded DNA ends for co-transformation cloning and the influence of single-stranded gaps in circular plasmids on SLIC cloning efficiency. Most importantly, our data show that single-stranded gaps in double-stranded plasmids, which occur in typical SLIC protocols, can drastically decrease the efficiency at which the DNA transforms competent E. coli bacteria. Accordingly, filling-in of single-stranded gaps using DNA polymerase resulted in increased transformation efficiency. Ligation of the remaining nicks did not lead to a further increase in transformation efficiency. These findings demonstrate that highly efficient insert-plasmid assembly can be achieved by using only T5 exonuclease and Phusion DNA polymerase, without Taq DNA ligase from the original Gibson protocol, which significantly reduces the cost of the reactions. We successfully used this modified Gibson assembly protocol with two short insert-plasmid overlap regions, each counting only 15 nucleotides. |
format | Online Article Text |
id | pubmed-4830597 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2016 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-48305972016-04-22 Seamless Insert-Plasmid Assembly at High Efficiency and Low Cost Benoit, Roger M. Ostermeier, Christian Geiser, Martin Li, Julia Su Zhou Widmer, Hans Auer, Manfred PLoS One Research Article Seamless cloning methods, such as co-transformation cloning, sequence- and ligation-independent cloning (SLIC) or the Gibson assembly, are essential tools for the precise construction of plasmids. The efficiency of co-transformation cloning is however low and the Gibson assembly reagents are expensive. With the aim to improve the robustness of seamless cloning experiments while keeping costs low, we examined the importance of complementary single-stranded DNA ends for co-transformation cloning and the influence of single-stranded gaps in circular plasmids on SLIC cloning efficiency. Most importantly, our data show that single-stranded gaps in double-stranded plasmids, which occur in typical SLIC protocols, can drastically decrease the efficiency at which the DNA transforms competent E. coli bacteria. Accordingly, filling-in of single-stranded gaps using DNA polymerase resulted in increased transformation efficiency. Ligation of the remaining nicks did not lead to a further increase in transformation efficiency. These findings demonstrate that highly efficient insert-plasmid assembly can be achieved by using only T5 exonuclease and Phusion DNA polymerase, without Taq DNA ligase from the original Gibson protocol, which significantly reduces the cost of the reactions. We successfully used this modified Gibson assembly protocol with two short insert-plasmid overlap regions, each counting only 15 nucleotides. Public Library of Science 2016-04-13 /pmc/articles/PMC4830597/ /pubmed/27073895 http://dx.doi.org/10.1371/journal.pone.0153158 Text en © 2016 Benoit et al http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. |
spellingShingle | Research Article Benoit, Roger M. Ostermeier, Christian Geiser, Martin Li, Julia Su Zhou Widmer, Hans Auer, Manfred Seamless Insert-Plasmid Assembly at High Efficiency and Low Cost |
title | Seamless Insert-Plasmid Assembly at High Efficiency and Low Cost |
title_full | Seamless Insert-Plasmid Assembly at High Efficiency and Low Cost |
title_fullStr | Seamless Insert-Plasmid Assembly at High Efficiency and Low Cost |
title_full_unstemmed | Seamless Insert-Plasmid Assembly at High Efficiency and Low Cost |
title_short | Seamless Insert-Plasmid Assembly at High Efficiency and Low Cost |
title_sort | seamless insert-plasmid assembly at high efficiency and low cost |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4830597/ https://www.ncbi.nlm.nih.gov/pubmed/27073895 http://dx.doi.org/10.1371/journal.pone.0153158 |
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