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Streamlining recombination-mediated genetic engineering by validating three neutral integration sites in Synechococcus sp. PCC 7002

BACKGROUND: Synechococcus sp. PCC 7002 (henceforth Synechococcus) is developing into a powerful synthetic biology chassis. In order to streamline the integration of genes into the Synechococcus chromosome, validation of neutral integration sites with optimization of the DNA transformation protocol p...

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Autores principales: Vogel, Anne Ilse Maria, Lale, Rahmi, Hohmann-Marriott, Martin Frank
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5458483/
https://www.ncbi.nlm.nih.gov/pubmed/28592992
http://dx.doi.org/10.1186/s13036-017-0061-8
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author Vogel, Anne Ilse Maria
Lale, Rahmi
Hohmann-Marriott, Martin Frank
author_facet Vogel, Anne Ilse Maria
Lale, Rahmi
Hohmann-Marriott, Martin Frank
author_sort Vogel, Anne Ilse Maria
collection PubMed
description BACKGROUND: Synechococcus sp. PCC 7002 (henceforth Synechococcus) is developing into a powerful synthetic biology chassis. In order to streamline the integration of genes into the Synechococcus chromosome, validation of neutral integration sites with optimization of the DNA transformation protocol parameters is necessary. Availability of BioBrick-compatible integration modules is desirable to further simplifying chromosomal integrations. RESULTS: We designed three BioBrick-compatible genetic modules, each targeting a separate neutral integration site, A2842, A0935, and A0159, with varying length of homologous region, spanning from 100 to 800 nt. The performance of the different modules for achieving DNA integration were tested. Our results demonstrate that 100 nt homologous regions are sufficient for inserting a 1 kb DNA fragment into the Synechococcus chromosome. By adapting a transformation protocol from a related cyanobacterium, we shortened the transformation procedure for Synechococcus significantly. CONCLUSIONS: The optimized transformation protocol reported in this study provides an efficient way to perform genetic engineering in Synechococcus. We demonstrated that homologous regions of 100 nt are sufficient for inserting a 1 kb DNA fragment into the three tested neutral integration sites. Integration at A2842, A0935 and A0159 results in only a minimal fitness cost for the chassis. This study contributes to developing Synechococcus as the prominent chassis for future synthetic biology applications. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13036-017-0061-8) contains supplementary material, which is available to authorized users.
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spelling pubmed-54584832017-06-07 Streamlining recombination-mediated genetic engineering by validating three neutral integration sites in Synechococcus sp. PCC 7002 Vogel, Anne Ilse Maria Lale, Rahmi Hohmann-Marriott, Martin Frank J Biol Eng Research BACKGROUND: Synechococcus sp. PCC 7002 (henceforth Synechococcus) is developing into a powerful synthetic biology chassis. In order to streamline the integration of genes into the Synechococcus chromosome, validation of neutral integration sites with optimization of the DNA transformation protocol parameters is necessary. Availability of BioBrick-compatible integration modules is desirable to further simplifying chromosomal integrations. RESULTS: We designed three BioBrick-compatible genetic modules, each targeting a separate neutral integration site, A2842, A0935, and A0159, with varying length of homologous region, spanning from 100 to 800 nt. The performance of the different modules for achieving DNA integration were tested. Our results demonstrate that 100 nt homologous regions are sufficient for inserting a 1 kb DNA fragment into the Synechococcus chromosome. By adapting a transformation protocol from a related cyanobacterium, we shortened the transformation procedure for Synechococcus significantly. CONCLUSIONS: The optimized transformation protocol reported in this study provides an efficient way to perform genetic engineering in Synechococcus. We demonstrated that homologous regions of 100 nt are sufficient for inserting a 1 kb DNA fragment into the three tested neutral integration sites. Integration at A2842, A0935 and A0159 results in only a minimal fitness cost for the chassis. This study contributes to developing Synechococcus as the prominent chassis for future synthetic biology applications. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13036-017-0061-8) contains supplementary material, which is available to authorized users. BioMed Central 2017-06-05 /pmc/articles/PMC5458483/ /pubmed/28592992 http://dx.doi.org/10.1186/s13036-017-0061-8 Text en © The Author(s). 2017 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
spellingShingle Research
Vogel, Anne Ilse Maria
Lale, Rahmi
Hohmann-Marriott, Martin Frank
Streamlining recombination-mediated genetic engineering by validating three neutral integration sites in Synechococcus sp. PCC 7002
title Streamlining recombination-mediated genetic engineering by validating three neutral integration sites in Synechococcus sp. PCC 7002
title_full Streamlining recombination-mediated genetic engineering by validating three neutral integration sites in Synechococcus sp. PCC 7002
title_fullStr Streamlining recombination-mediated genetic engineering by validating three neutral integration sites in Synechococcus sp. PCC 7002
title_full_unstemmed Streamlining recombination-mediated genetic engineering by validating three neutral integration sites in Synechococcus sp. PCC 7002
title_short Streamlining recombination-mediated genetic engineering by validating three neutral integration sites in Synechococcus sp. PCC 7002
title_sort streamlining recombination-mediated genetic engineering by validating three neutral integration sites in synechococcus sp. pcc 7002
topic Research
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5458483/
https://www.ncbi.nlm.nih.gov/pubmed/28592992
http://dx.doi.org/10.1186/s13036-017-0061-8
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