Cargando…
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...
Autores principales: | , , |
---|---|
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 |
_version_ | 1783241771210244096 |
---|---|
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. |
format | Online Article Text |
id | pubmed-5458483 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2017 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
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 |
work_keys_str_mv | AT vogelanneilsemaria streamliningrecombinationmediatedgeneticengineeringbyvalidatingthreeneutralintegrationsitesinsynechococcussppcc7002 AT lalerahmi streamliningrecombinationmediatedgeneticengineeringbyvalidatingthreeneutralintegrationsitesinsynechococcussppcc7002 AT hohmannmarriottmartinfrank streamliningrecombinationmediatedgeneticengineeringbyvalidatingthreeneutralintegrationsitesinsynechococcussppcc7002 |