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A new recombineering system for Photorhabdus and Xenorhabdus

Precise and fluent genetic manipulation is still limited to only a few prokaryotes. Ideally the highly advanced technologies available in Escherichia coli could be broadly applied. Our efforts to apply lambda Red technology, widely termed ‘recombineering’, in Photorhabdus and Xenorhabdus yielded onl...

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Autores principales: Yin, Jia, Zhu, Hongbo, Xia, Liqiu, Ding, Xuezhi, Hoffmann, Thomas, Hoffmann, Michael, Bian, Xiaoying, Müller, Rolf, Fu, Jun, Stewart, A. Francis, Zhang, Youming
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2015
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4381043/
https://www.ncbi.nlm.nih.gov/pubmed/25539914
http://dx.doi.org/10.1093/nar/gku1336
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author Yin, Jia
Zhu, Hongbo
Xia, Liqiu
Ding, Xuezhi
Hoffmann, Thomas
Hoffmann, Michael
Bian, Xiaoying
Müller, Rolf
Fu, Jun
Stewart, A. Francis
Zhang, Youming
author_facet Yin, Jia
Zhu, Hongbo
Xia, Liqiu
Ding, Xuezhi
Hoffmann, Thomas
Hoffmann, Michael
Bian, Xiaoying
Müller, Rolf
Fu, Jun
Stewart, A. Francis
Zhang, Youming
author_sort Yin, Jia
collection PubMed
description Precise and fluent genetic manipulation is still limited to only a few prokaryotes. Ideally the highly advanced technologies available in Escherichia coli could be broadly applied. Our efforts to apply lambda Red technology, widely termed ‘recombineering’, in Photorhabdus and Xenorhabdus yielded only limited success. Consequently we explored the properties of an endogenous Photorhabdus luminescens lambda Red-like operon, Plu2934/Plu2935/Plu2936. Bioinformatic and functional tests indicate that Plu2936 is a 5’-3’ exonuclease equivalent to Redα and Plu2935 is a single strand annealing protein equivalent to Redβ. Plu2934 dramatically enhanced recombineering efficiency. Results from bioinformatic analysis and recombineering assays suggest that Plu2934 may be functionally equivalent to Redγ, which inhibits the major endogenous E. coli nuclease, RecBCD. The recombineering utility of Plu2934/Plu2935/Plu2936 was demonstrated by engineering Photorhabdus and Xenorhabdus genomes, including the activation of the 49-kb non-ribosomal peptide synthase (NRPS) gene cluster plu2670 by insertion of a tetracycline inducible promoter. After tetracycline induction, novel secondary metabolites were identified. Our work unlocks the potential for bioprospecting and functional genomics in the Photorhabdus, Xenorhabdus and related genomes.
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spelling pubmed-43810432015-04-03 A new recombineering system for Photorhabdus and Xenorhabdus Yin, Jia Zhu, Hongbo Xia, Liqiu Ding, Xuezhi Hoffmann, Thomas Hoffmann, Michael Bian, Xiaoying Müller, Rolf Fu, Jun Stewart, A. Francis Zhang, Youming Nucleic Acids Res Methods Online Precise and fluent genetic manipulation is still limited to only a few prokaryotes. Ideally the highly advanced technologies available in Escherichia coli could be broadly applied. Our efforts to apply lambda Red technology, widely termed ‘recombineering’, in Photorhabdus and Xenorhabdus yielded only limited success. Consequently we explored the properties of an endogenous Photorhabdus luminescens lambda Red-like operon, Plu2934/Plu2935/Plu2936. Bioinformatic and functional tests indicate that Plu2936 is a 5’-3’ exonuclease equivalent to Redα and Plu2935 is a single strand annealing protein equivalent to Redβ. Plu2934 dramatically enhanced recombineering efficiency. Results from bioinformatic analysis and recombineering assays suggest that Plu2934 may be functionally equivalent to Redγ, which inhibits the major endogenous E. coli nuclease, RecBCD. The recombineering utility of Plu2934/Plu2935/Plu2936 was demonstrated by engineering Photorhabdus and Xenorhabdus genomes, including the activation of the 49-kb non-ribosomal peptide synthase (NRPS) gene cluster plu2670 by insertion of a tetracycline inducible promoter. After tetracycline induction, novel secondary metabolites were identified. Our work unlocks the potential for bioprospecting and functional genomics in the Photorhabdus, Xenorhabdus and related genomes. Oxford University Press 2015-03-31 2014-12-24 /pmc/articles/PMC4381043/ /pubmed/25539914 http://dx.doi.org/10.1093/nar/gku1336 Text en © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research. 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 reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Methods Online
Yin, Jia
Zhu, Hongbo
Xia, Liqiu
Ding, Xuezhi
Hoffmann, Thomas
Hoffmann, Michael
Bian, Xiaoying
Müller, Rolf
Fu, Jun
Stewart, A. Francis
Zhang, Youming
A new recombineering system for Photorhabdus and Xenorhabdus
title A new recombineering system for Photorhabdus and Xenorhabdus
title_full A new recombineering system for Photorhabdus and Xenorhabdus
title_fullStr A new recombineering system for Photorhabdus and Xenorhabdus
title_full_unstemmed A new recombineering system for Photorhabdus and Xenorhabdus
title_short A new recombineering system for Photorhabdus and Xenorhabdus
title_sort new recombineering system for photorhabdus and xenorhabdus
topic Methods Online
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4381043/
https://www.ncbi.nlm.nih.gov/pubmed/25539914
http://dx.doi.org/10.1093/nar/gku1336
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