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Construction of stably maintained non-mobilizable derivatives of RSF1010 lacking all known elements essential for mobilization

BACKGROUND: RSF1010 is a well-studied broad-host-range plasmid able to be mobilized to different bacteria and plants. RSF1010-derived plasmid vectors are widely used in both basic research and industrial applications. In the latter case, exploiting of mobilizable plasmids or even the plasmids posses...

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Autores principales: Katashkina, Joanna I, Kuvaeva, Tatiana M, Andreeva, Irina G, Skorokhodova, Alexandra Yu, Biryukova, Irina V, Tokmakova, Irina L, Golubeva, Lubov I, Mashko, Sergey V
Formato: Texto
Lenguaje:English
Publicado: BioMed Central 2007
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2200642/
https://www.ncbi.nlm.nih.gov/pubmed/18028554
http://dx.doi.org/10.1186/1472-6750-7-80
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author Katashkina, Joanna I
Kuvaeva, Tatiana M
Andreeva, Irina G
Skorokhodova, Alexandra Yu
Biryukova, Irina V
Tokmakova, Irina L
Golubeva, Lubov I
Mashko, Sergey V
author_facet Katashkina, Joanna I
Kuvaeva, Tatiana M
Andreeva, Irina G
Skorokhodova, Alexandra Yu
Biryukova, Irina V
Tokmakova, Irina L
Golubeva, Lubov I
Mashko, Sergey V
author_sort Katashkina, Joanna I
collection PubMed
description BACKGROUND: RSF1010 is a well-studied broad-host-range plasmid able to be mobilized to different bacteria and plants. RSF1010-derived plasmid vectors are widely used in both basic research and industrial applications. In the latter case, exploiting of mobilizable plasmids or even the plasmids possessing negligible mobilization frequency, but containing DNA fragments that could promote conjugal transfer, is undesirable because of biosafety considerations. Previously, several mutations significantly decreasing efficiency of RSF1010 mobilization have been selected. Nevertheless, construction of the RSF1010 derivative lacking all known loci involved in the conjugal transfer has not been reported yet. RESULTS: Novel non-mobilizable derivatives of RSF1010 lacking all known DNA sequences involved in the mobilization process have been obtained due to the exploiting of λRed-driven recombination between the plasmid and a constructed in vitro linear DNA fragment. To provide auto-regulated transcription of the essential replication gene, repB, the plasmid loci oriT, mobC and mobA were substituted by the DNA fragment containing P(lacUV5)→lacI. Mobilization of the obtained RSFmob plasmid was not detected in standard tests. The derivative of RSFmob with increased copy number has been obtained after lacI elimination. High stability of both constructed plasmids has been demonstrated in Escherichia coli and Pantoea ananatis. Design of RSFmob allows easy substitution of P(lacUV5 )by any desirable promoter for construction of novel derivatives with changed copy number or host range. CONCLUSION: Novel non-mobilizable derivatives of RSF1010 lacking all known DNA sequences involved in the mobilization process and stably maintained at least in E. coli and P. ananatis have been constructed. The obtained plasmids became the progenitors of new cloning vectors answering all biosafety requirements of genetically modified organisms used in scale-up production.
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spelling pubmed-22006422008-01-16 Construction of stably maintained non-mobilizable derivatives of RSF1010 lacking all known elements essential for mobilization Katashkina, Joanna I Kuvaeva, Tatiana M Andreeva, Irina G Skorokhodova, Alexandra Yu Biryukova, Irina V Tokmakova, Irina L Golubeva, Lubov I Mashko, Sergey V BMC Biotechnol Research Article BACKGROUND: RSF1010 is a well-studied broad-host-range plasmid able to be mobilized to different bacteria and plants. RSF1010-derived plasmid vectors are widely used in both basic research and industrial applications. In the latter case, exploiting of mobilizable plasmids or even the plasmids possessing negligible mobilization frequency, but containing DNA fragments that could promote conjugal transfer, is undesirable because of biosafety considerations. Previously, several mutations significantly decreasing efficiency of RSF1010 mobilization have been selected. Nevertheless, construction of the RSF1010 derivative lacking all known loci involved in the conjugal transfer has not been reported yet. RESULTS: Novel non-mobilizable derivatives of RSF1010 lacking all known DNA sequences involved in the mobilization process have been obtained due to the exploiting of λRed-driven recombination between the plasmid and a constructed in vitro linear DNA fragment. To provide auto-regulated transcription of the essential replication gene, repB, the plasmid loci oriT, mobC and mobA were substituted by the DNA fragment containing P(lacUV5)→lacI. Mobilization of the obtained RSFmob plasmid was not detected in standard tests. The derivative of RSFmob with increased copy number has been obtained after lacI elimination. High stability of both constructed plasmids has been demonstrated in Escherichia coli and Pantoea ananatis. Design of RSFmob allows easy substitution of P(lacUV5 )by any desirable promoter for construction of novel derivatives with changed copy number or host range. CONCLUSION: Novel non-mobilizable derivatives of RSF1010 lacking all known DNA sequences involved in the mobilization process and stably maintained at least in E. coli and P. ananatis have been constructed. The obtained plasmids became the progenitors of new cloning vectors answering all biosafety requirements of genetically modified organisms used in scale-up production. BioMed Central 2007-11-21 /pmc/articles/PMC2200642/ /pubmed/18028554 http://dx.doi.org/10.1186/1472-6750-7-80 Text en Copyright © 2007 Katashkina et al; licensee BioMed Central Ltd. http://creativecommons.org/licenses/by/2.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( (http://creativecommons.org/licenses/by/2.0) ), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Research Article
Katashkina, Joanna I
Kuvaeva, Tatiana M
Andreeva, Irina G
Skorokhodova, Alexandra Yu
Biryukova, Irina V
Tokmakova, Irina L
Golubeva, Lubov I
Mashko, Sergey V
Construction of stably maintained non-mobilizable derivatives of RSF1010 lacking all known elements essential for mobilization
title Construction of stably maintained non-mobilizable derivatives of RSF1010 lacking all known elements essential for mobilization
title_full Construction of stably maintained non-mobilizable derivatives of RSF1010 lacking all known elements essential for mobilization
title_fullStr Construction of stably maintained non-mobilizable derivatives of RSF1010 lacking all known elements essential for mobilization
title_full_unstemmed Construction of stably maintained non-mobilizable derivatives of RSF1010 lacking all known elements essential for mobilization
title_short Construction of stably maintained non-mobilizable derivatives of RSF1010 lacking all known elements essential for mobilization
title_sort construction of stably maintained non-mobilizable derivatives of rsf1010 lacking all known elements essential for mobilization
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2200642/
https://www.ncbi.nlm.nih.gov/pubmed/18028554
http://dx.doi.org/10.1186/1472-6750-7-80
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