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

Katashkina JI, Kuvaeva TM, Andreeva IG, Skorokhodova AY, Biryukova IV, Tokmakova IL, Golubeva LI, Mashko SV - BMC Biotechnol. (2007)

Bottom Line: Design of RSFmob allows easy substitution of PlacUV5 by any desirable promoter for construction of novel derivatives with changed copy number or host range.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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Affiliation: Closed Joint-Stock Company Ajinomoto-Genetika Research Institute, 1st Dorozhny Pr, 1, Moscow 117545, Russia. joanna_katashkina@agri.ru

ABSTRACT

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 lambdaRed-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 PlacUV5-->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 PlacUV5 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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Map of the plasmid RSF1010. The loci involved in conjugal transfer are in red.
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Figure 1: Map of the plasmid RSF1010. The loci involved in conjugal transfer are in red.

Mentions: At present, the following loci involved in the plasmid vegetative replication and its mobilization, have been identified in RSF1010 [16,17] (see Fig. 1 as well):


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

Katashkina JI, Kuvaeva TM, Andreeva IG, Skorokhodova AY, Biryukova IV, Tokmakova IL, Golubeva LI, Mashko SV - BMC Biotechnol. (2007)

Map of the plasmid RSF1010. The loci involved in conjugal transfer are in red.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2200642&req=5

Figure 1: Map of the plasmid RSF1010. The loci involved in conjugal transfer are in red.
Mentions: At present, the following loci involved in the plasmid vegetative replication and its mobilization, have been identified in RSF1010 [16,17] (see Fig. 1 as well):

Bottom Line: Design of RSFmob allows easy substitution of PlacUV5 by any desirable promoter for construction of novel derivatives with changed copy number or host range.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Closed Joint-Stock Company Ajinomoto-Genetika Research Institute, 1st Dorozhny Pr, 1, Moscow 117545, Russia. joanna_katashkina@agri.ru

ABSTRACT

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 lambdaRed-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 PlacUV5-->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 PlacUV5 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.

Show MeSH
Related in: MedlinePlus