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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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Estimation of the relative copy numbers of the RSFmob and RSFmob-I plasmids. RSF1010, RSFmob and RSFmob-I plasmids were transformed to E. coli MG1655 strain. Three independently obtained transformants of each type were analyzed. 1, 2, 3 – 0.5, 2.0 and 5.0 μl of the DNA probe isolated from clone 1 of MG1655/RSF1010; 4, 5 – 2.0 μl of the DNA probes isolated from clones 2 and 3 of MG1655/RSF1010; 6, 7, 8 – 2.0 μl of the DNA probes isolated from 3 independent clones of MG1655/RSFmob; 9, 10, 11 – 2.0 μl of the DNA probes isolated from 3 independent clones of MG1655/RSFmob-I. Relative copy number was calculated as the ratio of the DNA amounts in the upper bands (5.9 kb EcoRV-EcoRV fragment). Electrophoretic patterns for stationary phase are represented as an example.
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Figure 3: Estimation of the relative copy numbers of the RSFmob and RSFmob-I plasmids. RSF1010, RSFmob and RSFmob-I plasmids were transformed to E. coli MG1655 strain. Three independently obtained transformants of each type were analyzed. 1, 2, 3 – 0.5, 2.0 and 5.0 μl of the DNA probe isolated from clone 1 of MG1655/RSF1010; 4, 5 – 2.0 μl of the DNA probes isolated from clones 2 and 3 of MG1655/RSF1010; 6, 7, 8 – 2.0 μl of the DNA probes isolated from 3 independent clones of MG1655/RSFmob; 9, 10, 11 – 2.0 μl of the DNA probes isolated from 3 independent clones of MG1655/RSFmob-I. Relative copy number was calculated as the ratio of the DNA amounts in the upper bands (5.9 kb EcoRV-EcoRV fragment). Electrophoretic patterns for stationary phase are represented as an example.

Mentions: Rough evaluation by electrophoresis of purified DNA (see, Methods) showed that RSFmob had approximately the same copy-number as RSF1010 in the stationary growth phase (Fig. 3, Table 1). During logarithmic growth, the copy-number of the obtained derivative was about two times lower than that of RSF1010. These data were in direct correlation with the levels of streptomycin resistance of the corresponding plasmid strains. Addition of 1 mM IPTG to the medium caused a 2.5-fold increase of RSFmob copy-number at the logarithmic stage of cell growing (Table 1). It was proposed that elimination of the lacI gene from the RSFmob could increase the plasmid copy-number due to derepression of repB and/or repAC transcription [26,27].


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)

Estimation of the relative copy numbers of the RSFmob and RSFmob-I plasmids. RSF1010, RSFmob and RSFmob-I plasmids were transformed to E. coli MG1655 strain. Three independently obtained transformants of each type were analyzed. 1, 2, 3 – 0.5, 2.0 and 5.0 μl of the DNA probe isolated from clone 1 of MG1655/RSF1010; 4, 5 – 2.0 μl of the DNA probes isolated from clones 2 and 3 of MG1655/RSF1010; 6, 7, 8 – 2.0 μl of the DNA probes isolated from 3 independent clones of MG1655/RSFmob; 9, 10, 11 – 2.0 μl of the DNA probes isolated from 3 independent clones of MG1655/RSFmob-I. Relative copy number was calculated as the ratio of the DNA amounts in the upper bands (5.9 kb EcoRV-EcoRV fragment). Electrophoretic patterns for stationary phase are represented as an example.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Estimation of the relative copy numbers of the RSFmob and RSFmob-I plasmids. RSF1010, RSFmob and RSFmob-I plasmids were transformed to E. coli MG1655 strain. Three independently obtained transformants of each type were analyzed. 1, 2, 3 – 0.5, 2.0 and 5.0 μl of the DNA probe isolated from clone 1 of MG1655/RSF1010; 4, 5 – 2.0 μl of the DNA probes isolated from clones 2 and 3 of MG1655/RSF1010; 6, 7, 8 – 2.0 μl of the DNA probes isolated from 3 independent clones of MG1655/RSFmob; 9, 10, 11 – 2.0 μl of the DNA probes isolated from 3 independent clones of MG1655/RSFmob-I. Relative copy number was calculated as the ratio of the DNA amounts in the upper bands (5.9 kb EcoRV-EcoRV fragment). Electrophoretic patterns for stationary phase are represented as an example.
Mentions: Rough evaluation by electrophoresis of purified DNA (see, Methods) showed that RSFmob had approximately the same copy-number as RSF1010 in the stationary growth phase (Fig. 3, Table 1). During logarithmic growth, the copy-number of the obtained derivative was about two times lower than that of RSF1010. These data were in direct correlation with the levels of streptomycin resistance of the corresponding plasmid strains. Addition of 1 mM IPTG to the medium caused a 2.5-fold increase of RSFmob copy-number at the logarithmic stage of cell growing (Table 1). It was proposed that elimination of the lacI gene from the RSFmob could increase the plasmid copy-number due to derepression of repB and/or repAC transcription [26,27].

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