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

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Results</p> <p>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 <it>in vitro </it>linear DNA fragment. To provide auto-regulated transcription of the essential replication gene, <it>repB</it>, the plasmid loci <it>oriT</it>, <it>mobC </it>and <it>mobA </it>were substituted by the DNA fragment containing P_{<it>lac</it>UV5}→<it>lacI</it>. Mobilization of the obtained RSFmob plasmid was not detected in standard tests. The derivative of RSFmob with increased copy number has been obtained after <it>lacI </it>elimination. High stability of both constructed plasmids has been demonstrated in <it>Escherichia coli </it>and <it>Pantoea ananatis</it>. Design of RSFmob allows easy substitution of P_{<it>lac</it>UV5}by any desirable promoter for construction of novel derivatives with changed copy number or host range.</p> <p>Conclusion</p> <p>Novel non-mobilizable derivatives of RSF1010 lacking all known DNA sequences involved in the mobilization process and stably maintained at least in <it>E. coli </it>and <it>P. ananatis </it>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.</p

Dual-In/Out strategy for genes integration into bacterial chromosome: a novel approach to step-by-step construction of plasmid-less marker-less recombinant E. coli strains with predesigned genome structure

<p>Abstract</p> <p>Background</p> <p>The development of modern producer strains with metabolically engineered pathways poses special problems that often require manipulating many genes and expressing them individually at different levels or under separate regulatory controls. The construction of plasmid-less marker-less strains has many advantages for the further practical exploitation of these bacteria in industry. Such producer strains are usually constructed by sequential chromosome modifications including deletions and integration of genetic material. For these purposes complex methods based on <it>in vitro </it>and <it>in vivo </it>recombination processes have been developed.</p> <p>Results</p> <p>Here, we describe the new scheme of insertion of the foreign DNA for step-by-step construction of plasmid-less marker-less recombinant <it>E. coli </it>strains with chromosome structure designed in advance. This strategy, entitled as Dual-In/Out, based on the initial Red-driven insertion of artificial φ80-<it>attB </it>sites into desired points of the chromosome followed by two site-specific recombination processes: first, the φ80 system is used for integration of the recombinant DNA based on selective marker-carrier conditionally-replicated plasmid with φ80-<it>attP</it>-site, and second, the λ system is used for excision of inserted vector part, including the plasmid <it>ori</it>-replication and the marker, flanked by λ-<it>attL/R</it>-sites.</p> <p>Conclusion</p> <p>The developed Dual-In/Out strategy is a rather straightforward, but convenient combination of previously developed recombination methods: phages site-specific and general Red/ET-mediated. This new approach allows us to detail the design of future recombinant marker-less strains, carrying, in particular, rather large artificial insertions that could be difficult to introduce by usually used PCR-based Recombineering procedure. The developed strategy is simple and could be particularly useful for construction of strains for the biotechnological industry.</p

Comprehensive Assessment of Promising Potato Hybrids of Breeding VSC RAS

Using the traditional and marker-assisted selection methods, a comprehensive assessment of promising hybrids from the collection of the All-Russian Scientific Center was carried out. The assessment was conducted in 2018–2019 in the Republic of North Ossetia-Alania. As a result of molecular genetic analysis, hybrids were found with complex resistance to potato nematode, virus Y and X viruses - 2 / V, 5 / V, 6 / V, 17 / V, 40 / V, 43 / V, 46 / V, 54 / V, 124 / V, 9 / VI, 22a / VI, 35 / VI, 130 / VI, 71 / VII and 118 / VIII. Use of these selected forms allows optimal protection of potatoes, limitation of the spread of pathogens and prevention of the emergence of more aggressive pathotypes (races and strains). The hybrids with resistance to potato virus Y (with the presence of R-gene markers - 1/I, 3/I, 10/I, 13/I, 11/II, 15/III, 2/V, 5/V, 6/V, 7/V, 10/V/1140, 17/V, 40/V, 43/V, 46/V, 54/V, 124/V, 9/VI, 22a/VI, 35/VI, 100/VI, 130/VI, 71/VII) are of interest for practical breeding, as well as the hybrids with resistance to Phytophthora infestans such as 15/III, 119/IX and the hybrids 15 / III, 35 / VI, 130 / VI and 71 / VII, which have high marketable yield and weight of tuber. Keywords: potato, interspecific hybrids, marker-assisted breeding, resistance gene

Construction of stably maintained non-mobilizable derivatives of RSF1010 lacking all known elements essential for mobilization-1

<p><b>Copyright information:</b></p><p>Taken from "Construction of stably maintained non-mobilizable derivatives of RSF1010 lacking all known elements essential for mobilization"</p><p>http://www.biomedcentral.com/1472-6750/7/80</p><p>BMC Biotechnology 2007;7():80-80.</p><p>Published online 21 Nov 2007</p><p>PMCID:PMC2200642.</p><p></p>e 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

"Flora of Russia" on iNaturalist: a dataset

The "Flora of Russia" project on iNaturalist brought together professional scientists and amateur naturalists from all over the country. Over 10,000 people are involved in the data collection.Within 20 months the participants accumulated over 750,000 photo observations of 6,853 species of the Russian flora. This constitutes the largest dataset of open spatial data on the country’s biodiversity and a leading source of data on the current state of the national flora. About 85% of all project data are available under free licenses (CC0, CC-BY, CC-BY-NC) and can be freely used in scientific, educational and environmental activities