Construction and evaluation of multisite recombinatorial (Gateway) cloning vectors for Gram-positive bacteria

<p>Abstract</p> <p>Background</p> <p>The Gateway recombinatorial cloning system allows easy and rapid joining of DNA fragments. Here we report the construction and evaluation of three different Gram-positive vectors that can be used with the Multisite Gateway cloning system to rapidly produce new gene arrangements in plasmid constructs for use in a variety of Gram-positive bacteria.</p> <p>Results</p> <p>Comparison of patterns of reporter gene expression with conventionally constructed clones show that the presence of residual recombination (att) sites does not have an effect on patterns of gene expression, although overall levels of gene expression may vary. Rapid construction of these new vectors allowed vector/gene combinations to be optimized following evaluation of plasmid constructs in different bacterial cells and demonstrates the benefits of plasmid construction using Gateway cloning.</p> <p>Conclusion</p> <p>The residual <it>att </it>sites present after Gateway cloning did not affect patterns of promoter induction in Gram-positive bacteria and there was no evidence of differences in mRNA stability of transcripts. However overall levels of gene expression may be reduced, possibly due to some post-transcriptional event. The new vectors described here allow faster, more efficient cloning in range of Gram-positive bacteria.</p

A Practical, One-Pot Synthesis of Highly Substituted Thiophenes and Benzo[b]thiophenes from Bromoenynes and o-Alkynylbromobenzenes

An efficient synthesis of thiophenes and benzo[b]thiophenes has been developed from easily available bromoenynes and o-alkynylbromobenzene derivatives. This novel one-pot procedure involves a Pd-catalyzed C–S bond formation using a hydrogen sulfide surrogate followed by a heterocyclization reaction. Moreover, in situ functionalization with selected electrophiles further expands the potential of this methodology to the preparation of the corresponding highly substituted sulfur heterocycles.Junta de Castilla y Leon (BU021A09 and GR-172) and Ministerio de Ciencia e Innovacion (MICINN) and FEDER (CTQ2010-15358 and CTQ2009-09949/BQU) for financial support. P.G.-G. and M.A.F.-R. thank MICINN for "Juan de la Cierva" and "Ramon y Cajal" contractsJunta de Castilla y Leon (BU021A09 and GR-172) and Ministerio de Ciencia e Innovacion (MICINN) and FEDER (CTQ2010-15358 and CTQ2009-09949/BQU) for financial support. P.G.-G. and M.A.F.-R. thank MICINN for "Juan de la Cierva" and "Ramon y Cajal" contractsThis document is the Accepted Manuscript version of a Published Work that appeared in final form in Organic Letters, copyright © American Chemical Society after peer review and technical editing by the publisher

"Fritillary Fever": Cultivating the self and gardening the world in the writing of Clara Coltman Vyvyan

This chapter is published within the context of book of essays that takes forward critical debates concerning women’s appropriations of private and public space by providing new readings of complex negotiations for traversing space in Anglo-American literature, written by or about women between the Victorian period and the Second World War. Whereas previous studies have tended to concentrate on a single aspect of women’s engagement with space, be it within the domestic, the urban or the natural world, this corpus of essays explores women’s transit through a multiplicity of spaces and its literary representation during a period that permanently changed gender relations. My contribution to the debate closes the book with an exploration of the gendered space of the post-Second World War English country house garden by examining the work of a little-known but prolific garden and travel writer, Clara Coltman Vyvyan. Although Vyvyan wrote extensively on gardening, Cornish landscape and European travel during the inter-war and post-war period, her work has received very little critical attention. This chapter addresses Vyvyan’s absence from critical accounts of women’s writing in this period, establishing the significance of her work in relation to other ‘conservative modernists’ such as her friend Daphne du Maurier. It provides both an original reading of the work of Vyvyan, and new insights into the contested space of the country house garden in literature. Challenging critical accounts that privilege the garden as a private and static arena, it shows how Vyvyan places herself in transit through the gendered spaces of garden and wilderness by negotiating male traditions of exploration, botany and writing

Construction and evaluation of multisite recombinatorial (Gateway) cloning vectors for Gram-positive bacteria-2

<p><b>Copyright information:</b></p><p>Taken from "Construction and evaluation of multisite recombinatorial (Gateway) cloning vectors for Gram-positive bacteria"</p><p>http://www.biomedcentral.com/1471-2199/8/80</p><p>BMC Molecular Biology 2007;8():80-80.</p><p>Published online 19 Sep 2007</p><p>PMCID:PMC2039747.</p><p></p> ●; no sites) were grown to mid-log phase in Tris Minimal Succinate medium with 5 μg mlCm and 0.5% xylose. Bacteria were washed and resuspended in an equal volume of medium containing 5 μg mlCm and 1% glucose. Aliquots were placed in a 96 well microtitre plate and incubated at 37°C in a Tecan Genios Pro. Panel A: fluorescence (solid symbols, (Relative Fluorescence Units; RFU) and absorbance (open symbols) were measured at 10 min intervals. Plasmids used were pSB3004 (□, ■; 4 sites), pSB3002 (□, ▲; 2 sites) and pSB3000 (○, ●; no sites). Panel B: luminescence (solid symbols, Relative Light Units; RLU) and absorbance (open symbols) were measured at 10 minute intervals. Plasmids used were pSB3014 (□, ■; 4 sites), pSB3012 (□, ▲; 2 sites) and pSB3010 (○, ●; no sites). Data is presented as % maximal signal to allow direct comparison of repression kinetics despite the fact that light levels from each construct were different