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Transformation Efficiency (transformation + efficiency)
Selected AbstractsNanogold-Loaded Sharp-Edged Carbon Bullets as Plant-Gene CarriersADVANCED FUNCTIONAL MATERIALS, Issue 15 2010Periyasamy S. Vijayakumar Abstract The higher DNA delivery efficiency into plants by gold nanoparticles embedded in sharp carbonaceous carriers is demonstrated. These nanogold-embedded carbon matrices are prepared by heat treatment of biogenic intracellular gold nanoparticles. The DNA-delivery efficiency is tested on a model plant, Nicotiana tabacum, and is further extended to the monocot, Oryza sativa, and a hard dicot tree species, Leucaena leucocephala. These materials reveal good dispersion of the transport material, producing a greater number of GUS foci per unit area. The added advantages of the composite carrier are the lower plasmid and gold requirements. Plant-cell damage with the carbon-supported particles is very minimal and can be gauged from the increased plant regeneration and transformation efficiency compared with that of the commercial micrometer-sized gold particles. This is ascribed to the sharp edges that the carbon supports possess, which lead to better piercing capabilities with minimum damage. [source] Germ-line transformation of the South American malaria vector, Anopheles albimanus, with a piggyBac/EGFP transposon vector is routine and highly efficientINSECT MOLECULAR BIOLOGY, Issue 4 2002O. P. Perera Abstract Stable and efficient germ-line transformation was achieved in the South American malaria vector, Anopheles albimanus, using a piggyBac vector marked with an enhanced green fluorescent protein gene regulated by the Drosophila melanogaster polyubiquitin promoter. Transgenic mosquitoes were identified from four independent experiments at frequencies ranging from 20 to 43% per fertile G0. Fluorescence was observable throughout the body of larvae and pupae, and abdominal segments of adults. Transgenic lines analysed by Southern hybridization had one to six germ-line integrations, with most lines having three or more integrations. Hybridized transposon vector fragments and insertion site sequences were consistent with precise piggyBac -mediated integrations, although this was not verified for all lines. The piggyBac/PUbnlsEGFP vector appears to be a robust transformation system for this anopheline species, in contrast to the use of a piggyBac vector in An. gambiae. Further tests are needed to determine if differences in anopheline transformation efficiency are due to the marker systems or to organismal or cellular factors specific to the species. [source] Efficient transformation of Lactococcus lactis IL1403 and generation of knock-out mutants by homologous recombinationJOURNAL OF BASIC MICROBIOLOGY, Issue 3 2007Simon D. Gerber Abstract Lactococcus lactis IL1403 is a Gram-positive bacterium of great biotechnological interest for food grade applications. Its use is however hampered by the difficulty to efficiently transform this strain. We here describe a detailed, optimized electrotransformation protocol which yields a transformation efficiency of 106 cfu/,g of DNA with the two E. coli Gram-positive shuttle vectors pC3 and pVA838. The utility of the protocol was demonstrated by the generation of single- and double-knock-out mutants by homologous recombination. (© 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim) [source] MOLECULAR GENETIC MANIPULATION OF THE DIATOM THALASSIOSIRA PSEUDONANA (BACILLARIOPHYCEAE),JOURNAL OF PHYCOLOGY, Issue 5 2006Nicole Poulsen Here, we describe the first system for genetic transformation of Thalassiosira pseudonana (Hustedt) Hasle et Heimdal, the only diatom for which a complete genome sequence is presently available. This method is based on microparticle bombardment followed by selection of transformants using the antibiotic nourseothricin. It exhibits the highest transformation efficiency compared with transformation systems for other diatom species. To achieve the high transformation efficiency, it is important to allow recovery of the bombarded T. pseudonana cells in non-selective suspension culture before spreading on nourseothricin containing agar plates. It is demonstrated that T. pseudonana is readily susceptible to co-transformation allowing for the simultaneous introduction of a non-selective gene together with the selection marker gene. Both introduced genes are stably inherited even in the absence of the antibiotic selection pressure. We have developed two T. pseudonana -specific expression vectors that can drive constitutive expression (vector pTpfcp) and inducible expression (vector pTpNR) of introduced genes. In combination with the available genome data the T. pseudonana transformation system is expected to provide a powerful tool for functional genomics in diatoms. [source] Influence of genetic background on transformation and expression of Green Fluorescent Protein in Actinobacillus actinomycetemcomitansMOLECULAR ORAL MICROBIOLOGY, Issue 5 2005W. Teughels Background/aims:, The development of an electro-transformation system and the construction of shuttle plasmids for Actinobacillus actinomycetemcomitans have enhanced the molecular analysis of virulence factors. However, inefficient transformation is frequently encountered. This study investigated the efficiency of electro-transformation and expression of Green Fluorescent Protein (GFP) in 12 different A. actinomycetemcomitans strains. The influence of the plasmid vector, serotype, and phenotype were the major factors taken into consideration. Material and methods:, Twelve serotyped A. actinomycetemcomitans strains were independently electro-transformed with two different Escherichia coli,A. actinomycetemcomitans shuttle plasmids (pVT1303 and pVT1304), both containing an identical ltx-GFPmut2 gene construct but a different backbone (pDMG4 and pPK1, respectively). The transformation efficiency, transformation frequency, and electro-transformation survival rate were determined by culture techniques. GFP expression was observed at the colony level by fluorescence microscopy. Results:, All strains could be transformed with both plasmids. However, major differences were observed for the transformation efficiency, transformation frequency, and electro-transformation survival rate between strains. The data demonstrated that plasmid vector, serotype, and phenotype are key players for obtaining a successful transformation. An inverted relationship between the electro-transformation survival rate and tranformation frequency was also observed. GFP expression was also influenced by phenotype, serotype and plasmid vector. Conclusions:, The serotype of A. actinomycetemcomitans has an important influence on its survival after electro-transformation and on transformation frequency. The expression of GFP is strain and plasmid vector dependent. [source] Efficient cloning of plant genomes into bacterial artificial chromosome (BAC) libraries with larger and more uniform insert sizePLANT BIOTECHNOLOGY JOURNAL, Issue 3 2004Boulos Chalhoub Summary The construction of bacterial artificial chromosome (BAC) libraries remains relatively complex and laborious, such that any technological improvement is considered to be highly advantageous. In this study, we addressed several aspects that improved the quality and efficiency of cloning of plant genomes into BACs. We set the ,single tube vector' preparation method with no precipitation or gel electrophoresis steps, which resulted in less vector DNA damage and a remarkable two- to threefold higher transformation efficiency compared with other known vector preparation methods. We used a reduced amount of DNA for partial digestion (up to 5 µg), which resulted in less BAC clones with small inserts. We performed electrophoresis in 0.25 × TBE (Tris, boric acid, ethylenediaminetetraacetic acid) buffer instead of 0.5 × TBE, which resulted in larger and more uniformly sized BAC inserts and, surprisingly, a two- to threefold higher transformation efficiency, probably due to less contamination with borate ions. We adopted a triple size selection that resulted in an increased mean insert size of up to 70 kb and a transformation efficiency comparable with that of double size selection. Overall, the improved protocol presented in this study resulted in a five- to sixfold higher cloning efficiency and larger and more uniformly sized BAC inserts. BAC libraries with the desired mean insert size (up to 200 kb) were constructed from several plant species, including hexaploid wheat. The improved protocol will render the construction of BAC libraries more available in plants and will greatly enhance genome analysis, gene mapping and cloning. [source] | ||||||||||