TY - CHAP. T1 - Agrobacterium tumefaciens-Mediated Transformation. AU - Frandsen, Rasmus John Normand. PY - 2015. Y1 - 2015. N2 - The use of Agrobacterium tumefaciens-mediated transformation for achieving genetic transformation of fungi has steadily increased over the last decade, and has proven to be almost universally applicable technique once suitable selection markers have been developed. In recent years the major technical advances has been made within the initial steps of the process, more specifically the efficient construction of plasmids for performing targeted genome modifications. This chapter provides a generic protocol for performing genetic transformation of ascomycetes via A. tumefaciens-mediated transformation (AMT) and guidelines for optimizing the AMT process with new fungal species. The chapter also includes a highly efficient vector construction system based on Uracil Specific Excisions Reagent (USER) cloning and specific PCR generated building blocks, which can be combined ...

6.Thaw frozen competent cells on ice. Effect of DNA incubation time on NEB Express competent E. coli transformation efficiency: 50 μl of competent cells were transformed with 100 pg of pUC19 control DNA following the provided High Efficiency Transformation Protocol except DNA incubation time varied from 0 to 40 minutes. Heat shock at exactly 42°C for exactly 30 seconds. Return to Protocols End Chemically Competent Cell Production ∅∅∅∅∅ ∅∅∅∅∅ Chemical Transformation modified from NEB transformation protocol. modification of the reported protocols used for preparation of chemical competent cells of Agrobacterium (McCormac et al., 1998) and E. coli (Green and Rogers 2013), and the freeze-thaw method for the genetic transformation of A. tumefaciens (Höfgen and Willmitzer,1988). a. Protocols BH3 Project. version 1.0 Updated:1/21/2013 Store competent cells at -80°C only! Bacterial transformation, as mentioned above, means the uptake of DNA molecules through the cell wall from ...

Uncorrected OCR) Abstract of thesis entitled PRODUCTION OF TRANSGENIC PLANT-DERIVED VACCINES VIA PLASTID TRANSFORMATION TECHNOLOGY Submitted by Lee Yuk Ting for the degree of Master of Philosophy at The University of Hong Kong in July 2004 With the advent of genetic engineering in higher plants, the need for very large quantities of therapeutic protein at low cost, and the desire to have heat-stable edible vaccines directed at human and animal diseases, transgenic plant-derived vaccines offer a new strategy for the development of safe, inexpensive vaccines against infectious diseases. The first success of plastid transformation in tobacco in 1990 has opened up the opportunities for genetically modifying plastids in higher plants for high level expression of biopharmaceuticals, such as antibodies and vaccines for oral administration. Since each plant cell contains up to 10,000 copies of identical plastid genome, plastid engineering should result in very high levels of transgene expression. In ...

A series of vectors has been constructed for the purpose of introducing cloned DNAs into plant genomes, using Agrobacterium tumefaciens-mediated transformation methods. One of these vectors, pCIT20, is a plasmid that contains a multiple cloning site (MCS), and a marker (Hph) that confers hygromycin resistance to plant cells. The others are all cosmid vectors which allow insertion of up to 46 kb of plant genomic DNA, and which also contain all of the necessary sequences for A. tumefaciens-mediated plant transformation. The cosmid vectors either contain a Hph marker (pCIT03), or a kanamycin-resistance marker (pCIT101-104). Three of the cosmid vectors (pCIT30, pCIT101, and pCIT103) carry bacteriophage T7 and SP6 promoters flanking the cloning Bg/II site, for synthesis of end-specific RNAs. The end-specific RNAs may be used as probes when labeled with radioactive or biotinylated nucleotides, for example, in a chromosome-walking experiment. The other two cosmid vectors (pCIT102 and pCIT104) carry ...

TY - JOUR. T1 - Co‐transformation of Lec1 CHO cells with N‐acetylgucosaminyltransferase 1 activity and a selectable marker. AU - Ripka, James. AU - Pierce, Michael. AU - Fregien, Nevis. PY - 1990/3. Y1 - 1990/3. N2 - In animal cells, the enzyme α(1,3)-mannoside-β(1,2)-N-acetylglucosaminyltransferase I (GlcNAc-TI, EC.2.4.1.101) catalyzes the addition of N-acetylglucosamine to the ASN-linked Man GlcNAc oligosaccharide. The Chinese hamster ovary (CHO) mutant cell line Lec1 is deficient in this enzyme activity and, therefore, accumulates mannose-terminating cell surface ASN-linked oligosaccharides. Consequently, Lec1 cells are sensitive to the cytotoxic effects of the mannose-binding lectin Concanavalin A (Con A). Lec1 cells were co-transformed with human DNA form A431 cells and eukaryotic expression plasmids containing the bacterial neo gene by calcium phosphate/DNA-mediated transformation. Co-transformants were selected for resistance to Con A and G-418. DNA from a primary co-transformant ...

The use of traditional breeding for improvement of avocado cultivars is time consuming, hence other methods such as genetic transformation by Agrobacterium is indispensable to adopt. The strain GV3850/pBI121gave best transformation outcome compared to five other binary vectors (AGL1/pCGP904; AGL1/pBI121; GV3850/pCGP904; LBA4404/pCG-P904 and LBA4404/pBI121) under different pH and acetosyringone concentrations. The optimal condition for reliable transformation was by using 200 μM acetosyringone and a pH of 5.2. Transformed embryonic shoots co-cultivated with GV3850/pBI121 were tested using the histochemical x-gluc assay. Further analysis was conducted by polymerase chain reaction using specific primers for the reporter gene (GUS).

Plastid transformation technology has been well established and widely utilized in plant transgenic research. In comparison with conventional nuclear gene transformation techniques, plastid engineering offers several potential advantages such as (i) more than 10-100 times greater expression levels than the conventional nuclear transformation system, (ii) a more convenient methodology for transferring multiple genes into plants via gene stacking methods, (iii) elimination of position effects in chloroplasts, which thereby reduces the chances for transgene silencing, (iv) minimal chance for transgene flow by pollen contamination due to maternal inheritance (Verma and Daniell, 2007).. One of the key aspects for the plastid transformation system is to employ a plastidic sequence to exchange exogenous genes into the chloroplast genome via homologous recombination. The usage of genetic markers, which enable the selective enrichment of ptDNA copies, is also a critical component for plastid ...

Bacterial transformation studies have shown that - Biology - Lab 4 (the Bacterial Transformation Lab). Bowtrol Probiotic improve gastrointestinal function & intestinal good bacterial microbial balance.

Researchers at UC Davis have produced a non-dormant alfalfa line highly amenable to transformation, allowing direct improvement of the line. Higher transformation efficiency and a non-dormant life-cycle make this line of alfalfa a valuable tool for research and breeding.

Although the Agrobacterium tumefaciens-mediated transformation efficiency was only a fraction of 1%, it was possible to exploit the transposition frequency of a single T0 line to initiate the development of a functional resource for activation tagging in tomato. The practice of using micropropagation to produce many clonal plants from a single tissue culture regenerant proved valuable, as it multiplied T1 seed production by up to 25 times. This strategy also capitalized on the behavior of transposase in Ac/Ds-ATag-Bar_gosGFP by isolating chimeric tissue from the original transformant into separate plantlets, allowing germinal transposition from multiple sites of Ds integration. The selection of a self-fertile, true breeding tomato cultivar allowed crossing to nontransgenic cv M82, thus maximizing T1 seed production. Pollen could be collected from transgenic flowers and distributed to multiple nontransgenic plants, all while still obtaining transgenic self-progeny.. Modifications made to the ...

Citation: Malnoy, M., Boresjza-Wysocka, E., Norelli, J.L., Flaishman, M., Gidoni, D., Aldwinckle, H.S. 2010. Genetic transformation of apple (Malus x domestica) without use of a selectable marker gene. Tree Genetics and Genomes. 6:423-433. Interpretive Summary: Antibiotic and herbicide resistance genes are widely used as selectable markers to facilitate the efficient transformation of crop plants. Due to the negative public connotations associated with the use of selectable markers, a completely marker-free transformation technology would be desirable for the commercialization of genetically transformed plants. With this goal in mind, a technique was developed to genetically transform apple without the use of selectable marker genes. The technique takes advantage of the apples capacity for high efficiency transformation and allows for the generation of marker-free transgenic plants without the need for repeated transformation or sexual crossing. When two different marker-gene free vectors ...

A forward genetics approach was applied in order to investigate the molecular basis of morphological transition in the wheat pathogenic fungus Zymoseptoria tritici. Z. tritici is a dimorphic plant pathogen displaying environmentally regulated morphogenetic transition between yeast-like and hyphal growth. Considering the infection mode of Z. tritici, the switching to hyphal growth is essential for pathogenicity allowing the fungus the host invasion through natural openings like stomata. We exploited a previously developed Agrobacterium tumefaciens-mediated transformation (ATMT) to generate a mutant library by insertional mutagenesis including more than 10,000 random mutants. To identify genes involved in dimorphic switch, a plate-based screening system was established. With this approach eleven dimorphic switch deficient random mutants were recovered, ten of which exhibited a yeast-like mode of growth and one mutant predominantly growing filamentously, producing high amount of mycelium under different

The plasmid vector pGreenII, which is widely used in the production of stable plant transformants, is shown herein to predispose constructs to the acquisition of mutations (Figure 1) despite its earlier revision (Hellens and Mullineaux 2000). This predisposition arises from pGreenII having an adverse effect on the growth of E. coli. It perturbs normal cell division resulting in the production of long filaments (Figure 1), a phenomenon associated with stressed cells (Justice et al. 2008), and causes a dramatic reduction in cell viability following overnight incubation (Table 1). This is far from ideal as the insertion of DNA into plasmids can itself affect the growth of E. coli through increased metabolic burden and the acquisition of activities that perturb cellular functions (Bentley et al. 2009; Rosano and Ceccarelli 2014). In our case, the insertion of a 4605 bp fragment containing the cDNA of the plant DNA methyltransferase 1 (MET1) into pGreenII generated sufficient selective pressure for ...

A method for the integration of linear DNA into the Dictyostelium genome is described. Restriction enzyme-mediated integration, or REMI, involves the transformation of cells with a mixture of plasmid DNA, linearized with a restriction enzyme, along with a restriction enzyme that is capable of genera …

With the help of sepiolite, a unique method for transforming DNA into bacteria, based on the Yoshida effect, has been developed recently. However, we confronted many problems when this newest method was tried. Only a few transformants could be obtained even when 100 ng of plasmid pET15b was used, and a successful result seemed difficult to repeat. To address this problem, we optimized the operating method and could achieve about 15,000 transformants using the same amount of plasmid, which could match the efficiency gained using the calcium chloride transformation method. Meanwhile, the results could also be reproduced well. In the same way, carbon nanotubes were used to attain more than 15,000 transformants in the same situation. Therefore, the transformation method could be extended to other nanomaterials. Meanwhile, compared with the mechanism previously reported, we verified quite a different principle for the mechanism responsible for such a transformation. In sum, this unique transformation can be

uncut plasmid dna vs linearized plasmid gel - posted in Molecular Cloning: Hello, I am going to run a gel comparing my uncut plasmid dna vs linearized plasmid. My insert is 135 bps and my vector is 3Kb. What can I expect to see on my gel, and how many bands can I expect to see. I am assuming the uncut plasmid will have several bands at different sizes and the linearized will have only one, is that right ? I am assuming the total size of my product will now be 3135 bps. Pls advise.

We have established an efficient Agrobacterium-mediated transformation procedure for Arabidopsis thaliana genotype C24 using the chimeric bialaphos resistance gene (bar) coding for phosphinothricin ac

Citation: Malnoy, M., Borejsza-Wysocka, E.E., Abbott, P., Lewis, S., Norelli, J.L., Flaishman, M., Gidoni, D., Aldwinckle, H.S. 2007. Genetic transformation of apple without use of a selectable marker. Acta Horticulturae. 738:319-322. Interpretive Summary: Technical Abstract: Selectable marker genes are widely used for the efficient transformation of crop plants. In most cases, selection is based on antibiotic or herbicide resistance. These marker genes are preferred because they tend to be most efficient (e.g. in apple up to 80% transformation). Due mainly to consumer and grower concerns, considerable effort is being put into developing a suite of strategies (site-specific recombination, homologous recombination, transposition and co-transformation) to eliminate the marker gene from the nuclear or chloroplast genome after selection. Current efforts concentrate on systems where the marker genes are eliminated efficiently soon after transformation. These methods, however, are laborious and of ...

Poplar is a model system for the regeneration and genetic transformation of woody plants. increased the regeneration rate. The integration of the desired gene into transgenic poplars was detected using selective medium containing kanamycin, followed by southern blot analysis. The expression of the transgene in the transgenic lines was confirmed by northern blot analysis. have focused on a variety of parameters, including different strains and culture densities, various acetosyringone (AS) concentrations and other factors[1,8,9,10,11]. The expression of high-copy number transgenes in transgenic plants buy 1265229-25-1 may be more or less than only a single copy of transgene, and also, sometimes, this is good for molecular analysis and genetic engineering. Furthermore, Husaini [12] reported that strawberry transgenic plants with a high transgene copy number (four copies or more) have become best for molecular evaluation and genetic executive. Moreover, Bartlett [13] reported an improved technique ...

TY - JOUR. T1 - Putative mechanism of natural transformation as deduced from genome data. AU - Yura, Kei. AU - Toh, Hiroyuki. AU - Go, Mitiko. PY - 1999. Y1 - 1999. N2 - Genetic transformation is widely utilized in molecular biology as a tool for gene cloning in Escherichia coli and for gene mapping in Bacillus subtilis. Several strains of eubacteria can naturally take up exogenous DNA and integrate the DNA into their own genomes. Molecular details of natural transformation, however, remained to be elucidated. The complete genome of a cyanobacterium, Synechocystis sp. PCC6803, has been sequenced. This bacterium has been used to examine functions of a particular gene. The genome is considered to carry information on natural transformable characteristics of Synechocystis. The first step in genetic transformation is the uptake of exogenous DNA. Proteins with non-specific DNA binding features are required, because specificity in the exogenous DNA has not been demonstrated. Such proteins have modules ...

Nyaboga, E., Njiru, J., Nguu, E., Gruissem, W., Vanderschuren, H. & Tripathi, L. (2013). Unlocking the potential of tropical root crop biotechnology in east Africa by establishing a genetic transformation platform for local farmer-preferred cassava cultivars. Frontiers in Plant Science, 4(526), 1-11 ...

The following protocol is designed for NEB 10-beta Competent E. coli (NEB #C3019 ) which are included in the NEB PCR Cloning Kit (NEB #E1202 ) only

What Does Digital Transformation Look Like to Airbus? What Does A Mobile-First Digital Transformation Strategy Look Like? Published on January 11, 2019 January 11, 2019 â ¢ 29 Likes â ¢ 2 Comments Successful digital transformation depends on speed and the ability to navigate between strategy development, planning, testing and learning with ease and crucially, without too many meetings. Without that â air coverâ from the board and from shareholders who understand the change that youâ re taking the organization through, it is very, very hard to do it successfully. Digital transformation frameworks are what separate successful transformation efforts from the unsuccessful. Shares (Image credit: Shutterstock) The digital transformation journey involves more than just launching an app orâ ¦ A comprehensive digital transformation roadmap is the key to driving change in a coordinated and effective way â whether you are a small company or a multi-national enterprise. No insurance company has ...

Genomic DNA isolation Creative Genomics has extensive experience on genomic DNA isolation. Serve for SNP genotyping and genome sequencing, genomic DNA was extracted from different species including human, plants and microbes. A wide range of starting material can be accepted which contains blood, buccal swaps, FTA paper etc. Plating and plasmid DNA extraction. High quality plasmid DNA is the basis for many molecular biology applications. Creative Genomics offers cost-effective, high-throughput DNA extraction from plasmid, BAC, and fosmid samples. Isolation procedures are performed using commercially available alkaline lysis kits on Apricot personal pipettor. To save researchers time, we can start from the ligation products and perform the following Transformation by Electroporation and plating. Samples will be returned with a detailed yield report ready for downstream applications. Ordering Information:. ...

This study is based on the overexpression of endogenous diacylglycerol acyltransferase type 2 (NeoDGAT2) in N. oleoabundans to improve triacylglycerol (TAG) accumulation for potential biodiesel production. The important prerequisites for NeoDGAT2 overexpression in N. oleoabundans are the availabilities of: (i) the stable nuclear transformation system [25], (ii) the NeoDGAT2 cDNA encoding a functional DGAT protein. NeoDGAT2 fused with His tag at the C-terminus for facilitating Western blot analysis has been shown to reduce the NeoDGAT2 activity [11]. NeoDGAT2 without tag was therefore used in this study, and (iii) the functional promoter that can drive the expression of the NeoDGAT2. Because no data concerning N. oleoabundans endogenous promoter are available, promoters AR and β2-Tub from C. reinhardtii [28, 29] that have been shown to function in N. oleoabundans with similar activity [25] were utilized in this study. Among the transformants, AR-DGAT2-40 showed the highest neutral lipid content ...

IPTG is also commonly used in blue/white screening experiments to determine the presence of absence of bacterial recombinant vectors following transformation. If the insert of interest is cloned into the vectors lacZ gene, it disrupts B-Gal and prevents its expression. Bacterial colonies are grown on media containing X-gal (5-bromo-4-chloro-3-indolyl- beta-D-galactopyranoside), a substrate for B-Gal that turns into an insoluble blue product upon being cleaved by the enzyme. Without B-Gal, bacterial colonies exposed to X-gal cannot process the substrate and remain white, evidence that they are transformed with recombinant vector and not non-recombinant vector. These white bacterial colonies can be selected for subsequent recombinant vector amplification.. Many regulatory elements of the lac operon are used in inducible recombinant protein systems; IPTG is an effective inducer when used in the concentration range of 100 micromolar to 1.5 millimolar. Inducer concentration depends on the required ...

Transformation Protocol. BY-2 (NT1) Cell Transformation with Agrobactrium. Day 1: 1. Grow up 1 ml of the Agrobacterium overnight in LB + all selective drugs. This culture may be started from frozen glycerol cultures if necessary. Day 2: 2. NT cells are used 3 days after splitting the NT cell culture. 4 ml of NT cells are required for each transformation with an additional 4 ml for the control culture which receives no bacteria. 3. 1 ul Acetosyringone (20 mM in ethanol) is added per ml of NT cells. Typically treat the whole 50 ml culture at this point and discard any that is left over when Im finished. 4. Using a 10 ml pipette, the NT cells are pipetted in and out about 20 times. This helps to induce small lesions in the cells and increases the efficiency of the transformation. 5. 75 ul of bacteria (dense growth) or 100 ul (moderate growth) are added to a petri dish containing 4 ml of NT cells (from step 4) and mixed thoroughly. REMEMBER TO INCLUDE A CONTROL HAVING NO BACTERIA. 6. Wrap plates ...

Gene transfer to plants was first achieved more than twenty years ago. Since then, plant transformation technology has developed rapidly and in the last few years the benefits of this research have become apparent in the commercial production of engineered plants that are resistant to pests and diseases, plants with enhanced or modified traits and plants that are used as factories to produce valuable molecules. As with any expanding technology, it becomes difficult to find a concise and comprehensive source of information that explains all the underlying principles and brings together disparate techniques ...

Agrobacterium tumefaciens is the preferred method for transformation of a wide range of plant species. Commonly, the genes to be transferred are cloned between the left and right T-DNA borders of so-called binary T-DNA vectors that can replicate both in E. coli and Agrobacterium. Because these vecto …

This protocol is a variant of the Hanahan protocol [1] using CCMB80 buffer for DH10B, TOP10 and MachI strains. It builds on Example 2 of the Bloom05 patent as well. This protocol has been tested on TOP10, MachI and BL21(DE3) cells. See Bacterial Transformation for a more general discussion of other techniques. The Jesse 464 patent describes using this buffer for DH5α cells. The Bloom04 patent describes the use of essentially the same protocol for the Invitrogen Mach 1 cells. This is the chemical transformation protocol used by Tom Knight and the Registry of Standard Biological Parts. ...

This protocol is a variant of the Hanahan protocol [1] using CCMB80 buffer for DH10B, TOP10 and MachI strains. It builds on Example 2 of the Bloom05 patent as well. This protocol has been tested on TOP10, MachI and BL21(DE3) cells. See Bacterial Transformation for a more general discussion of other techniques. The Jesse 464 patent describes using this buffer for DH5α cells. The Bloom04 patent describes the use of essentially the same protocol for the Invitrogen Mach 1 cells. This is the chemical transformation protocol used by Tom Knight and the Registry of Standard Biological Parts. ...

The aim of this work was to develop a method to evaluate the kinetics of bainite transformation by theoretical deduction and thermal dilatation curve analysis. A Gleeble-3500 thermomechanical simulator and dilatometer (DIL805A) were employed to study the isothermal transformation in deformed (360 ∘ C , 600 ∘ C , and 860 ∘ C ) and undeformed conditions. The thermal dilatation information during isothermal transformation was recorded, and the dilatation curves were well smoothed. By taking a derivative of the dilation curve with respect to the transformation time, the peak time of transformation rate (PTTR) was obtained, which can serve as the essence of isothermal transformation time. The relative change of length ( Δ L / L ) due to phase transformation was theoretically deduced, and the effect of temperature was taken into consideration. Combing experimental data, the volume fraction of bainite in isothermal transformation was calculated. Making a graph of

This report presents studies on the growth conditions necessary for transformation to prototrophy of 14 auxotrophs of B. licheniformis. The unexpected finding of different growth requirements by each auxotroph for the development of transformable cells is discussed. Under optimum growth conditions for a serine-deficient mutant, transformation frequencies of 0.1% were obtained. A defined medium for tube transformation of competent cells is described. In addition, this report presents evidence for the transformation of three non-encapsulated mutants of B. licheniformis for the ability to synthesize polyglutamic acid (capsular material).

The state of Penang is an example of successful transformation in Asia based on strengthening the rule of law. Open and Distance Learning (ODL) is an important vehicle for the education that can underpin this process by expanding the freedoms that people can enjoy. Using technology can not only cut costs but also enhance the…

Our own rapid and highly successful transformation from a product company to a leading cloud services provider powered by the complete suite of Oracle Cloud services demonstrates that the Oracle Cloud is the only platform available to reimagine your possibilities and leapfrog others.

Hilal Deri, within the scope of its strategy in 2021, took care to balance its profitability level with its market share.. The company considers the development and transformation it deems necessary among its priorities in order to increase the service quality and strengthen its business processes in the rapidly growing sector globally.. The digital transformation studies carried out in coordination with the aim of transitioning to an agile and analytical organizational structure, aims to reach a structure that uses data-based analytics, quickly detects and adapts changes, and develops and implements digital business models by disseminating machine learning under the umbrella of Hilal Deri.. In the upcoming period, it will move forward in line with the goal of strengthening cost management in line with its vision and mission, implementing digital transformation in all possible business processes, creating fan customers and being an efficient and sustainable company for all our employees, ...

Existing methods for cloning and recombination of DNA enable construction of arbitrary sequences. However, the sequential nature of these techniques makes them time-consuming and expensive. Furthermore, while the transformation of an existing plasmid into a host strain can be reliable when a selection marker is used, there are many current limitations: the number of different plasmids that can be co-transformed is limited by the choice of markers and compatible origins of replication; plasmids are less stable than chromosomal DNA and are difficult to maintain indefinitely without mutation; and cistronic interactions cannot be designed since each new nucleotide sequence added is on an unconnected DNA molecule. To overcome these limitations, we are designing reconfigurable chromosomes consisting of both fixed and variable regions. While the fixed region is carefully optimized and tuned ahead of time, the variable region can be modified in the field, at the point-of-use, leading to rapid and ...

Existing methods for cloning and recombination of DNA enable construction of arbitrary sequences. However, the sequential nature of these techniques makes them time-consuming and expensive. Furthermore, while the transformation of an existing plasmid into a host strain can be reliable when a selection marker is used, there are many current limitations: the number of different plasmids that can be co-transformed is limited by the choice of markers and compatible origins of replication; plasmids are less stable than chromosomal DNA and are difficult to maintain indefinitely without mutation; and cistronic interactions cannot be designed since each new nucleotide sequence added is on an unconnected DNA molecule. To overcome these limitations, we are designing reconfigurable chromosomes consisting of both fixed and variable regions. While the fixed region is carefully optimized and tuned ahead of time, the variable region can be modified in the field, at the point-of-use, leading to rapid and ...

An efficient variety-independent method for producing transgenic eggplant (Solanum melongena L.) via Agrobacterium tumefaciens-mediated genetic transformation was developed. Root explants were transformed by co-cultivation with Agrobacterium tumefaciens strain LBA4404 harbouring a binary vector pBAL2 carrying the reporter gene \beta-glucuronidase intron (GUS-INT) and the marker gene neomycin phosphotransferase (NPTII). Transgenic calli were induced in media containing 0.1 mg$ l-^{1}$ thidiazuron (TDZ), 3.0 mg $l-^{1} N^{6}$-benzylaminopurine, 100 mg$ l-^{1}$ kanamycin and 500 mg l? cefotaxime. The putative transgenic shoot buds elongated on basal selection medium and rooted efficiently on Soilrite irrigated with water containing 100 mg$ l-^{1} $kanamycin sulphate. Transgenic plants were raised in pots and seeds subsequently collected from mature fruits. Histochemical GUS assay and polymerase vchain reaction analysis of field-established transgenic plants and their offsprings confirmed the ...

Ice plant (Mesembryanthemum crystallinum L.) is a model plant for studying salt-tolerant mechanisms in higher plants. Many salt stress-responsive ice plant genes have been identified with molecular and biochemical approaches. However, no further functional characterization of these genes in host plant due to lack of easy and effective transformation protocols. To establish efficient transformation system of ice plants, three types of ice plant materials, hypocotyl-derived callus, aseptically-grown seedlings and pot-grown juvenile plants, were used to develop Agrobacterium-mediated transformation protocols. The highest transient transformation efficiency was with 5-day-old ice plant callus co-incubated with an Agrobacterium tumefaciens at 2.5 × 109 cells mL−1 for 48 h. The 3-day-old ice plant seedlings with root tip removed were successfully infected with A. tumefaciens or A. rhizogenes, and obtained 85% and 33-100% transient transformation rates, respectively. The transient transformation assays in

Sarker, R.H. and Biswas, A. (2002) In Vitro Plantlet Regeneration and Agrobacterium-Mediated Genetic Transformation of Wheat (Triticum aestivum L.) Plant Tissue Culture, 12, 155-165.

Transformation Protocol. BY-2 (NT1) Cell Transformation with Agrobactrium. Day 1: 1. Grow up 1 ml of the Agrobacterium overnight in LB + all selective drugs. This culture may be started from frozen glycerol cultures if necessary. Day 2: 2. NT cells are used 3 days after splitting the NT cell culture. 4 ml of NT cells are required for each transformation with an additional 4 ml for the control culture which receives no bacteria. 3. 1 ul Acetosyringone (20 mM in ethanol) is added per ml of NT cells. Typically treat the whole 50 ml culture at this point and discard any that is left over when Im finished. 4. Using a 10 ml pipette, the NT cells are pipetted in and out about 20 times. This helps to induce small lesions in the cells and increases the efficiency of the transformation. 5. 75 ul of bacteria (dense growth) or 100 ul (moderate growth) are added to a petri dish containing 4 ml of NT cells (from step 4) and mixed thoroughly. REMEMBER TO INCLUDE A CONTROL HAVING NO BACTERIA. 6. Wrap plates ...

The developments in transformation technology have enabled the scientists to incorporate, mutate or substitute gene(s) leading to a particular trait; advancing it to a point where only few technical limitations remain. Genotype...

Plasmid transformation in Leuconostoc carnosum 4010 was analyzed. A successful transformation protocol for L. carnosum was established by modifying an existing protocol for Lactococcus lactis. Several parameters, including the number of generations that the cells had grown at the time of harvest, glycine concentration, the time of incubation for phenotypic expression, and the electrical field strength, were investigated and proved to have influence on the transformation frequency. Electrocompetence was found to be transient and to peak in the early exponential growth phase. Optimized conditions resulted in transformation frequencies of up to 6.7 x 10(5) transformants per microgram of plasmid DNA. A total of five plasmids in L. carnosum were successfully introduced and maintained. Interestingly, we discovered that DNA uptake was of a frequency of 3 x 10(-6) to 19 x 10(-6) transformants per CFU in the absence of an applied electrical field. We concluded that L. carnosum is naturally competent ...

Transgenic tomato plants of south Indian cultivar Arka Vikas were developed using Agrobacterium strain EHA 105, harbouring Bt Cry2A gene with a construct containing 35S CaMV promoter, OCS terminator and nptII selectable marker, through Agrobacterium-mediated transformation. This study was conducted to improve the regeneration and transformation protocol for south Indian cultivar Arka Vikas. Hypocotyl was used as explant source for transformation due to high regeneration efficiency, molecular analysis through PCR for putative transformants in T0 generation and qualitative ELISA method was performed for Bt protein expression followed by insect bioassays. Insect bioassay studies was conducted using neonate larva of helicoverpa armigera to screen the plants and the plants expressing good resistance with molecular and phenotypic characters were carried further for successive generations. The experimental results concluded that Bt gene was deployed in tomato cultivar successfully and had developed resistance

In ,i,Vanda,/i, orchids, it is important to produce cultivars with economically important traits such as disease and pest resistances and novel flower colors, which are difficult to achieve by conventional cross breeding methods. To realize these breeding objectives, it is now expected to apply genetic transformation technology to introduce useful foreign genes into ,i,Vanda,/i, orchids. However, there has been almost no information on the genetic transformation of ,i,Vanda,/i,. Transgenic plants were successfully regenerated after co-cultivating protocorm-like bodies (PLBs) with ,i,Agrobacterium tumefaciens,/i, strain EHA101 (pIG121Hm) that harbored genes for β-glucuronidase (,i,gus,/i,), hygromycin phosphotransferase (,i,hpt,/i,) and neomycin phosphotransferase II (,i,nptII,/i,). PLBs of Tokyo Blue maintained in liquid New Dogashima medium (NDM) under dark condition, were subjected to transformation experiments. The PLBs inoculated with ,i,Agrobacterium,/i, produced secondary PLBs 4 weeks ...

The study was carried out to evaluate the amenability of tropical inbred and hybrid maize lines, using Agrobacterium mediated transformation technique. Agrobacteriumtumefaciens strains EHA101 harbouri

Traditional image enhancement techniques produce different types of noise such as unnatural effects, over-enhancement, and artifacts, and these drawbacks become more prominent in enhancing dark images. To overcome these drawbacks, we propose a dark image enhancement technique where local transformation of the pixels have been performed. Here, we apply a transformation method of different parts of the histogram of an input image to get a desired histogram. Afterwards, histogram specification technique has been done on the input image using this transformed histogram. The performance of the proposed technique has been evaluated in both qualitative and quantitative manner, which shows that the proposed method improves the quality of the image with minimal unexpected artifacts as compared to the other techniques.

Zumba fitness total body transformation system dvd set does not offer t and nutrition advice or zumba fitness workout zumba fitness total body transformation system dvd set does not offer t and nutrition advice or

Sorghum is the fifth most widely planted cereal crop in the world and is commonly cultivated in arid and semi-arid regions such as Africa. Despite its importance as a food source, sorghum genetic improvement through transgenic approaches has been limited because of an inefficient transformation system. Here we report a ternary vector (also known as co-habitating vector) system using a recently described pVIR accessory plasmid that facilitates efficient Agrobacterium-mediated transformation of sorghum. We report regeneration frequencies ranging from 6-29% in Tx430 using different selectable markers and single copy, backbone free quality events ranging from 45-66% of the total events produced ...

DOBV RNA (strain DOB-90/5) was extracted from infected Vero E6 cells (CRL 1586; ATCC, USA). The genomic RNA of DOBV was reverse-transcribed and PCR amplified in order to generate the entire S-segment, using following oligonucleotide primers: 5-GCGAATTCGCAACACTAGAGGAACTCCAAAAGG-3 and 5-CGAAGCTTAGTGGTGGTGGTGGTGGTGAAGTTTGAGCGGCTCC-3. The amino-terminal part encoding the first 118 amino acids was generated using 5-CTGGCGCCTAACCGACGTGGTGGTGGTGGTGGTGATTCGAAGC-3 as reverse primer. In all constructs, a histidine (His) tag was introduced at the C-terminal end. PCR fragments were cloned in plasmids pTEX(rP40) or pTEXmp18 respectively with or without the inclusion of the P40 sequence in the construct. Following transformation of the E. coli ICONE 200 strain, the recombinant proteins were produced as intracellular inclusion bodies, recovered, and renatured. The recombinant proteins were purifi ed by metal chelate affinity chromatography using a HisTrap kit (Pharmacia, Puurs, Belgium). Using this ...

PubMed comprises more than 30 million citations for biomedical literature from MEDLINE, life science journals, and online books. Citations may include links to full-text content from PubMed Central and publisher web sites.

Plasmids, strains and primers used in this study are listed in Additional file 7: Table S1, Additional file 8: Table S2, Additional file 9: S3 1. Oligonucleotides and gBlocks were ordered from IDT and Eurofins. All fragments obtained by PCR were gel- or column purified (Nucleospin® Gel and PCR Clean-up columns) before cloning, and resulting plasmids were verified by sequencing (Eurofins). Yeast transformations were done using lithium acetate and PEG3350, and genomic integrations were performed with various helper plasmids and pre-expressed iCas9 from plasmid pCT (Addgene #60620) and plated on Sc-Leu+cloNAT. Strains were cured for pCT and helper plasmids after genome engineering and before proceeding to transcriptional regulation using dCas9.. EasyClone-MarkerFree vectors pCfB2909, pCfB3035, pCfB3037 and helper plasmids pCfB3042, pCfB3046, and pCfB3050 as well as genomic integration verification primers were adapted as previously described [46]. Yeast strains were plated according to ...

Author(s): Jain, Rashmi; Jenkins, Jerry; Shu, Shengqiang; Chern, Mawsheng; Martin, Joel A; Copetti, Dario; Duong, Phat Q; Pham, Nikki T; Kudrna, David A; Talag, Jayson; Schackwitz, Wendy S; Lipzen, Anna M; Dilworth, David; Bauer, Diane; Grimwood, Jane; Nelson, Catherine R; Xing, Feng; Xie, Weibo; Barry, Kerrie W; Wing, Rod A; Schmutz, Jeremy; Li, Guotian; Ronald, Pamela C | Abstract: BACKGROUND:The availability of thousands of complete rice genome sequences from diverse varieties and accessions has laid the foundation for in-depth exploration of the rice genome. One drawback to these collections is that most of these rice varieties have long life cycles, and/or low transformation efficiencies, which limits their usefulness as model organisms for functional genomics studies. In contrast, the rice variety Kitaake has a rapid life cycle (9 weeks seed to seed) and is easy to transform and propagate. For these reasons, Kitaake has emerged as a model for studies of diverse monocotyledonous species. RESULTS

I dont know how to choose helper plasmid.Would u please give me some advice on which helper plasmid better and how I can get it?thirst for ur reply as soon as possible ...

The techniques of plant transformation are discussed. They are bacterial-mediated, electroporation transformation, biolistic (particle bombardment), and microbeam laser. By using different strategies, foreign gene(s) from various organisms can be inserted into plant genome and expressed. Bacteria-mediated transformation uses Agrobacterium as a vector to insert genes of interest. The other three directly deliver the plasmid into the plant. While Bacteria-mediated method is relatively simple, the other three can transform both monocot and dicot plant, and use various plant tissues as target cells. Public concerns about the impact that transgenic plant might have on the environment have partly slowed down the large-scale use of transgenic plants. These concerns are discussed. Some suggestions regarding the releasing of the transgenic plant under field conditions are also discussed.

Radioactive Elements: Elements which exhibit atomic emission due to natural or artificial nuclear transformation. These elements spontaneously undergo radioactive decay.

Nuclear medicine Pet/Spect. Chapters 18 to 22. Activity. Number of radioactive atoms undergoing nuclear transformation per unit time. Change in radioactive atoms N in time dt Number of radioactive atoms decreases with time (- minus sign). Activity. Expressed in Curie Slideshow 83492 by...

PubMed comprises more than 30 million citations for biomedical literature from MEDLINE, life science journals, and online books. Citations may include links to full-text content from PubMed Central and publisher web sites.

Tony Zapata, a NearShore Technology, Project Services Manager, discusses the various implications of developing a Test Automation culture. The first step to a successful transformation involves a commitment to automation across all silos within the organization. In reality, automation testing benefits both developers and testers.. NearShore Technologys Automation Culture. The testing phase of an implementation project is where DevOps and QA teams truly come together. And, as Mr. Zapata suggests, the NearShore team abides by the words of George, E.P. Box, Discovering the unexpected is more important than confirming the known. Everyone needs to be aware of the technology stack and how each piece of the framework affects their roles in the organization. What does this mean for your team? As developers code new environments, tests need to be pulled automatically. Before these reports can be pulled without a human, all teams need to understand the applications and develop their own code, a place ...

Author(s): Propp, Douglas A | Abstract: Our emergency department had always relied on a paper-based infrastructure. Our goal was to convert to a paperless, efficient, easily accessible, technologically advanced system to support optimal care. We outline our sequential successful transformation, and describe the resistance, costs, incentives and benefits of the change. Critical factors contributing to the significant change included physician leadership, training and the rate of the endorsed change. We outline various tactics, tools, challenges and unintended benefits and problems. [West J Emerg Med. 2012;13(4):358-361.]

Trans5α Chemically Competent Cell,Cloning Competent Cells,Cloning and Mutagenesis System,Products,Beijing TransGen Biotech Co.Ltd,OverviewContents& storageCitations & referencesRelated ImagesDownloadOverviewDescriptionTrans5&alpha Chemically Co

ALPHA-ARBUTIN is one of the most efficient skin lightener, widely accepted and has been rated well by the consumers. Using specific enzyme transformation technology high purity is guaranteed ...

The soil organism Bacillus subtilis has the ability to take up DNA from its environment and, provided there is homology, recombine it into its genome. This process is called transformation. In order for the bacterium to be transformed it must be in a specific physiological state called competence. During the competent state specific proteins are synthesized which are required for the binding and transport of the DNA molecule into the competent cell. It has been found that as transformability increases many competence specific proteins localize to the poles of the rod shaped Bacillus subtilis bacterium and as transformability declines the competence proteins delocalize. These proteins colocalize and interact and seem to form a complex that helps internalize the DNA molecule, preferentially at the poles of the cell. Jeanette Hahn s focus is understanding how the polar localization and delocalization of the competence proteins occurs. Localization seems to occur via a diffusion and capture ...

Hello all- I am in need of a good all around human endothelial cell line for use in viral transformation studies, as well as studies of certain viral genes. I have looked through some of the thousands at ATCC, but most have been derived from leukemias, etc. I would prefer one that is normal (as normal as a culturable cell line can be). The big restriction is that I am looking for an endothelial line. Thanks in advance. Kajetan -------------- next part -------------- A non-text attachment was scrubbed... Name: Kajetan-Groicher.vcf Type: text/x-vcard Size: 427 bytes Desc: Card for Kajetan H. Groicher Url : http://iubio.bio.indiana.edu/bionet/mm/methods/attachments/19991110/240b4f7f/Kajetan-Groicher.bin ...

293 Cell Line; Recommended culture Medium: EMEM (EBSS) + 2 mM glutamine + 1% non essential amino acids (NEAA) + 10% fetal bovine serum (FBS). 293 Cell Line; Transformation studies. Bulk and Prepack available at Sigmaaldrich.com.

A little while back someone posted that Strategenes SURE cells harbour a Tn5 transposon. Has anyone had any problems with these cells? We have been having problems with what looks like homologous recombination events with pBIN-based plasmids and with another group of plasmids which have an insert repeat. To date we have been using Novagens Novablue competent cells. Has anyone has any problems with these cells? Our transformation efficiency is not really a problem. Its just that we appear to be losing chunks between sequences that are repeated in the plasmids. The NovaBlue cells are supposed to be recA1 mutants so Im at a quandry as to what is going on. My druthers at present are to switch to a massively recombination defficient strain like SURE; hoping that that may solve the problem. Problem is I dont know anyone with experience with SURE cells. Any advice for what should be routine subcloning experiments most appreciated. Best regards, Malcolm ...

For transformation of PCR-TRAP and pAPtag-5 vectors. The GH Competent cells can be used with both our PCR-TRAP PCR product cloning system & with our pAPtag-5 AP fusion cloning vector (AP-TAG Kit B). The pAPtag-5 vector contains the ampicillin resistance gene. It can be easily and efficiently transformed and propagated in GH Competent cells.. 3 mLs (6 x 0.5 mL tubes) Detailed protocol included.. ...

First, lets define it. Digital transformation utilizes technology and software to transform the way a business operates. We are currently undergoing a cloud-based transformation, with 69% of enterprise organizations migrating ERP applications data to the cloud.(1) A digital transformation can offer an elevated customer experience, increase productivity, and create a wealth of new business opportunities. The IDC predicts that spending on such technologies will hit $1.97 trillion in 2022.(2). Why the Resistance? Its not an easy task to fundamentally rethink the way a business has been operating for years. Leaders and people within an organization get comfortable with processes, future projections, and predicable revenue streams. Thought leaders worry their colleagues may not want to change and have to ask themselves if now the time is to adopt new practices. These thoughts are completely understandable but will leave you in a mediocre state of business. The businesses that truly thrive and ...

Patient flow at a crawl, outcome measures flagging and clinician tensions running high. Sound like an average day in your hospitals emergency department? If so, it might be time for a total ED transformation.. Transformation can be complex and dependent on a multitude of factors. Fill out this form to receive a whitepaper on the universal strategies to consider when taking on an ED transformation project and address the problem at its root - and see how Sharp Grossmont Hospital in La Mesa, Calif., committed to ED overhaul and succeeded.. Please fill out the form to download the whitepaper.. ...

Transformation is a process by which recipient calls acquire genes from free DNA molecules in the surrounding medium. In other words, this is the transfer of genes from free DNA molecules in the surrounding medium into a recipient cell. Transformation starts the uptake of a DNA fragment from the neighbouring medium by the recipients cell and ends with one strand of donor DNA substituting the homologous segment in the recipient DNA. It has to be noted that even amongst cells of a species that is generally capable of transformation, only some cells in a growing population are efficient in the uptake of the free genetic information. The main experiment linked to this was Griffiths work with smooth virulent (Deadly) strain of Streptococcus pneumonia and rough (non-deadly) strain on Mice. The smooth appearance of the deadly colony is caused by a carbohydrate coating on the surface of each cell, which acts as protection against the mouses immune system and therefore allows the colony to thrive. ...

Quantitation of β-galactosidase activity. In yeast cells, co-transformed with pGADT7 (AD) and pGBKT7 (BD) constructs as indicated, β-galactosidase activity wa

I Have Thin Hair and These Are My 7 Most Effective Tips to Care for It the scalp and are looking for a solution to your perpetual question of how to thicken hair.. Get Price ...

Krech, K.; Fu, H.-Y.; Thiele, W.; Ruf, S.; Schöttler, M. A.; Bock, R.: Reverse genetics in complex multigene operons by co-transformation of the plastid genome and its application to the open reading frame previously designated psbN. The Plant Journal 75 (6), pp. 1062 - 1074 (2013 ...

HI-Control™ BL21(DE3) and HI-Control™ 10G Competent Cells Induce high level protein expression from lac based promoters (T7, T5-lac, tac, trc, lac) Tightly regulate expression from T7 promoters with HI-Control BL21(DE3) Chemically Competent Cells Clone targets and reduce background protein expression with HI-Control 10

[106 Pages Report] Check for Discount on United States Clone Competent Cell Market Report 2017 report by QYResearch Group. In this report, the United States Clone Competent Cell market...

[183 Pages Report] Check for Discount on Global Expression Competent Cells Market Professional Survey 2019 by Manufacturers, Regions, Countries, Types and Applications, Forecast to 2024 report by HJ Research. The Expression Competent Cells market was valued at XX Million...

Revision history for the Hematopoietic and Lymphoid Neoplasm Coding Manual and Database: diagnostic confirmation, M rules, primary site/histology coding, grade.

Well, my stupid friends, I can somewhat see where your logic originates from. On the surface, a baby does indeed seem like a parasite. It feeds off of the host mommys body and intakes her food supply in order to grow healthy and strong. It can sometimes make the host mommy sick. Even the placenta masks itself from the host mommys immune system like a parasite does.. But thats where the similarities end, and the glaring fact that a baby is not a parasite begins.. Like a cloud in the sky is not actually floating cotton because its white and fluffy, a baby is not a parasite just because its a growing organism inside someones body. Bearing some similarities does not a perfect comparison make, especially due to the starkness of its differences. Just because I can kill the electricity by shooting at powerlines, it doesnt make my AR-15 a light switch.. For one, a baby in the womb is homospecific. This means that both mommy and baby are the same species, and mommys body is specifically geared to ...

Agrobacterium-medieret transformation ved hjælp af en blomster-dip metode kan anvendes med succes at skabe stabile transgene linjer af...

Read how Accentures Digital Workplace Transformation can help transform IT infrastructure to a flexible, efficient and responsive workplace environment.

Accentures Digital Workplace Transformation can help transform IT infrastructure to a flexible, efficient and responsive workplace. Find out how.