TY - JOUR. T1 - Automated Yeast Transformation Protocol to Engineer Saccharomyces cerevisiae Strains for Cellulosic Ethanol Production with Open Reading Frames That Express Proteins Binding to Xylose Isomerase Identified Using a Robotic Two-Hybrid Screen. AU - Hughes, Stephen R.. AU - Rich, Joseph O.. AU - Bischoff, Kenneth M.. AU - Hector, Ronald E.. AU - Qureshi, Nasib. AU - Saha, Badal C.. AU - Dien, Bruce S.. AU - Liu, Siqing. AU - Jackson, John S.. AU - Sterner, David E.. AU - Butt, Tauseef R.. AU - LaBaer, Joshua. AU - Cotta, Michael A.. PY - 2009/8. Y1 - 2009/8. N2 - Commercialization of fuel ethanol production from lignocellulosic biomass has focused on engineering the glucose-fermenting industrial yeast Saccharomyces cerevisiae to use pentose sugars. Because S. cerevisiae naturally metabolizes xylulose, one approach involves introducing xylose isomerase (XI), which catalyzes conversion of xylose to xylulose. In this study, an automated two-hybrid interaction protocol was used to find ...
Plant transformation provides a promising methodology of introducing new genes that encode desirable traits to a wide range of crop plants. Success in genetic transformation has been achieved in many of the important crop species, such as soybean, cotton, rice, corn. However, wheat, one of the major crops of the world, has been considered to be difficult to transform via either Agrobacterium or biolistic bombardment (Rakszegi et al., 2001). There have been limited studies on A. tumefaciens-mediated transformation of cereals, including wheat, because of the overall refractory character of host-pathogen interactions between Agrobacterium and the cereal plants (Gould et al., 1991; Hiei et al., 1994; Cheng et al., 1997). While the genetic transformation of rice using Agrobacterium has become routine, only a few successful studies of Agrobacterium- mediated transformation of wheat have been reported, and these involved a model spring wheat, Triticum aestivum cultivar Bobwhite (Cheng et al., 1997). ...
Transformation efficiency is the efficiency by which cells can take up extracellular DNA and express genes encoded by it. This is based on the competence of the cells. It can be calculated by dividing the number of successful transformants by the amount of DNA used during a transformation procedure. Transformants are cells that have taken up DNA (foreign, artificial or modified) and which can express genes on the introduced DNA. Transformation efficiency should be determined under conditions of cell excess. The number of viable cells in a preparation for a transformation reaction may range from 2×108 to 1011; most common methods of E. coli preparation yield around 1010 viable cells per reaction. The standard plasmids used for determination of transformation efficiency in Escherichia coli are pBR322 or other similarly-sized or smaller vectors, such as the pUC series of vectors. Different vectors however may be used to determine their transformation efficiency. 10-100 pg of DNA may be used for ...
Frozen-EZ Yeast Transformation II Kit from Zymo Research,The transformation of Saccharomyces cerevisiae, as well as other yeasts, has become a routine procedure in many molecular biology laboratories. The ability to transform whole cells at high efficiencies with plasmid and linear DNAs using lithium based buffers and polyethylene glycol has made routine,biological,biology supply,biology supplies,biology product
Nuclear transformation occurs when the atoms of one element change to become atoms of another element. This is most commonly seen with...
DNA-mediated transformation provides a powerful tool for both genome analysis and gene manipulation. Advances in transformation procedures of filamentous fungi have played a key role in making these...
Various plant species are typically transformed by one of three methods. Arguably, the simplest and most preferable of these methods is transformation using a species of bacteria, Agrobacterium tumefasciens. Agrobacterium naturally transforms its host plants with DNA that causes tumors or galls to grow on the host. It accomplishes this by altering hormone levels in the host plant. The tumorous growth produces ideal tissue for the bacteria to infect. In order to use Agrobacterium for plant biotechnology, researchers replace the tumor-inducing piece of DNA, or plasmid, with DNA that encodes the genes they want to engineer into the host plant. Depending on the host species, this type of transformation can be very simple or can be quite challenging. A general advantage of this transformation strategy is that it typically leads to only one or a few copies of the engineered DNA being introduced to the plant genome, which helps to ensure stable gene expression (i.e. the engineered genes will usually ...
Track 11 : Genetic Transformation. Genetic Transformation is the genetic alteration of cell resulting from the direct uptake and incorporation of exogenous genetic material from its surroundings through the cell membrane. Transformation is one of three processes for horizontal gene transfer, in which exogenous genetic material passes from bacterium to another, the other two being conjugation transfer of genetic material between two bacterial cells in direct contact and Transduction injection of foreign DNA by a bacteriophage virus into the host bacterium. And about 80 species of bacteria were known to be capable of transformation, in 2014, about evenly divided between Gram-positive and Gram-negative Transformation may also be used to describe the insertion of new genetic material into non-bacterial cells, including animal and plant cells.. Related Molecular Biology Conferences ,Genetics Conferences, Gene Therapy Conferences, Biotechnology Conferences. 13th European Pathology Congress, Milan, ...
Artificial competence of Synechococcus PCC 6301 cells was induced by lysozyme treatment and the cells were transformed to chloramphenicol resistance with foreign plasmid pBR325 at a frequency of approximately 5 times 10-5 or 5 times 10-4 with the transformant DNA. The transformation frequencies were higher than those reported by other workers for the same strain with cloned DNA employing a physiological transformation system. Analyses of DNA electrophoresis, secondary transformation and dot blotting demonstrated that foreign plasmid had integrated into the recipient chromosome by a single crossover event. The results showed that the artificial transformation system was efficient and reproducible. Conditions that affected transformation, such as, incubation time of cells with DNA, age of the cells, light or dark incubation were also studied ...
Creation of a Bacterial Cell Controlled by a Chemically Synthesized Genome: http://www.sciencemag.org/content/329/5987/52.abstract Automated seamless DNA co-transformation cloning with direct expression vectors applying positive or negative insert selection: http://www.biomedcentral.com/1472-6750/10/56 Reports the use of Seamless Enzyme-Free Cloning (or in their words, Co-transformation cloning to produce a large library. The E.coli strain Mach1 yields most colonies, but a few other strains like DH5alpha and Top10 work also. Co-transformation employs chemically competent cells [7] yielding 107 or more colonies per μg plasmid. Per co-transformation 200 ng of vector plus 50-500 ng of insert were mixed and the competent cells added to the DNA mixture which was less than 10% of the cell volume (50 μL cells). ...
Molecular Cloning, also known as Maniatis, has served as the foundation of technical expertise in labs worldwide for 30 years. No other manual has been so popular, or so influential.
Hi John! We are currently using a lithium acetate-DMSO method - grow O/N culture in 2 ml - spin down in a 2 ml-Eppi - remove supernatant, add 0.01 ml carrier DNA (= 0.1 mg) and transforming DNA in minimal volume, vortex briefly - add 0.5 ml PEG (40% 3350, 0.05 ml 10xLiOAc, 0.05 ml 10xTE, H2O ad 0.5 ml) - add 0.051 ml DMSO - shake 15 min RT - shock cells for 15 min at 42C - spin down, wash the pellet with TE, resuspend in TE and plate on selective plates Hope this helps Anne ...
This Driving Digital: The Leaders Guide to Business Transformation Through Technology book is not really ordinary book, you have it then the world is in your hands. The benefit you get by reading this book is actually information inside this reserve incredible fresh, you will get information which is getting deeper an individual read a lot of information you will get. This kind of Driving Digital: The Leaders Guide to Business Transformation Through Technology without we recognize teach the one who looking at it become critical in imagining and analyzing. Dont be worry Driving Digital: The Leaders Guide to Business Transformation Through Technology can bring any time you are and not make your tote space or bookshelves grow to be full because you can have it inside your lovely laptop even cell phone. This Driving Digital: The Leaders Guide to Business Transformation Through Technology having great arrangement in word and layout, so you will not really feel uninterested in reading.. ...
Science Behind Cotton Transformation. By Abdul Qayyum Rao, Muhammad Azam Ali, Muhammad Azmat Ullah Khan, Kamran Shahzad Bajwa, Adnan Iqbal, Tahir Iqbal, Ahmad Ali Shahid, Idrees Ahmad Nasir and Tayyab Husnain. The introduction of foreign genes into plant has made possible to bring out desired traits into crop of our own interest. With the advancement in cell biology, regeneration of plants from single cell and advent of different procedures for gene transformation to the plants have opened new avenues for the efficient and applicable implementation of biotechnology for the modifications of desired crop characteristics. Identifications and isolation of different genes for various traits from different organisms have made possible to get the crop plants with modified characters. Over time improvement has been made in transformation technology depending upon the crop of interest. The efficiency of plant transformation has been increased with advances in plant transformation vectors and ...
If enterprises are going to succeed in their digital transformations, they need to be able to shake-up and challenge the status quo within their
Pamela A. Trail, Ph.D., to transition from Chief Scientific Officer to strategic consultant; Daniel Steiner, Ph.D., to assume leadership of research organizatio
Hello researchers, , ,I am study one gene promoter and fused it to GUS in pCAMBIA1391Z, in which ,CaMV35S drives the selectable marker Hyg gene. Among the 10 independent T3 ,lines, which were confirmed by PCR, some show very strong GUS staining, ,some no, and some weak staining. , ,I knew from this paper that vectors containing 35S promoter driving ,selectable marker gene are not good for promoter study. ,The 35S promoter used in a selectable marker gene of a plant transformation ,vector affects the expression of the transgene. Planta. Volume 221, Number ,4 / June, 2005 , ,But I found that quite many published papers used vectors (like ,pCAMBIA1391, pCAMBIA1301, etc) containing 35S promoter driving selectable ,marker. , ,As the staining patterns of my promoter fusion lines are not consistent at ,al, I want to use another vector pBI121 or pCGN1547. Does anyone know where ,to get pCGN1547? ,How do you deal with those lines from 35S containing vector? CAMBIA ,suggests doing co-transformation ...
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HMS174(DE3) Competent Cells - Novagen HMS174 strains provide high transformation efficiencies and the recA mutation in a K-12 background. Strain may stabilize certain target genes whose products may cause the loss of the DE3 prophage. - Find MSDS or SDS, a COA, data sheets and more information.
The transformation efficiency (TE) for the 96-well plate format NEB 5-alpha Competent |em|E.coli|/em| (NEB #C2987P) is as high as the two other formats, NEB #C2987H and NEB #C2987I.
As vaccines roll out across the world and cities and countries begin to lift restrictions and open up, it may seem like the world is ready to move on. However, the healthcare industry in particular will continue to face new challenges and unexpected, long-lasting impacts from the COVID-19 pandemic that will take time and innovation to recover from. In order to bounce back from such an unprecedented event, the medical industry is going to need to find ways to improve efficiency without cutting corners or lowering standards. Digital transformation technology offers a lot of opportunities for just that. By enabling automation of mundane and repetitive tasks, centralizing communication, and making information more readily accessible across vast distances, digital technology is revolutionizing the way business is done, and may hold the key to getting the healthcare industry back on track post-pandemic.. A full-scale digital transformation can be a massive undertaking, so it often helps to break it ...
An image processing method is provided for reducing noise in a sampled image, particularly for reducing noise in an image divided into blocks of sampled image elements that are transformed by a linear procedure, such as the Walsh-Hadamard transform, and improved regarding visible noise by non-linear thresholding of the transform coefficients. By operating the process in a hierarchy of stages, each stage employing a block operating on image signals derived from a preceding stage, and by overlapping the blocks processed in each stage, the processed signal from each image element is the linear combination of many transform coefficients from each stage and from each overlapped block within each stage. Such a large number of contributions making up each processed image element assures that the processed image is generated without a characteristic block-like structure due to block transform processing while the wanted components of the image are rendered with minimal image loss or distortion.
is provided, focused on the role played by the different components of the virulence system. The general assessments for the establishment of efficient transformation protocols are discussed with an emphasis in the application of this methodology to monocotyledonous plants. Based on our own experience, we present the establishment of sugarcane transformation by A. tumefaciens as a model of application of this methodology to an important culture plant species, previously considered recalcitrant and inaccessible for this type of genetic manipulation ...
Fluorescent Protein Transformation Student Background Genetic transformation occurs when a cell takes up (i.e. takes inside) and expresses a new piece of genetic material DNA. Genetic transformation literally
The expression of these genes is regulated by an insertion sequences. This could be due to a random mutation and would not affect the overall effectiveness of the antibiotic. In Salmonella there are two genes which code for two antigenically different flagellar antigens. In the recipient a generalized recombination event can occur which substitutes the donor DNA and recipient DNA (See Figure 2). Others are interested in creating genetically modified cells to further scientific understanding of genetics, or for new fields of medical treatments. Transduction. Transformation - you absorb DNA from around you and transform (could be ⠦ The ability of a phage to mediated transduction is related to the life cycle of the phage. β-lactamase, b) Alteration of target site - e.g. Transduction is the transfer of genetic information from a donor to a recipient by way of a bacteriophage. Conjugation occurs between two living cells, involves cell to cell contact, and requires mobilization of either a ...
This was surprising, as Id expected that the low transformation frequencies of such partially-induced cultures would be because the cells were all only a little bit competent. We see this max-or-nothing pattern in not only in wild-type cells under poorly-inducing conditions, but also in low-competence mutants under fully inducing conditions and in hypercompetent mutants under what are otherwise non-inducing conditions. Ive had this puzzling result hanging around in the back of my brain for about 15 years ...
Since 1976, CGI has helped more than 2,000 government clients in 15 countries improve citizen service and operational efficiency through successful transformation programs.
The B73 inbred line is the source of our community reference genome. However, B73 cannot be transformed, making it difficult to make use genomic data for genetics studies that involve transformation. Our motivation was to identify an inbred line similar to B73 for plant transformation. Both B73 and B104 are derived from the Iowa Stiff Stalk Synthetic lines. Unlike B73, B104 is readily transformed. The ISU Plant Transformation Facility now offers transformation services for the B104 inbred line. They share about 93% similarity as calculated using TIPSimSelector at MaizeGDB, a tool that assess genetics similarity based on methods described in Romay et al., ...
Edvotek Series 200 Experiments. For 10 Transformations and controls. Time Required: Transformation -- 45 min.; Plating -- 5 min.; Incubation -- overnight; Transformation Efficiency …
Edvotek Series 200 Experiments. For 10 transformations and controls. Time required: Transformation 45 min., Plating 5 min., Incubation overnight, Transformation efficiency 15 min. …
Yesterdays transformed competent cells of the 014C, 020C, 405C, 327C and 304C ligation mixes (using the BioBrick assembly method) yielded only red colonies, indicating the lack of positive colonies. It is interesting to note the presence of numerous red colonies on the digest control plate, whereas the ligation control plate (Digested pSB1C3 + ligase buffer + ligase) yielded but 1 red colony (at similar trasnsformation mix concentrations). Based on the information gathered thus far a few hypotheses were made: Hypothesis I: The ligase buffer inhibits transformation efficiency in some way. Hypothesis II: The ligation time of 20 mins was too short. In order to test hypothesis I, the following mixes will be transformed into commercial competent cells: ...
talk , contribs) (Created page with In prokaryotes, a cell that has been genetically altered through the uptake of foreign DNA. In higher eukaryotes, a cultured cell that has acquired a malignant phenotype.) ...
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 ...
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. ...
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 ...