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What Is Transformation In Biology

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What Is An Example Of Bacterial Transformation

Transformation in Bacteria

Examples of Bacterial Transformation The first and most prominent example of bacterial transformation is the transformation of DNA from smooth capsule-positive colonies of Streptococcus pneumonia to the rough capsule-negative colonies. This was the first mechanism of bacterial genetic exchange to be recognized.

Attachment Of A Tumefaciens To Plant Cells

Bacterial attachment to host cells is a crucial early step in disease development for many plant and animal pathogens. The isolation of A. tumefaciens mutants unable to bind to plant cells led to the identification of several chromosomal virulence genes, chvA, chvB, and pscA, required for the attachment processes. chvA, chvB, and pscA are involved in the synthesis, processing, and export of cyclic Ã-1,2-glucan and other sugars . These mutants are either avirulent or highly attenuated in virulence under many inoculation conditions . During attachment, the A. tumefaciens cells synthesize cellulose fibrils that entrap large numbers of bacteria at the wounded sites . The cellulose deficient A. tumefaciens mutant is still able to attach to the carrot cell surface but cannot form aggregates . These mutants are virulent but are more susceptible to being washed off the plant cells . Thus, the major role of cellulose fibrils may be in aiding attachment of A. tumefaciens to the plant cell.

Selection Of Bacterial Transformants

Transformants can be selected from non-transformant by the property of antibiotic resistance.

For example, you can ligate a foreign DNA at the BamH I site of the tetracycline resistance gene in the vector pBR322. The recombinant plasmids will lose tetracycline resistance due to the insertion of foreign DNA but can still be selected out from non-transformants ones by plating the transformants on an ampicillin-containing medium. The transformants growing on an ampicillin-containing medium are then transferred on a medium containing tetracycline. The recombinants will grow in an ampicillin-containing medium but not on that containing tetracycline. But, nonrecombinants will grow on the medium containing both the antibiotics

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Reverse Genetic Screening For Plant Genes Involved In Agrobacterium

Plant genes encoding several proteins that interact with virulence proteins have been identified using a yeast two-hybrid system. Such interactions are at best suggestive of a role for these genes in plant transformation. Their roles must be shown directly. One way to accomplish this is to inhibit gene expression in planta using techniques such as antisense RNA, RNAi, or mutagenesis. I have discussed above the use of antisense RNA and RNAi to show that VIP1 , a Rab GTPase, and several proteins of unidentified function are involved in Agrobacterium-mediated transformation. Suppression of expression of these genes may be one method to generate plants resistant to crown gall disease.

Escobar et al. have recently described a novel reverse genetic strategy to produce crown gall-resistant plants. They generated transgenic Arabidopsis and tomato plants expressing double-stranded RNA constructions targeted to T-DNA-encoded auxin and cytokinin biosynthetic oncogenes. These genes are highly homologous among a large variety of different Agrobacterium strains. Many transgenic plants expressing these RNAi constructions were highly resistant to crown gall disease directed by a broad range of oncogenic strains, although they were not generally resistant to Agrobacterium-mediated transformation per se. A similar approach has been used to generate crown gall disease-resistant tobacco and apple plants .

Cytoplasmic Trafficking And Nuclear Import Of T

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Both VirD2 and VirE2 proteins contain plant-active nuclear localization signal sequences that are believed to be involved in T-DNA import into the nucleus. By the yeast interaction trap approach, several plant proteins that interact with either VirD2 or VirE2 proteins were identified . Two VirD2-interacting proteins, ROC1 and the CyP A proteins of Arabidopsis, belong to a large cyclophilin family of peptidyl-prolyl cis-trans isomerases, which are highly conserved in plants, animals, and prokaryotes . The VirD2 domain interacting with the cyclophilins is in the central region of VirD2 protein, which is distinct from the nuclear localization signal domains . Although the biological role of cyclophilins in A. tumefaciens infection is unclear, they were proposed to maintain the proper conformation of VirD2 within the host cell cytoplasm and/or nucleus during T-DNA nuclear import and/or integration . Another cellular factor encoded by a tomato protein phosphatase 2C interacted with the VirD2 NLS region . An Arabidopsis abi1 mutant showed higher sensitivity to A. tumefaciens transformation than did the wild-type plant, whereas over-expression of PP2C in tobacco protoplasts inhibited nuclear import of a Ã-glucuronidase-VirD2 NLS fusion protein . These data suggested that phosphorylation of the VirD2 protein near the NLS region may influence the import of the T-complex.

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Integration Into The Plant Genome And Expression Of The T

T-DNA integration into the plant cell genome is the final step of the Agrobacterium-mediated transformation process. Unlike other mobile DNA elements such as transposons and retroviruses, the T-DNA does not encode enzymatic activities required for integration. Thus, T-DNA insertion into the plant DNA must be mediated by proteins transported from A. tumefaciens itself, namely VirD2 and VirE2, and/or host cell factors.

VirD2 might play a dual role in the T-DNA integration process, ensuring both its fidelity and efficiency. The integration of the 5â² end of the T-strand into plant DNA is generally precise. This may result from the linkage of VirD2 to the 5â² end of the T-strand, which provides protection from exonucleases inside the plant cells . A region in the C-terminal portion of VirD2 called the Ï domain is implicated in the integration process . However, it is not clear how exactly the Ï domain of VirD2 is involved in T-DNA integration because the pattern of integration of the T-DNA is not affected by deletion of the Ï sequence . Involvement of VirD2 in protecting the integrity of the right border was also demonstrated in a fungal transformation system this study further showed that VirE2 similarly protects the left end of the T-DNA, while VirC2 helps ensure correct processing. Importantly, VirC2 is also required for single-copy integration in this system .

Examples Of Bacterial Transformation

  • The first and most prominent example of bacterial transformation is the transformation of DNA from smooth capsule-positive colonies of Streptococcus pneumonia to the rough capsule-negative colonies. This was the first mechanism of bacterial genetic exchange to be recognized.
  • Neisseria and H. influenzae take up DNA from their own species which occurs by species-specific recognition.
  • Natural bacterial transformation is also observed in the case of B. subtilis.
  • Verma PS and Agarwal VK . Cell Biology, Genetics, Molecular Biology, Evolution and Ecology. Multicolored Edition.
  • McGee, David & Coker, Christopher & Harro, Janette & Mobley, Harry. . Bacterial Genetic Exchange. Doi: 10.1038/npg.els.0001416.
  • Griffiths AJF, Miller JH, Suzuki DT, et al. An Introduction to Genetic Analysis. 7th edition. New York: W. H. Freeman 2000. Bacterial transformation.Available from: https://www.ncbi.nlm.nih.gov/books/NBK21993/

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Definition Of Transformation In Biology

Transformation is an important component of molecular genetics studies into the process began in the 1920s when a physician named F.Griffith realized that Streptococcus pneumoniae could convert between being harmless and disease-causing.What is transformation in biology? The theory he had back then was that a transforming principle released by dead S.pneumoniae cells caused living cells around them to form a capsule membrane that turns the pathogen into its disease-causing form. This theory has since been expanded upon with the discovery of DNA.

So, what is the meaning of transformation in biology? Transformation is one of the three processes where genetic material is transferred from one microbial cell to another, the other two being conjugation and archaeal DNA transfer.

Selection And Screening In Plasmid Transformation

DNA transformation in bacteria

Because transformation usually produces a mixture of relatively few transformed cells and an abundance of non-transformed cells, a method is necessary to select for the cells that have acquired the plasmid. The plasmid therefore requires a selectable marker such that those cells without the plasmid may be killed or have their growth arrested. Antibiotic resistance is the most commonly used marker for prokaryotes. The transforming plasmid contains a gene that confers resistance to an antibiotic that the bacteria are otherwise sensitive to. The mixture of treated cells is cultured on media that contain the antibiotic so that only transformed cells are able to grow. Another method of selection is the use of certain auxotrophic markers that can compensate for an inability to metabolise certain amino acids, nucleotides, or sugars. This method requires the use of suitably mutated strains that are deficient in the synthesis or utility of a particular biomolecule, and the transformed cells are cultured in a medium that allows only cells containing the plasmid to grow.

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Transforming Bacteria With Recombinant Plasmid

Inserting a gene into a plasmid vector is an important first step in the gene cloning process. However, if the ultimate goal is to produce a large amount of a particular protein, the plasmid must replicate to make sure that there are many copies of the gene and the gene of interest must be expressed, meaning the gene is utilized to produce the encoded protein. Both activities can only occur inside a cell. Therefore, in this lab we will put a recombinant plasmid into E. coli bacteria through a process that is called transformation, so named because it changes the DNA content of the bacteria.

The plasmid will be taken up by bacteria where it replicates, and its genes will be expressed using the bacterial cellular machinery. If a gene of interest has been inserted into the plasmid vector, the bacteria produces the product encoded by that gene.

In this exercise, you will carry out the transformation of E. coli bacteria using a recombinant plasmid that contains a gene that produces colored proteins.

What Is Transformation In Biology

Transformation is the specific process where exogenous genetic material is directly taken up and incorporated by a cell through its cell membrane. This usually occurs when the cell is in a state of competence, which is a state where the cell can uptake exogenous material. A state of competence is typically a time-limited one caused by environmental conditions around the cell.

It can occur naturally in the environment or laboratory conditions referred to as natural competence. It can also be artificially induced by treating laboratory cultures to make cell membranes permeable to the exogenous genetic material around them.

Combining the knowledge of colony microbiology and transformation is a crucial part of molecular cloning and synthetic biology.

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What Dna Is Transferred From Agrobacterium To Plants

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T-DNA was initially defined as the portion of the Ti plasmid that was transferred from Agrobacterium to plant cells to form crown gall tumors. T-DNA border repeat sequences defined the T-region , and regions of the Ti plasmid outside these borders were not initially found in tumor cells . However, the transfer of Ti-plasmid sequences outside the conventional T-region may at first have been missed because of a lack of known selectable or screenable markers. Ooms et al. observed the incorporation into plant DNA of regions of the Ti plasmid later shown to be outside the classical T-DNA borders. Ramanathan and Veluthambi also showed that a nos-nptII cassette, placed outside the T-DNA left border, could be transferred to and confer kanamycin resistance on infected tobacco cells.

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Natural Competence And Transformation

As of 2014 about 80 species of bacteria were known to be capable of transformation, about evenly divided between Gram-positive and Gram-negative bacteria the number might be an overestimate since several of the reports are supported by single papers.

Naturally competent bacteria carry sets of genes that provide the protein machinery to bring DNA across the cell membrane. The transport of the exogenous DNA into the cells may require proteins that are involved in the assembly of type IV pili and type II secretion system, as well as DNA translocase complex at the cytoplasmic membrane.

Formation Of Bacterial Transformants

  • The foreign DNA is linked with the bacterium host with the help of DNA ligase. This process is called ligation.
  • The bacteria are made to take up plasmid by giving heat shocks. These heat shocks make the bacterial membrane-permeable by forming pores which help in the easy uptake of DNA molecules.
  • Plasmids used in Cloning contain an antibiotic resistance gene. Thus, all of the bacteria are placed on an antibiotic plate to select for ones that took up a Bacteria without a plasmid die. Each bacterium with a plasmid gives rise to a cluster of identical, plasmid-containing bacterim called a colony.
  • Several colonies are checked to identify one with the right plasmid .
  • A colony containing the right plasmid is grown in bulk and used for plasmid or proein production.
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    What Is Bacterial Transformation

    The horizontal gene transfer process is in which a foreign DNA gets linked with the plasmid DNA of the host bacterium. It is the most crucial step in DNA cloning. This was first performed by Griffith in 1928 in streptococcus pneumonia. The linking up of foreign DNA is done with help of DNA ligase. The organism from which the foreign DNA and host bacterium should be closely related. The foreign DNA replicates using the new hosts DNA polymerase enzyme and makes multiple copies.

    The foreign DNA doesnt have to be from the living donor but the host bacterium should be persistent. The host bacterium should be competent and able to uptake extracellular DNA. The nature of competence is based on the genus of the organism. If the foreign DNA is similar to bacterial DNA, it may integrate into the chromosome.

    Transient Transformation In Adult Leaves Via Agrofiltration

    The Mechanism of Transformation with Competent Cells

    Plant defense responses also play a key role in limiting transient transformation. Arabidopsis senses A. tumefaciens elongation factor EF-Tu through a transmembrane Leucine-rich-repeat receptor kinase, EFR , to trigger downstream immune responses. A. thaliana efr mutants fail to elicit defence responses effectively against A. tumefaciens, and as a result the mutants are more susceptible to transient transformation in agroinfiltrated Arabidopsis leaves, as assessed using GUS as a reporter . Effector AvrPto from the plant bacterial pathogen Pseudomonas syringae weakens the immunity of Arabidopsis by targeting multiple pattern recognition receptors . The effector was engineered into A. thaliana, such that expression was driven by a dexamethasone -inducible promoter. In the transgenic line, AvrPto was expressed only when DEX was applied. Pre-treating the plant with DEX before A. tumefaciens infiltration suppressed the plant immunity and hence the transient expression efficiency was significantly enhanced . Salicylic acid plays an important role in defense against bacteria, a recent report shows that use of nahG expressing Arabidopsis plants can also enhance the transient transformation efficiency in Arabidopsis leaves.

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    Quick Answer: What Is Bacterial Transformation In Biology

    Bacterial transformation is a process of horizontal gene transfer by which some bacteria take up foreign genetic material from the environment. The prerequisite for bacteria to undergo transformation is its ability to take up free, extracellular genetic material. Such bacteria are termed as competent cells.

    The Biology Of Agrobacterium

    The initial research interest in the genus Agrobacterium resulted from the search for the causative agent of plant diseases including crown gall, cane gall, and hairy root. Agrobacterium tumefaciens harboring the tumor-inducing plasmid induces galls on roots and crowns of numerous dicot angiosperm species and some gymnosperms whereas Agrobacterium rhizogenes harboring the root-inducing plasmid induces abnormal root production on host plants. The neoplastic tumor-like cell growth is induced by expression of the oncogenes residing in the transferred-DNA transported from these bacteria into the plant nucleus and integrated into the plant genome.

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    What Is A Transformation

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    In this article we will discuss about the meaning of transformation.

    Transformation is the process of adding a foreign DNA fragment from a donor genome into genome of a recipient cell. The donor fragment passes through the cell membrane of the recipient cell and becomes incorporated into the latters genome through recombination. Transformation is detected by the presence of new cell phenotypes in the recipient cells progeny.

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    The actual process of transformation is achieved in at least 3 steps as follows:

    Interaction between a transforming DNA fragment and the recipient bacterium. For successful transformation of a Pneumococcus cell the DNA fragment must have a minimal size of 800 nucleotide pairs for Bacillus 16000 nucleotide pairs are necessary.

    The number of DNA fragments which can interact with a recipient cell is about 10, as these many receptor sites are present on the cell membrane. The recipient cell must be physiologically competent to receive a donor DNA fragment. This state of competence is acquired during the brief period when the recipient cells are dividing at a maximal rate.

    The next step involves movement of DNA fragment in the recipient cell cytoplasm by association with an enzyme system. At the time of its initial contact with the recipient cell, the donor DNA fragment is double stranded but inside the recipient cell it becomes single stranded.

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    Transformation Systems Based On Transposable Elements

    Transformation in Bacteria

    Genetic transformation involves the transfer and incorporation of foreign DNA into a host genome. In order for this transferred DNA to be transmitted to later generations, transformation of germline or other appropriate cells of the recipient species is essential. The transfer of DNA sequences into eukaryotic species usually requires a vector such as a bacterium, virus, or transposable element.

    Considerable effort has been expended recently in the development of TE-based transformation systems, particularly in insects, and such systems are now being used in many areas of biology. For example, between 1998 and 2004, four new TE-based vector systems were successfully developed for stable germline transformation of nondrosophilid insects. These systems are based on the Mos-1 element from Drosophila mauritiana, the Hermes element from Musca domestica, the Minos element from Drosophila hydei, and the piggyBac element from Trichoplusia ni. In addition to Drosophila species, successful transformation of mosquitoes, tephritid fruit flies, and other dipteran species has been achieved. In fact, insect transformation can now be considered routine .

    Saurabh Bhatia, Tanmoy Bera, in, 2015

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