Genetic transformation - Knowledge (XXG)

832:. The bacteria will attach to many of the plant cells exposed by the cut. The plant cells secrete wound-related phenolic compounds which in turn act to upregulate the virulence operon of the Agrobacterium. The virulence operon includes many genes that encode for proteins that are part of a Type IV secretion system that exports from the bacterium proteins and DNA (delineated by specific recognition motifs called border sequences and excised as a single strand from the virulence plasmid) into the plant cell through a structure called a pilus. The transferred DNA (called T-DNA) is piloted to the plant cell nucleus by nuclear localization signals present in the Agrobacterium protein VirD2, which is covalently attached to the end of the T-DNA at the Right border (RB). Exactly how the T-DNA is integrated into the host plant genomic DNA is an active area of plant biology research. Assuming that a selection marker (such as an antibiotic resistance gene) was included in the T-DNA, the transformed plant tissue can be cultured on selective media to produce shoots. The shoots are then transferred to a different medium to promote root formation. Once roots begin to grow from the transgenic shoot, the plants can be transferred to soil to complete a normal life cycle (make seeds). The seeds from this first plant (called the T1, for first transgenic generation) can be planted on a selective (containing an antibiotic), or if an 1052:. The cells are incubated on ice with the DNA, and then briefly heat-shocked (e.g., at 42 °C for 30–120 seconds). This method works very well for circular plasmid DNA. Non-commercial preparations should normally give 10 to 10 transformants per microgram of plasmid; a poor preparation will be about 10/μg or less, but a good preparation of competent cells can give up to ~10 colonies per microgram of plasmid. Protocols, however, exist for making supercompetent cells that may yield a transformation efficiency of over 10. The chemical method, however, usually does not work well for linear DNA, such as fragments of chromosomal DNA, probably because the cell's native 633:

especially effective for repairing double-strand damages, such as double-strand breaks. This process depends on a second homologous chromosome in addition to the damaged chromosome. During logarithmic growth, a DNA damage in one chromosome may be repaired by HRR using sequence information from the other homologous chromosome. Once cells approach stationary phase, however, they typically have just one copy of the chromosome, and HRR requires input of homologous template from outside the cell by transformation.

658: 620:, Charpentier et al. tested 64 toxic molecules to determine which of these induce competence. Of these, only six, all DNA damaging agents, caused strong induction. These DNA damaging agents were mitomycin C (which causes DNA inter-strand crosslinks), norfloxacin, ofloxacin and nalidixic acid (inhibitors of DNA gyrase that cause double-strand breaks), bicyclomycin (causes single- and double-strand breaks), and hydroxyurea (induces DNA base oxidation). UV light also induced competence in 42: 933:, which are, if at all, separated by internal cell walls interrupted by pores big enough to enable nutrients and organelles, sometimes even nuclei, to travel through each hypha. As a result, individual cells usually cannot be separated. This is problematic as neighbouring transformed cells may render untransformed ones immune to selection treatments, e.g. by delivering nutrients or proteins for antibiotic resistance. 4933: 807:, and thus may not be optimal for other species. Even within one species, different strains have different transformation efficiencies, sometimes different by three orders of magnitude. For instance, when S. cerevisiae strains were transformed with 10 ug of plasmid YEp13, the strain DKD-5D-H yielded between 550 and 3115 colonies while strain OS1 yielded fewer than five colonies. 641:

likely HRR. Transformation in bacteria can be viewed as a primitive sexual process, since it involves interaction of homologous DNA from two individuals to form recombinant DNA that is passed on to succeeding generations. Bacterial transformation in prokaryotes may have been the ancestral process that gave rise to meiotic sexual reproduction in eukaryotes (see

879:: Also referred to as particle bombardment, microprojectile bombardment, or biolistics. Particles of gold or tungsten are coated with DNA and then shot into young plant cells or plant embryos. Some genetic material will stay in the cells and transform them. This method also allows transformation of plant plastids. The 689:

bonds by increasing the ratio of ionic to covalent bonds, which increases membrane fluidity, facilitating transformation. The role of lipopolysaccharides here are verified from the observation that shorter O-side chains are more effectively transformed – perhaps because of improved DNA accessibility.

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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

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irradiated by UV light as the damaging agent (reviewed by Michod et al. and Bernstein et al.) The results of these experiments indicated that transforming DNA acts to repair potentially lethal DNA damages introduced by UV light in the recipient DNA. The particular process responsible for repair was

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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

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Natural transformation is a bacterial adaptation for DNA transfer that depends on the expression of numerous bacterial genes whose products appear to be responsible for this process. In general, transformation is a complex, energy-requiring developmental process. In order for a bacterium to bind,

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In a cloning experiment, a gene may be inserted into a plasmid used for transformation. However, in such experiment, not all the plasmids may contain a successfully inserted gene. Additional techniques may therefore be employed further to screen for transformed cells that contain plasmid with the

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Due to the differences in structure of the cell envelope between Gram-positive and Gram-negative bacteria, there are some differences in the mechanisms of DNA uptake in these cells, however most of them share common features that involve related proteins. The DNA first binds to the surface of the

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It appears to be an ancient process inherited from a common prokaryotic ancestor that is a beneficial adaptation for promoting recombinational repair of DNA damage, especially damage acquired under stressful conditions. Natural genetic transformation appears to be an adaptation for repair of DNA

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It is suggested that exposing the cells to divalent cations in cold condition may also change or weaken the cell surface structure, making it more permeable to DNA. The heat-pulse is thought to create a thermal imbalance across the cell membrane, which forces the DNA to enter the cells through

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process. During logarithmic growth, two or more copies of any particular region of the chromosome may be present in a bacterial cell, as cell division is not precisely matched with chromosome replication. The process of homologous recombinational repair (HRR) is a key DNA repair process that is

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the length of the transferred DNA is greater than 1271 kb (more than 1 million bases). The length transferred is likely double stranded DNA and is often more than a third of the total chromosome length of 4215 kb. It appears that about 7-9% of the recipient cells take up an entire chromosome.

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method decreases with plasmid size, and electroporation therefore may be a more effective method for the uptake of large plasmid DNA. Cells used in electroporation should be prepared first by washing in cold double-distilled water to remove charged particles that may create sparks during the

859:): Package the desired genetic material into a suitable plant virus and allow this modified virus to infect the plant. If the genetic material is DNA, it can recombine with the chromosomes to produce transformant cells. However, genomes of most plant viruses consist of single stranded 717:(which also uses these channels) was found to competitively inhibit DNA uptake. Another type of channel implicated in DNA uptake consists of poly (HB):poly P:Ca. In this poly (HB) is envisioned to wrap around DNA (itself a polyphosphate), and is carried in a shield formed by Ca ions. 848:

was isolated from a cherry tree in an orchard at Cornell University in Ithaca, New York). Though many plants remain recalcitrant to transformation by this method, research is ongoing that continues to add to the list the species that have been successfully modified in this

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on its cell surface, and the DNA is also negatively charged. One function of the divalent cation therefore would be to shield the charges by coordinating the phosphate groups and other negative charges, thereby allowing a DNA molecule to adhere to the cell surface.

682:) under cold conditions, before being exposed to a heat pulse (heat shock). Calcium chloride partially disrupts the cell membrane, which allows the recombinant DNA to enter the host cell. Cells that are able to take up the DNA are called competent cells. 1189:

to emit light. The recombinant DNA may also be detected using other methods such as nucleic acid hybridization with radioactive RNA probe, while cells that expressed the desired protein from the plasmid may also be detected using immunological methods.

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in fungal cells became possible. In 2016 the USDA declared that it will not regulate a white button mushroom strain edited with CRISPR/CAS9 to prevent fruit body browning causing a broad discussion about placing CRISPR/CAS9-edited crops on the

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Artificial competence can be induced in laboratory procedures that involve making the cell passively permeable to DNA by exposing it to conditions that do not normally occur in nature. Typically the cells are incubated in a solution containing

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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

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Naturally competent bacteria carry sets of genes that provide the protein machinery to bring DNA across the cell membrane(s). The transport of the exogenous DNA into the cells may require proteins that are involved in the assembly of

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Agrobacterium is not only capable of infecting plants but also fungi, however, unlike plants, fungi do not secrete the phenolic compounds necessary to trigger Agrobacterium so that they have to be added, e.g. in the form of

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requires expression of about 40 genes. The DNA integrated into the host chromosome is usually (but with rare exceptions) derived from another bacterium of the same species, and is thus homologous to the resident chromosome.

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that parts of some fungi are in; dikaryotic cells contain two haploid nuclei, one of each parent fungus. If only one of these gets transformed, which is the rule, the percentage of transformed nuclei decreases after each

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may be required for competence, but its role is uncertain. The uptake of DNA is generally non-sequence specific, although in some species the presence of specific DNA uptake sequences may facilitate efficient DNA uptake.

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via DNA translocase. Only single-stranded DNA may pass through, the other strand being degraded by nucleases in the process. The translocated single-stranded DNA may then be integrated into the bacterial chromosomes by a

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and not a real transformation, since the inserted genes never reach the nucleus of the cell and do not integrate into the host genome. The progeny of the infected plants is virus-free and also free of the inserted

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Hayama Y, Fukuda Y, Kawai S, Hashimoto W, Murata K (2002). "Extremely simple, rapid and highly efficient transformation method for the yeast Saccharomyces cerevisiae using glutathione and early log phase cells".

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gene, and no functional β-galactosidase can form, resulting in white colonies. Cells containing successfully ligated insert can then be easily identified by its white coloration from the unsuccessful blue ones.

326:, in which a gene is passed through direct contact between bacteria. In transformation, the genetic material passes through the intervening medium, and uptake is completely dependent on the recipient bacterium. 627:

Logarithmically growing bacteria differ from stationary phase bacteria with respect to the number of genome copies present in the cell, and this has implications for the capability to carry out an important

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Albertini S, Chételat AA, Miller B, Muster W, Pujadas E, Strobel R, Gocke E (July 1995). "Genotoxicity of 17 gyrase- and four mammalian topoisomerase II-poisons in prokaryotic and eukaryotic test systems".

828:-mediated transformation is the easiest and most simple plant transformation. Plant tissue (often leaves) are cut into small pieces, e.g. 10x10mm, and soaked for ten minutes in a fluid containing suspended 735:/cm, which is thought to create holes in the cell membrane through which the plasmid DNA may enter. After the electric shock, the holes are rapidly closed by the cell's membrane-repair mechanisms. 787:, and single-stranded DNA. In these protocols, the single-stranded DNA preferentially binds to the yeast cell wall, preventing plasmid DNA from doing so and leaving it available for transformation. 3840: 3056: 2219:

Saito Y, Taguchi H, Akamatsu T (March 2006). "Fate of transforming bacterial genome following incorporation into competent cells of Bacillus subtilis: a continuous length of incorporated DNA".

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Competence for transformation is typically induced by high cell density and/or nutritional limitation, conditions associated with the stationary phase of bacterial growth. Transformation in

138:. Griffith was interested in determining whether injections of heat-killed bacteria could be used to vaccinate mice against pneumonia. However, he discovered that a non-virulent strain of 554:

The capacity for natural transformation appears to occur in a number of prokaryotes, and thus far 67 prokaryotic species (in seven different phyla) are known to undergo this process.

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In this image, a gene from one bacterial cell is moved to another bacterial cell. This process of the second bacterial cell taking up new genetic material is called transformation.

616:, ciprofloxacin, which interacts with DNA gyrase and introduces double-strand breaks, induces expression of competence genes, thus enhancing the frequency of transformation Using 2422: 3743:

Rivera, Ana Leonor; Magaña-Ortíz, Denis; Gómez-Lim, Miguel; Fernández, Francisco; Loske, Achim M. (June 2014). "Physical methods for genetic transformation of fungi and yeast".

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Dohmen RJ, Strasser AW, Höner CB, Hollenberg CP (October 1991). "An efficient transformation procedure enabling long-term storage of competent cells of various yeast genera".

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virus into the host bacterium). In transformation, the genetic material passes through the intervening medium, and uptake is completely dependent on the recipient bacterium.

522:-dependent process. In Gram-negative cells, due to the presence of an extra membrane, the DNA requires the presence of a channel formed by secretins on the outer membrane. 2485:

Angelov, Angel; Bergen, Paul; Nadler, Florian; Hornburg, Philipp; Lichev, Antoni; Ãœbelacker, Maria; Pachl, Fiona; Kuster, Bernhard; Liebl, Wolfgang (10 February 2015).

803:– Different yeast genera and species take up foreign DNA with different efficiencies. Also, most transformation protocols have been developed for baker's yeast, 4184: 3968: 1553:

Wirth R, Friesenegger A, Fiedler S (March 1989). "Transformation of various species of gram-negative bacteria belonging to 11 different genera by electroporation".

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enzymes rapidly degrade linear DNA. In contrast, cells that are naturally competent are usually transformed more efficiently with linear DNA than with plasmid DNA.

81:, which might occur in nature as a time-limited response to environmental conditions such as starvation and cell density, and may also be induced in a laboratory. 713:

cells is through channels known as zones of adhesion or Bayer's junction, with a typical cell carrying as many as 400 such zones. Their role was established when

474:"Transformation" may also be used to describe the insertion of new genetic material into nonbacterial cells, including animal and plant cells; however, because " 118:"Transformation" may also be used to describe the insertion of new genetic material into nonbacterial cells, including animal and plant cells; however, because " 4122: 152:" from the heat-killed strain was responsible for making the harmless strain virulent. In 1944 this "transforming principle" was identified as being genetic by 906:

most of them being analogous to those used for plants. However, fungi have to be treated differently due to some of their microscopic and biochemical traits:

753:, may be transformed by exogenous DNA in the environment. Several methods have been developed to facilitate this transformation at high frequency in the lab. 4958: 4757: 3413:

Spencer, F.; Ketner, G.; Connelly, C.; Hieter, P. (1 August 1993). "Targeted Recombination-Based Cloning and Manipulation of Large DNA Segments in Yeast".

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Saito Y, Taguchi H, Akamatsu T (April 2006). "DNA taken into Bacillus subtilis competent cells by lysed-protoplast transformation is not ssDNA but dsDNA".

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take up and recombine exogenous DNA into its chromosome, it must become competent, that is, enter a special physiological state. Competence development in

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that occur in nature among bacteria, in which DNA encoding for a trait passes from one bacterium to another and is integrated into the recipient genome by

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Fungal cell walls are quite thick hindering DNA uptake so (partial) removal is often required; complete degradation, which is sometimes necessary, yields

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by the DNA damaging agents mitomycin C (a DNA cross-linking agent) and fluoroquinolone (a topoisomerase inhibitor that causes double-strand breaks). In

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and using just this DNA were able to make a harmless strain virulent. They called this uptake and incorporation of DNA by bacteria "transformation" (See

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Johnston C, Martin B, Fichant G, Polard P, Claverys JP (March 2014). "Bacterial transformation: distribution, shared mechanisms and divergent control".

893:: Formation of transient holes in cell membranes using electric pulses of high field strength; this allows DNA to enter as described above for bacteria. 4840: 1149:

cells, they form a functional β-galactosidase. The presence of an active β-galactosidase may be detected when cells are grown in plates containing

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gene was used, could alternatively be planted in soil, then later treated with herbicide to kill wildtype segregants. Some plants species, such as

172:) The results of Avery et al.'s experiments were at first skeptically received by the scientific community and it was not until the development of 4630: 3343:

Gietz RD, Schiestl RH, Willems AR, Woods RA (April 1995). "Studies on the transformation of intact yeast cells by the LiAc/SS-DNA/PEG procedure".

2538:"Amino acids as nutritional factors and (p)ppGpp as an alarmone of the stringent response regulate natural transformation in Micrococcus luteus" 997:

to be used as a convenient host for the manipulation of DNA as well as expressing proteins. Typically plasmids are used for transformation in

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treatment is also effective for transformation of plasmid DNA. The method of transformation by Mandel and Higa was later improved upon by

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Schiestl, Robert H.; Manivasakam, P.; Woods, Robin A.; Gietzt, R.Daniel (1 August 1993). "Introducing DNA into Yeast by Transformation".

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Claverys JP, Prudhomme M, Martin B (2006). "Induction of competence regulons as a general response to stress in gram-positive bacteria".

3961: 1141:) in the cell. Both genes by themselves produce non-functional peptides, however, when expressed together, as when a plasmid containing 953: 1883:"Cues and regulatory pathways involved in natural competence and transformation in pathogenic and environmental Gram-negative bacteria" 4787: 936:

Additionally, growth (and thereby mitosis) of these fungi exclusively occurs at the tip of their hyphae which can also deliver issues.

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being created by injecting a gene for a rat growth hormone into a mouse embryo in 1982. In 1897 a bacterium that caused plant tumors,

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Sanford JC, Klein TM, Wolf ED, Allen N (1987). "Delivery of substances into cells and tissues using a particle bombardment process".

197:, a commonly used laboratory organism, was refractory to transformation. However, in 1970, Morton Mandel and Akiko Higa showed that 4797: 4115: 3319: 793:: Formation of transient holes in the cell membranes using electric shock; this allows DNA to enter as described above for bacteria. 274:. By removing the genes in the plasmid that caused the tumor and adding in novel genes, researchers were able to infect plants with 1803:

Klein RM, Wolf ED, Wu R, Sanford JC (1992). "High-velocity microprojectiles for delivering nucleic acids into living cells. 1987".

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and is measured in colony forming unit (cfu) per μg DNA used. A transformation efficiency of 1×10 cfu/μg for a small plasmid like

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To test whether the adaptive function of transformation is repair of DNA damages, a series of experiments were carried out using

478:" has a special meaning in relation to animal cells, indicating progression to a cancerous state, the process is usually called " 278:

and let the bacteria insert their chosen DNA into the genomes of the plants. Not all plant cells are susceptible to infection by

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Gietz RD, Woods RA (2002). "Transformation of yeast by lithium acetate/single-stranded carrier DNA/polyethylene glycol method".

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that uses cavitation of gas bubbles produced by ultrasound to penetrate the cell membrane, etc. are also applicable to fungi.

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occurs most efficiently at the end of exponential growth as bacterial growth approaches stationary phase. Transformation in

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refers to a temporary state of being able to take up exogenous DNA from the environment; it may be induced in a laboratory.

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At the Max Planck Institute for Molecular Plant Physiology in Potsdam-Golm plant cells are 'bombarded' using a particle gun

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phylum), competence develops during the mid-late exponential growth phase and is also triggered by amino acids starvation.

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Solomon JM, Grossman AD (April 1996). "Who's competent and when: regulation of natural genetic competence in bacteria".

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Poyedinok, N. L.; Blume, Ya. B. (March 2018). "Advances, Problems, and Prospects of Genetic Transformation of Fungi".

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is induced toward the end of logarithmic growth, especially under conditions of amino acid limitation. Similarly, in

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Aspiras MB, Ellen RP, Cvitkovitch DG (September 2004). "ComX activity of Streptococcus mutans growing in biofilms".

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was developed in the late 1980s, increasing the efficiency of in-vitro transformation and increasing the number of

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Inoue H, Nojima H, Okayama H (November 1990). "High efficiency transformation of Escherichia coli with plasmids".

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As of 2014 about 80 species of bacteria were known to be capable of transformation, about evenly divided between

4051: 3981: 3946: 1598:"Dramatic growth of mice that develop from eggs microinjected with metallothionein-growth hormone fusion genes" 749: 475: 415: 311: 307: 140: 119: 85: 62: 31: 1451:"Nonchromosomal antibiotic resistance in bacteria: genetic transformation of Escherichia coli by R-factor DNA" 1005:, which allows it to be replicated in the cell independently of the replication of the cell's own chromosome. 149: 3934: 1654: 624:. Charpentier et al. suggested that competence for transformation probably evolved as a DNA damage response. 4007: 3985: 2977: 2291:"Incorporation of the whole chromosomal DNA in protoplast lysates into competent cells of Bacillus subtilis" 856: 433: 315: 181: 97: 604:

Competence is specifically induced by DNA damaging conditions. For instance, transformation is induced in

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The efficiency with which a competent culture can take up exogenous DNA and express its genes is known as

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that could be transformed. Transformation of animal and plant cells was also investigated with the first

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Palmiter RD, Brinster RL, Hammer RE, Trumbauer ME, Rosenfeld MG, Birnberg NC, Evans RM (December 1982).

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It has been found that growth of Gram-negative bacteria in 20 mM Mg reduces the number of protein-to-

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allows the cells to take up plasmid DNA. Later protocols adapted this transformation method, using

467: 349: 157: 115:; the number might be an overestimate since several of the reports are supported by single papers. 3785: 3637:

He, Liya; Feng, Jiao; Lu, Sha; Chen, Zhiwen; Chen, Chunmei; He, Ya; Yi, Xiuwen; Xi, Liyan (2017).

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is another method of promoting competence. In this method the cells are briefly shocked with an

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created an efficient and convenient procedure for transforming bacteria which allows for simpler

135: 54: 88:, in which exogenous genetic material passes from one bacterium to another, the other two being 1835:

Michod RE, Bernstein H, Nedelcu AM (May 2008). "Adaptive value of sex in microbial pathogens".

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which replicates in the cytoplasm of infected cell. For such genomes this method is a form of

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Case, Christine; Funke, Berdell; Tortora, Gerard. Microbiology An Introduction(tenth edition)

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Schematic of bacterial transformation – for which artificial competence must first be induced

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Thanks to development of an expression system for small RNAs in fungi the introduction of a

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As stated earlier, an array of methods used for plant transformation do also work in fungi:

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Enzymatic digestion or agitation with glass beads may also be used to transform yeast cells.

679: 569:, as well as in many other streptococci, occurs at high cell density and is associated with 257: 253: 193: 185: 2655: 2593:

Keen EC, Bliskovsky VV, Malagon F, Baker JD, Prince JS, Klaus JS, Adhya SL (January 2017).

4704: 4588: 4547: 4506: 4416: 4079: 4041: 3792: 3105: 2978:"Chapter 1: DNA repair as the primary adaptive function of sex in bacteria and eukaryotes" 890: 790: 780: 724: 394: 379: 295: 283: 249: 226: 173: 41: 2595:"Novel "Superspreader" Bacteriophages Promote Horizontal Gene Transfer by Transformation" 3756: 3703: 3211: 3057:"Large-volume transformation with high-throughput efficiency chemically competent cells" 3025: 2882: 2553: 2142: 2034: 1848: 1613: 1466: 1001:. In order to be stably maintained in the cell, a plasmid DNA molecule must contain an 134:

Transformation in bacteria was first demonstrated in 1928 by the British bacteriologist

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such that those cells without the plasmid may be killed or have their growth arrested.

946: 844:, typically strain C58 (C=Cherry, 58=1958, the year in which this particular strain of 728: 585: 445: 287: 266:, was discovered and in the early 1970s the tumor-inducing agent was found to be a DNA 161: 66: 3909: 3884: 3546: 3311: 3279: 3254: 3230: 3195: 2462: 2437: 2161: 2126: 2102: 2077: 2053: 2018: 1706: 1681: 1531: 1508:

Hanahan D (June 1983). "Studies on transformation of Escherichia coli with plasmids".

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after being exposed to heat-killed virulent strains. Griffith hypothesized that some "

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is roughly equivalent to 1 in 2000 molecules of the plasmid used being transformed.

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The discovery of artificially induced competence in bacteria allow bacteria such as

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Mandel M, Higa A (October 1970). "Calcium-dependent bacteriophage DNA infection".

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Lichev, Antoni; Angelov, Angel; Cucurull, Inigo; Liebl, Wolfgang (30 July 2019).

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