Genetic transformation

Last updated
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. Bacterial Transformation.svg
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.

In molecular biology and genetics, transformation is the genetic alteration of a cell resulting from the direct uptake and incorporation of exogenous genetic material from its surroundings through the cell membrane(s). For transformation to take place, the recipient bacterium must be in a state of competence, 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. [1]

Contents

Transformation is one of three processes that lead to horizontal gene transfer, in which exogenous genetic material passes from one 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). [1] In transformation, the genetic material passes through the intervening medium, and uptake is completely dependent on the recipient bacterium. [1]

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. [1]

"Transformation" may also be used to describe the insertion of new genetic material into nonbacterial cells, including animal and plant cells; however, because "transformation" has a special meaning in relation to animal cells, indicating progression to a cancerous state, the process is usually called "transfection". [2]

History

Transformation in bacteria was first demonstrated in 1928 by the British bacteriologist Frederick Griffith. [3] 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 Streptococcus pneumoniae could be made virulent after being exposed to heat-killed virulent strains. Griffith hypothesized that some "transforming principle" from the heat-killed strain was responsible for making the harmless strain virulent. In 1944 this "transforming principle" was identified as being genetic by Oswald Avery, Colin MacLeod, and Maclyn McCarty. They isolated DNA from a virulent strain of S. pneumoniae 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 Avery-MacLeod-McCarty experiment) [4] 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 genetic markers and the discovery of other methods of genetic transfer (conjugation in 1947 and transduction in 1953) by Joshua Lederberg that Avery's experiments were accepted. [5]

It was originally thought that Escherichia coli , a commonly used laboratory organism, was refractory to transformation. However, in 1970, Morton Mandel and Akiko Higa showed that E. coli may be induced to take up DNA from bacteriophage λ without the use of helper phage after treatment with calcium chloride solution. [6] Two years later in 1972, Stanley Norman Cohen, Annie Chang and Leslie Hsu showed that CaCl
2 treatment is also effective for transformation of plasmid DNA. [7] The method of transformation by Mandel and Higa was later improved upon by Douglas Hanahan. [8] The discovery of artificially induced competence in E. coli created an efficient and convenient procedure for transforming bacteria which allows for simpler molecular cloning methods in biotechnology and research, and it is now a routinely used laboratory procedure.

Transformation using electroporation was developed in the late 1980s, increasing the efficiency of in-vitro transformation and increasing the number of bacterial strains that could be transformed. [9] Transformation of animal and plant cells was also investigated with the first transgenic mouse being created by injecting a gene for a rat growth hormone into a mouse embryo in 1982. [10] In 1897 a bacterium that caused plant tumors, Agrobacterium tumefaciens , was discovered and in the early 1970s the tumor-inducing agent was found to be a DNA plasmid called the Ti plasmid. [11] By removing the genes in the plasmid that caused the tumor and adding in novel genes, researchers were able to infect plants with A. tumefaciens and let the bacteria insert their chosen DNA into the genomes of the plants. [12] Not all plant cells are susceptible to infection by A. tumefaciens, so other methods were developed, including electroporation and micro-injection. [13] Particle bombardment was made possible with the invention of the Biolistic Particle Delivery System (gene gun) by John Sanford in the 1980s. [14] [15] [16]

Definitions

Transformation is one of three forms of horizontal gene transfer 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 homologous recombination; the other two are transduction, carried out by means of a bacteriophage, and conjugation, in which a gene is passed through direct contact between bacteria. [1] In transformation, the genetic material passes through the intervening medium, and uptake is completely dependent on the recipient bacterium. [1]

Competence refers to a temporary state of being able to take up exogenous DNA from the environment; it may be induced in a laboratory. [1]

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 damage that also generates genetic diversity. [1] [17]

Transformation has been studied in medically important Gram-negative bacteria species such as Helicobacter pylori , Legionella pneumophila , Neisseria meningitidis , Neisseria gonorrhoeae , Haemophilus influenzae and Vibrio cholerae . [18] It has also been studied in Gram-negative species found in soil such as Pseudomonas stutzeri , Acinetobacter baylyi, and Gram-negative plant pathogens such as Ralstonia solanacearum and Xylella fastidiosa . [18] Transformation among Gram-positive bacteria has been studied in medically important species such as Streptococcus pneumoniae , Streptococcus mutans , Staphylococcus aureus and Streptococcus sanguinis and in Gram-positive soil bacterium Bacillus subtilis . [17] It has also been reported in at least 30 species of Pseudomonadota distributed in several different classes. [19] The best studied Pseudomonadota with respect to transformation are the medically important human pathogens Neisseria gonorrhoeae , Haemophilus influenzae , and Helicobacter pylori . [17]

"Transformation" may also be used to describe the insertion of new genetic material into nonbacterial cells, including animal and plant cells; however, because "transformation" has a special meaning in relation to animal cells, indicating progression to a cancerous state, the process is usually called "transfection". [2]

Natural competence and transformation

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 type IV pili and type II secretion system, as well as DNA translocase complex at the cytoplasmic membrane. [20]

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 competent cells on a DNA receptor, and passes through the cytoplasmic membrane via DNA translocase. [21] 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 RecA-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. Pilin may be required for competence, but its role is uncertain. [22] 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. [23]

Natural transformation

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. [20] [19] In general, transformation is a complex, energy-requiring developmental process. In order for a bacterium to bind, take up and recombine exogenous DNA into its chromosome, it must become competent, that is, enter a special physiological state. Competence development in Bacillus subtilis requires expression of about 40 genes. [24] 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.

In B. subtilis the length of the transferred DNA is greater than 1271 kb (more than 1 million bases). [25] The length transferred is likely double stranded DNA and is often more than a third of the total chromosome length of 4215 kb. [26] It appears that about 7-9% of the recipient cells take up an entire chromosome. [27]

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. [19]

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 Haemophilus influenzae occurs most efficiently at the end of exponential growth as bacterial growth approaches stationary phase. [28] Transformation in Streptococcus mutans , as well as in many other streptococci, occurs at high cell density and is associated with biofilm formation. [29] Competence in B. subtilis is induced toward the end of logarithmic growth, especially under conditions of amino acid limitation. [30] Similarly, in Micrococcus luteus (a representative of the less well studied Actinomycetota phylum), competence develops during the mid-late exponential growth phase and is also triggered by amino acids starvation. [31] [32]

By releasing intact host and plasmid DNA, certain bacteriophages are thought to contribute to transformation. [33]

Transformation, as an adaptation for DNA repair

Competence is specifically induced by DNA damaging conditions. For instance, transformation is induced in Streptococcus pneumoniae by the DNA damaging agents mitomycin C (a DNA cross-linking agent) and fluoroquinolone (a topoisomerase inhibitor that causes double-strand breaks). [34] In B. subtilis, transformation is increased by UV light, a DNA damaging agent. [35] In Helicobacter pylori, ciprofloxacin, which interacts with DNA gyrase and introduces double-strand breaks, induces expression of competence genes, thus enhancing the frequency of transformation [36] Using Legionella pneumophila, Charpentier et al. [37] 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 [38] ), bicyclomycin (causes single- and double-strand breaks [39] ), and hydroxyurea (induces DNA base oxidation [40] ). UV light also induced competence in L. pneumophila. Charpentier et al. [37] suggested that competence for transformation probably evolved as a DNA damage response.

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 DNA repair 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 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. [41]

To test whether the adaptive function of transformation is repair of DNA damages, a series of experiments were carried out using B. subtilis irradiated by UV light as the damaging agent (reviewed by Michod et al. [42] and Bernstein et al. [41] ) 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 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 Evolution of sexual reproduction; Meiosis.)

Methods and mechanisms of transformation in laboratory

Schematic of bacterial transformation - for which artificial competence must first be induced Artificial Bacterial Transformation.svg
Schematic of bacterial transformation – for which artificial competence must first be induced

Bacterial

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. [43] Typically the cells are incubated in a solution containing divalent cations (often calcium chloride) 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.

It has been found that growth of Gram-negative bacteria in 20 mM Mg reduces the number of protein-to-lipopolysaccharide bonds by increasing the ratio of ionic to covalent bonds, which increases membrane fluidity, facilitating transformation. [44] 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.

The surface of bacteria such as E. coli is negatively charged due to phospholipids and lipopolysaccharides 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.

DNA entry into E. coli 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 cobalamine (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. [44]

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 either cell pores or the damaged cell wall.

Electroporation is another method of promoting competence. In this method the cells are briefly shocked with an electric field of 10-20 kV/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.

Yeast

Most species of yeast, including Saccharomyces cerevisiae , may be transformed by exogenous DNA in the environment. Several methods have been developed to facilitate this transformation at high frequency in the lab. [45]

Efficiency– Different yeast genera and species take up foreign DNA with different efficiencies. [53] Also, most transformation protocols have been developed for baker's yeast, S. cerevisiae, 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. [54]

Plants

A number of methods are available to transfer DNA into plant cells. Some vector-mediated methods are:

Some vector-less methods include:

Fungi

There are some methods to produce transgenic fungi 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:

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

Animals

Introduction of DNA into animal cells is usually called transfection, and is discussed in the corresponding article.

Practical aspects of transformation in molecular biology

The discovery of artificially induced competence in bacteria allow bacteria such as Escherichia coli to be used as a convenient host for the manipulation of DNA as well as expressing proteins. Typically plasmids are used for transformation in E. coli. In order to be stably maintained in the cell, a plasmid DNA molecule must contain an origin of replication, which allows it to be replicated in the cell independently of the replication of the cell's own chromosome.

The efficiency with which a competent culture can take up exogenous DNA and express its genes is known as transformation efficiency and is measured in colony forming unit (cfu) per μg DNA used. A transformation efficiency of 1×108 cfu/μg for a small plasmid like pUC19 is roughly equivalent to 1 in 2000 molecules of the plasmid used being transformed.

In calcium chloride transformation, the cells are prepared by chilling cells in the presence of Ca2+
 (in CaCl
2 solution), making the cell become permeable to plasmid DNA. 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 106 to 107 transformants per microgram of plasmid; a poor preparation will be about 104/μg or less, but a good preparation of competent cells can give up to ~108 colonies per microgram of plasmid. [60] Protocols, however, exist for making supercompetent cells that may yield a transformation efficiency of over 109. [61] The chemical method, however, usually does not work well for linear DNA, such as fragments of chromosomal DNA, probably because the cell's native exonuclease enzymes rapidly degrade linear DNA. In contrast, cells that are naturally competent are usually transformed more efficiently with linear DNA than with plasmid DNA.

The transformation efficiency using the CaCl
2 method decreases with plasmid size, and electroporation therefore may be a more effective method for the uptake of large plasmid DNA. [62] 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 electroporation process.

Selection and screening in plasmid transformation

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. [63] 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.

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 insert. Reporter genes can be used as markers, such as the lacZ gene which codes for β-galactosidase used in blue-white screening. This method of screening relies on the principle of α-complementation, where a fragment of the lacZ gene (lacZα) in the plasmid can complement another mutant lacZ gene (lacZΔM15) in the cell. Both genes by themselves produce non-functional peptides, however, when expressed together, as when a plasmid containing lacZ-α is transformed into a lacZΔM15 cells, they form a functional β-galactosidase. The presence of an active β-galactosidase may be detected when cells are grown in plates containing X-gal, forming characteristic blue colonies. However, the multiple cloning site, where a gene of interest may be ligated into the plasmid vector, is located within the lacZα gene. Successful ligation therefore disrupts the lacZα 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.

Other commonly used reporter genes are green fluorescent protein (GFP), which produces cells that glow green under blue light, and the enzyme luciferase, which catalyzes a reaction with luciferin 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.

Related Research Articles

<span class="mw-page-title-main">Bacterial conjugation</span> Method of bacterial gene transfer

Bacterial conjugation is the transfer of genetic material between bacterial cells by direct cell-to-cell contact or by a bridge-like connection between two cells. This takes place through a pilus. It is a parasexual mode of reproduction in bacteria.

<span class="mw-page-title-main">Plasmid</span> Small DNA molecule within a cell

A plasmid is a small, extrachromosomal DNA molecule within a cell that is physically separated from chromosomal DNA and can replicate independently. They are most commonly found as small circular, double-stranded DNA molecules in bacteria; however, plasmids are sometimes present in archaea and eukaryotic organisms. Plasmids often carry useful genes, such as for antibiotic resistance. While chromosomes are large and contain all the essential genetic information for living under normal conditions, plasmids are usually very small and contain additional genes for special circumstances.

A bacterial artificial chromosome (BAC) is a DNA construct, based on a functional fertility plasmid, used for transforming and cloning in bacteria, usually E. coli. F-plasmids play a crucial role because they contain partition genes that promote the even distribution of plasmids after bacterial cell division. The bacterial artificial chromosome's usual insert size is 150–350 kbp. A similar cloning vector called a PAC has also been produced from the DNA of P1 bacteriophage.

<span class="mw-page-title-main">Horizontal gene transfer</span> Transfer of genes from unrelated organisms

Horizontal gene transfer (HGT) or lateral gene transfer (LGT) is the movement of genetic material between organisms other than by the ("vertical") transmission of DNA from parent to offspring (reproduction). HGT is an important factor in the evolution of many organisms. HGT is influencing scientific understanding of higher-order evolution while more significantly shifting perspectives on bacterial evolution.

<i>Agrobacterium tumefaciens</i> Bacterium, genetic engineering tool

Agrobacterium tumefaciens is the causal agent of crown gall disease in over 140 species of eudicots. It is a rod-shaped, Gram-negative soil bacterium. Symptoms are caused by the insertion of a small segment of DNA, from a plasmid into the plant cell, which is incorporated at a semi-random location into the plant genome. Plant genomes can be engineered by use of Agrobacterium for the delivery of sequences hosted in T-DNA binary vectors.

<i>Agrobacterium</i> Genus of bacteria

Agrobacterium is a genus of Gram-negative bacteria established by H. J. Conn that uses horizontal gene transfer to cause tumors in plants. Agrobacterium tumefaciens is the most commonly studied species in this genus. Agrobacterium is well known for its ability to transfer DNA between itself and plants, and for this reason it has become an important tool for genetic engineering.

<span class="mw-page-title-main">Transfer DNA</span> Type of DNA in bacterial genomes

The transfer DNA is the transferred DNA of the tumor-inducing (Ti) plasmid of some species of bacteria such as Agrobacterium tumefaciens and Agrobacterium rhizogenes . The T-DNA is transferred from bacterium into the host plant's nuclear DNA genome. The capability of this specialized tumor-inducing (Ti) plasmid is attributed to two essential regions required for DNA transfer to the host cell. The T-DNA is bordered by 25-base-pair repeats on each end. Transfer is initiated at the right border and terminated at the left border and requires the vir genes of the Ti plasmid.

Triparental mating is a form of bacterial conjugation where a conjugative plasmid present in one bacterial strain assists the transfer of a mobilizable plasmid present in a second bacterial strain into a third bacterial strain. Plasmids are introduced into bacteria for such purposes as transformation, cloning, or transposon mutagenesis. Triparental matings can help overcome some of the barriers to efficient plasmid mobilization. For instance, if the conjugative plasmid and the mobilizable plasmid are members of the same incompatibility group they do not need to stably coexist in the second bacterial strain for the mobilizable plasmid to be transferred.

<span class="mw-page-title-main">Ti plasmid</span> Circular plasmid used in creation of transgenic plants

A tumour inducing (Ti) plasmid is a plasmid found in pathogenic species of Agrobacterium, including A. tumefaciens, A. rhizogenes, A. rubi and A. vitis.

<span class="mw-page-title-main">Natural competence</span> Ability of cells to take up extracellular DNA

In microbiology, genetics, cell biology, and molecular biology, competence is the ability of a cell to alter its genetics by taking up extracellular DNA from its environment through a process called transformation. Competence can be differentiated between natural competence and induced or artificial competence. Natural competence is a genetically specified ability of bacteria that occurs under natural conditions as well as in the laboratory. Artificial competence arises when cells in laboratory cultures are treated to make them transiently permeable to DNA. Competence allows for rapid adaptation and DNA repair of the cell.

Microbial genetics is a subject area within microbiology and genetic engineering. Microbial genetics studies microorganisms for different purposes. The microorganisms that are observed are bacteria and archaea. Some fungi and protozoa are also subjects used to study in this field. The studies of microorganisms involve studies of genotype and expression system. Genotypes are the inherited compositions of an organism. Genetic Engineering is a field of work and study within microbial genetics. The usage of recombinant DNA technology is a process of this work. The process involves creating recombinant DNA molecules through manipulating a DNA sequence. That DNA created is then in contact with a host organism. Cloning is also an example of genetic engineering.

<span class="mw-page-title-main">Gene delivery</span> Introduction of foreign genetic material into host cells

Gene delivery is the process of introducing foreign genetic material, such as DNA or RNA, into host cells. Gene delivery must reach the genome of the host cell to induce gene expression. Successful gene delivery requires the foreign gene delivery to remain stable within the host cell and can either integrate into the genome or replicate independently of it. This requires foreign DNA to be synthesized as part of a vector, which is designed to enter the desired host cell and deliver the transgene to that cell's genome. Vectors utilized as the method for gene delivery can be divided into two categories, recombinant viruses and synthetic vectors.

Plant transformation vectors are plasmids that have been specifically designed to facilitate the generation of transgenic plants. The most commonly used plant transformation vectors are T-DNA binary vectors and are often replicated in both E. coli, a common lab bacterium, and Agrobacterium tumefaciens, a plant-virulent bacterium used to insert the recombinant DNA into plants.

Transformation efficiency refers to the ability of a cell to take up and incorporate exogenous DNA, such as plasmids, during a process called transformation. The efficiency of transformation is typically measured as the number of transformants per microgram of DNA added to the cells. A higher transformation efficiency means that more cells are able to take up the DNA, and a lower efficiency means that fewer cells are able to do so.

A P1-derived artificial chromosome, or PAC, is a DNA construct derived from the DNA of P1 bacteriophages and Bacterial artificial chromosome. It can carry large amounts of other sequences for a variety of bioengineering purposes in bacteria. It is one type of the efficient cloning vector used to clone DNA fragments in Escherichia coli cells.

In biology, an autoinducer is a signaling molecule that enables detection and response to changes in the population density of bacterial cells. Synthesized when a bacterium reproduces, autoinducers pass outside the bacterium and into the surrounding medium. They are a key component of the phenomenon of quorum sensing: as the density of quorum-sensing bacterial cells increases, so does the concentration of the autoinducer. A bacterium’s detection of an autoinducer above some minimum threshold triggers altered gene expression.

Calcium chloride (CaCl2) transformation is a laboratory technique in prokaryotic (bacterial) cell biology. The addition of calcium chloride to a cell suspension promotes the binding of plasmid DNA to lipopolysaccharides (LPS). Positively charged calcium ions attract both the negatively charged DNA backbone and the negatively charged groups in the LPS inner core. The plasmid DNA can then pass into the cell upon heat shock, where chilled cells (+4 degrees Celsius) are heated to a higher temperature (+42 degrees Celsius) for a short time.

<span class="mw-page-title-main">History of genetic engineering</span>

Genetic engineering is the science of manipulating genetic material of an organism. The concept of genetic engineering was first proposed by Nikolay Timofeev-Ressovsky in 1934. The first artificial genetic modification accomplished using biotechnology was transgenesis, the process of transferring genes from one organism to another, first accomplished by Herbert Boyer and Stanley Cohen in 1973. It was the result of a series of advancements in techniques that allowed the direct modification of the genome. Important advances included the discovery of restriction enzymes and DNA ligases, the ability to design plasmids and technologies like polymerase chain reaction and sequencing. Transformation of the DNA into a host organism was accomplished with the invention of biolistics, Agrobacterium-mediated recombination and microinjection. The first genetically modified animal was a mouse created in 1974 by Rudolf Jaenisch. In 1976, the technology was commercialised, with the advent of genetically modified bacteria that produced somatostatin, followed by insulin in 1978. In 1983, an antibiotic resistant gene was inserted into tobacco, leading to the first genetically engineered plant. Advances followed that allowed scientists to manipulate and add genes to a variety of different organisms and induce a range of different effects. Plants were first commercialized with virus resistant tobacco released in China in 1992. The first genetically modified food was the Flavr Savr tomato marketed in 1994. By 2010, 29 countries had planted commercialized biotech crops. In 2000 a paper published in Science introduced golden rice, the first food developed with increased nutrient value.

<span class="mw-page-title-main">Genetic engineering techniques</span> Methods used to change the DNA of organisms

Genetic engineering techniques allow the modification of animal and plant genomes. Techniques have been devised to insert, delete, and modify DNA at multiple levels, ranging from a specific base pair in a specific gene to entire genes. There are a number of steps that are followed before a genetically modified organism (GMO) is created. Genetic engineers must first choose what gene they wish to insert, modify, or delete. The gene must then be isolated and incorporated, along with other genetic elements, into a suitable vector. This vector is then used to insert the gene into the host genome, creating a transgenic or edited organism.

<span class="mw-page-title-main">Competence factor</span>

The ability of a cell to successfully incorporate exogenous DNA, or competency, is determined by competence factors. These factors consist of certain cell surface proteins and transcription factors that induce the uptake of DNA.

References

  1. 1 2 3 4 5 6 7 8 Johnston C, Martin B, Fichant G, Polard P, Claverys JP (March 2014). "Bacterial transformation: distribution, shared mechanisms and divergent control". Nature Reviews. Microbiology. 12 (3): 181–96. doi:10.1038/nrmicro3199. PMID   24509783. S2CID   23559881.
  2. 1 2 Alberts B, Johnson A, Lewis J, Raff M, Roberts K, Walter P (2002). Molecular Biology of the Cell. New York: Garland Science. p. G:35. ISBN   978-0-8153-4072-0.
  3. ↑ Griffith F (1928). "The Significance of Pneumococcal Types". The Journal of Hygiene. 27 (2): 113–59. doi:10.1017/s0022172400031879. PMC   2167760 . PMID   20474956.
  4. ↑ Case, Christine; Funke, Berdell; Tortora, Gerard. Microbiology An Introduction(tenth edition)
  5. ↑ Lederberg, Joshua (1994). "The Transformation of Genetics by DNA: An Anniversary Celebration of AVERY, MACLEOD and MCCARTY(1944) in Anecdotal, Historical and Critical Commentaries on Genetics". Genetics. 136 (2): 423–6. doi:10.1093/genetics/136.2.423. PMC   1205797 . PMID   8150273.
  6. ↑ Mandel M, Higa A (October 1970). "Calcium-dependent bacteriophage DNA infection". Journal of Molecular Biology. 53 (1): 159–62. doi:10.1016/0022-2836(70)90051-3. PMID   4922220.
  7. ↑ Cohen SN, Chang AC, Hsu L (August 1972). "Nonchromosomal antibiotic resistance in bacteria: genetic transformation of Escherichia coli by R-factor DNA". Proceedings of the National Academy of Sciences of the United States of America. 69 (8): 2110–4. Bibcode:1972PNAS...69.2110C. doi: 10.1073/pnas.69.8.2110 . PMC   426879 . PMID   4559594.
  8. ↑ Hanahan D (June 1983). "Studies on transformation of Escherichia coli with plasmids". Journal of Molecular Biology. 166 (4): 557–80. CiteSeerX   10.1.1.460.2021 . doi:10.1016/S0022-2836(83)80284-8. PMID   6345791.
  9. ↑ Wirth R, Friesenegger A, Fiedler S (March 1989). "Transformation of various species of gram-negative bacteria belonging to 11 different genera by electroporation". Molecular & General Genetics. 216 (1): 175–7. doi:10.1007/BF00332248. PMID   2659971. S2CID   25214157.
  10. ↑ Palmiter RD, Brinster RL, Hammer RE, Trumbauer ME, Rosenfeld MG, Birnberg NC, Evans RM (December 1982). "Dramatic growth of mice that develop from eggs microinjected with metallothionein-growth hormone fusion genes". Nature. 300 (5893): 611–5. Bibcode:1982Natur.300..611P. doi:10.1038/300611a0. PMC   4881848 . PMID   6958982.
  11. ↑ Nester, Eugene. "Agrobacterium: The Natural Genetic Engineer (100 Years Later)". APS. The American Phytopathological Society. Retrieved 14 January 2011.
  12. ↑ Zambryski P, Joos H, Genetello C, Leemans J, Montagu MV, Schell J (1983). "Ti plasmid vector for the introduction of DNA into plant cells without alteration of their normal regeneration capacity". The EMBO Journal. 2 (12): 2143–50. doi:10.1002/j.1460-2075.1983.tb01715.x. PMC   555426 . PMID   16453482.
  13. ↑ Peters, Pamela. "Transforming Plants - Basic Genetic Engineering Techniques". Access Excellence. Retrieved 28 January 2010.
  14. ↑ "Biologists invent gun for shooting cells with DNA" (PDF). Cornell Chronicle. 14 May 1987. p. 3.
  15. ↑ Sanford JC, Klein TM, Wolf ED, Allen N (1987). "Delivery of substances into cells and tissues using a particle bombardment process". Journal of Particulate Science and Technology. 5: 27–37. doi:10.1080/02726358708904533.
  16. ↑ Klein RM, Wolf ED, Wu R, Sanford JC (1992). "High-velocity microprojectiles for delivering nucleic acids into living cells. 1987". Biotechnology (Reading, Mass.). 24: 384–6. PMID   1422046.
  17. 1 2 3 Michod RE, Bernstein H, Nedelcu AM (May 2008). "Adaptive value of sex in microbial pathogens". Infection, Genetics and Evolution. 8 (3): 267–85. Bibcode:2008InfGE...8..267M. doi:10.1016/j.meegid.2008.01.002. PMID   18295550.
  18. 1 2 Seitz P, Blokesch M (May 2013). "Cues and regulatory pathways involved in natural competence and transformation in pathogenic and environmental Gram-negative bacteria" (PDF). FEMS Microbiology Reviews. 37 (3): 336–63. doi: 10.1111/j.1574-6976.2012.00353.x . PMID   22928673.
  19. 1 2 3 Johnsborg O, Eldholm V, Håvarstein LS (December 2007). "Natural genetic transformation: prevalence, mechanisms and function". Research in Microbiology. 158 (10): 767–78. doi: 10.1016/j.resmic.2007.09.004 . PMID   17997281.
  20. 1 2 Chen I, Dubnau D (March 2004). "DNA uptake during bacterial transformation". Nature Reviews. Microbiology. 2 (3): 241–9. doi:10.1038/nrmicro844. PMID   15083159. S2CID   205499369.
  21. ↑ Lacks S, Greenberg B, Neuberger M (June 1974). "Role of a deoxyribonuclease in the genetic transformation of Diplococcus pneumoniae". Proceedings of the National Academy of Sciences of the United States of America. 71 (6): 2305–9. Bibcode:1974PNAS...71.2305L. doi: 10.1073/pnas.71.6.2305 . PMC   388441 . PMID   4152205.
  22. ↑ Long CD, Tobiason DM, Lazio MP, Kline KA, Seifert HS (November 2003). "Low-level pilin expression allows for substantial DNA transformation competence in Neisseria gonorrhoeae". Infection and Immunity. 71 (11): 6279–91. doi:10.1128/iai.71.11.6279-6291.2003. PMC   219589 . PMID   14573647.
  23. ↑ Sisco KL, Smith HO (February 1979). "Sequence-specific DNA uptake in Haemophilus transformation". Proceedings of the National Academy of Sciences of the United States of America. 76 (2): 972–6. Bibcode:1979PNAS...76..972S. doi: 10.1073/pnas.76.2.972 . PMC   383110 . PMID   311478.
  24. ↑ Solomon JM, Grossman AD (April 1996). "Who's competent and when: regulation of natural genetic competence in bacteria". Trends in Genetics. 12 (4): 150–5. doi:10.1016/0168-9525(96)10014-7. PMID   8901420.
  25. ↑ 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". Journal of Bioscience and Bioengineering. 101 (3): 257–62. doi:10.1263/jbb.101.257. PMID   16716928.
  26. ↑ Saito Y, Taguchi H, Akamatsu T (April 2006). "DNA taken into Bacillus subtilis competent cells by lysed-protoplast transformation is not ssDNA but dsDNA". Journal of Bioscience and Bioengineering. 101 (4): 334–9. doi:10.1263/jbb.101.334. PMID   16716942.
  27. ↑ Akamatsu T, Taguchi H (April 2001). "Incorporation of the whole chromosomal DNA in protoplast lysates into competent cells of Bacillus subtilis". Bioscience, Biotechnology, and Biochemistry. 65 (4): 823–9. doi: 10.1271/bbb.65.823 . PMID   11388459. S2CID   30118947.
  28. ↑ Goodgal SH, Herriott RM (July 1961). "Studies on transformations of Hemophilus influenzae. I. Competence". The Journal of General Physiology. 44 (6): 1201–27. doi:10.1085/jgp.44.6.1201. PMC   2195138 . PMID   13707010.
  29. ↑ Aspiras MB, Ellen RP, Cvitkovitch DG (September 2004). "ComX activity of Streptococcus mutans growing in biofilms". FEMS Microbiology Letters. 238 (1): 167–74. doi:10.1016/j.femsle.2004.07.032 (inactive 2024-09-17). PMID   15336418.{{cite journal}}: CS1 maint: DOI inactive as of September 2024 (link)
  30. ↑ Anagnostopoulos C, Spizizen J (May 1961). "Requirements for Transformation in Bacillus Subtilis". Journal of Bacteriology. 81 (5): 741–6. doi:10.1128/JB.81.5.741-746.1961. PMC   279084 . PMID   16561900.
  31. ↑ Angelov, Angel; Bergen, Paul; Nadler, Florian; Hornburg, Philipp; Lichev, Antoni; Ãœbelacker, Maria; Pachl, Fiona; Kuster, Bernhard; Liebl, Wolfgang (10 February 2015). "Novel Flp pilus biogenesis-dependent natural transformation". Frontiers in Microbiology. 6: 84. doi: 10.3389/fmicb.2015.00084 . PMC   4322843 . PMID   25713572.
  32. ↑ Lichev, Antoni; Angelov, Angel; Cucurull, Inigo; Liebl, Wolfgang (30 July 2019). "Amino acids as nutritional factors and (p)ppGpp as an alarmone of the stringent response regulate natural transformation in Micrococcus luteus". Scientific Reports. 9 (1): 11030. Bibcode:2019NatSR...911030L. doi:10.1038/s41598-019-47423-x. PMC   6667448 . PMID   31363120.
  33. ↑ Keen EC, Bliskovsky VV, Malagon F, Baker JD, Prince JS, Klaus JS, Adhya SL (January 2017). "Novel "Superspreader" Bacteriophages Promote Horizontal Gene Transfer by Transformation". mBio. 8 (1): e02115–16. doi:10.1128/mBio.02115-16. PMC   5241400 . PMID   28096488.
  34. ↑ Claverys JP, Prudhomme M, Martin B (2006). "Induction of competence regulons as a general response to stress in gram-positive bacteria". Annual Review of Microbiology. 60: 451–75. doi:10.1146/annurev.micro.60.080805.142139. PMID   16771651.
  35. ↑ Michod RE, Wojciechowski MF, Hoelzer MA (January 1988). "DNA repair and the evolution of transformation in the bacterium Bacillus subtilis". Genetics. 118 (1): 31–9. doi:10.1093/genetics/118.1.31. PMC   1203263 . PMID   8608929.
  36. ↑ Dorer MS, Fero J, Salama NR (July 2010). Blanke SR (ed.). "DNA damage triggers genetic exchange in Helicobacter pylori". PLOS Pathogens. 6 (7): e1001026. doi: 10.1371/journal.ppat.1001026 . PMC   2912397 . PMID   20686662.
  37. 1 2 Charpentier X, Kay E, Schneider D, Shuman HA (March 2011). "Antibiotics and UV radiation induce competence for natural transformation in Legionella pneumophila". Journal of Bacteriology. 193 (5): 1114–21. doi:10.1128/JB.01146-10. PMC   3067580 . PMID   21169481.
  38. ↑ 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". Mutagenesis. 10 (4): 343–51. doi:10.1093/mutage/10.4.343. PMID   7476271.
  39. ↑ Washburn RS, Gottesman ME (January 2011). "Transcription termination maintains chromosome integrity". Proceedings of the National Academy of Sciences of the United States of America. 108 (2): 792–7. Bibcode:2011PNAS..108..792W. doi: 10.1073/pnas.1009564108 . PMC   3021005 . PMID   21183718.
  40. ↑ Sakano K, Oikawa S, Hasegawa K, Kawanishi S (November 2001). "Hydroxyurea induces site-specific DNA damage via formation of hydrogen peroxide and nitric oxide". Japanese Journal of Cancer Research. 92 (11): 1166–74. doi:10.1111/j.1349-7006.2001.tb02136.x. PMC   5926660 . PMID   11714440.
  41. 1 2 Bernstein H, Bernstein C, Michod RE (2012). "Chapter 1: DNA repair as the primary adaptive function of sex in bacteria and eukaryotes". In Kimura S, Shimizu S (eds.). DNA Repair: New Research. Nova Sci. Publ., Hauppauge, N.Y. pp. 1–49. ISBN   978-1-62100-808-8.
  42. ↑ Michod RE, Bernstein H, Nedelcu AM (May 2008). "Adaptive value of sex in microbial pathogens" (PDF). Infection, Genetics and Evolution. 8 (3): 267–85. Bibcode:2008InfGE...8..267M. doi:10.1016/j.meegid.2008.01.002. PMID   18295550.
  43. ↑ Donahue RA, Bloom FR (July 1998). "Large-volume transformation with high-throughput efficiency chemically competent cells" (PDF). Focus. Vol. 20, no. 2. pp. 54–56. OCLC   12352630. Archived from the original (PDF) on 2013-03-06 – via Invitrogen.[ unreliable source? ]
  44. 1 2 Srivastava S (2013). Genetics of Bacteria (PDF). India: Springer-Verlag. doi:10.1007/978-81-322-1090-0. ISBN   978-81-322-1089-4. S2CID   35917467.
  45. ↑ Kawai S, Hashimoto W, Murata K (1 November 2010). "Transformation of Saccharomyces cerevisiae and other fungi: methods and possible underlying mechanism". Bioengineered Bugs. 1 (6): 395–403. doi:10.4161/bbug.1.6.13257. PMC   3056089 . PMID   21468206.
  46. ↑ Hinnen A, Hicks JB, Fink GR (April 1978). "Transformation of yeast". Proceedings of the National Academy of Sciences of the United States of America. 75 (4): 1929–33. Bibcode:1978PNAS...75.1929H. doi: 10.1073/pnas.75.4.1929 . PMC   392455 . PMID   347451.
  47. ↑ Ito H, Fukuda Y, Murata K, Kimura A (January 1983). "Transformation of intact yeast cells treated with alkali cations". Journal of Bacteriology. 153 (1): 163–8. doi:10.1128/JB.153.1.163-168.1983. PMC   217353 . PMID   6336730.
  48. ↑ Gietz RD, Woods RA (2002). "Transformation of yeast by lithium acetate/single-stranded carrier DNA/polyethylene glycol method". Guide to Yeast Genetics and Molecular and Cell Biology - Part B. Methods in Enzymology. Vol. 350. pp. 87–96. doi:10.1016/S0076-6879(02)50957-5. ISBN   9780121822538. PMID   12073338.
  49. ↑ 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". Yeast. 11 (4): 355–60. doi:10.1002/yea.320110408. PMID   7785336. S2CID   22611810.
  50. ↑ Schiestl, Robert H.; Manivasakam, P.; Woods, Robin A.; Gietzt, R.Daniel (1 August 1993). "Introducing DNA into Yeast by Transformation". Methods. 5 (2): 79–85. doi:10.1006/meth.1993.1011.
  51. ↑ Spencer, F.; Ketner, G.; Connelly, C.; Hieter, P. (1 August 1993). "Targeted Recombination-Based Cloning and Manipulation of Large DNA Segments in Yeast". Methods. 5 (2): 161–175. doi:10.1006/meth.1993.1021.
  52. ↑ Costanzo MC, Fox TD (November 1988). "Transformation of yeast by agitation with glass beads". Genetics. 120 (3): 667–70. doi:10.1093/genetics/120.3.667. PMC   1203545 . PMID   3066683.
  53. ↑ 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". Yeast. 7 (7): 691–2. doi:10.1002/yea.320070704. PMID   1776359. S2CID   7108750.
  54. ↑ 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". Journal of Bioscience and Bioengineering. 94 (2): 166–71. doi:10.1016/s1389-1723(02)80138-4. PMID   16233287.
  55. ↑ V.Singh and D.K.Jain (2014). "Applications of recombinant DNA". ISC BIOLOGY. Nageen Prakashan. p. 840.
  56. 1 2 3 4 5 6 Poyedinok, N. L.; Blume, Ya. B. (March 2018). "Advances, Problems, and Prospects of Genetic Transformation of Fungi". Cytology and Genetics. 52 (2): 139–154. doi:10.3103/S009545271802007X. ISSN   0095-4527. S2CID   4561837.
  57. ↑ He, Liya; Feng, Jiao; Lu, Sha; Chen, Zhiwen; Chen, Chunmei; He, Ya; Yi, Xiuwen; Xi, Liyan (2017). "Genetic transformation of fungi". The International Journal of Developmental Biology. 61 (6–7): 375–381. doi: 10.1387/ijdb.160026lh . ISSN   0214-6282. PMID   27528043.
  58. ↑ Waltz, Emily (April 2016). "Gene-edited CRISPR mushroom escapes US regulation". Nature. 532 (7599): 293. Bibcode:2016Natur.532..293W. doi: 10.1038/nature.2016.19754 . ISSN   0028-0836. PMID   27111611.
  59. ↑ 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". Physics of Life Reviews. 11 (2): 184–203. Bibcode:2014PhLRv..11..184R. doi:10.1016/j.plrev.2014.01.007. PMID   24507729.
  60. ↑ Bacterial Transformation Archived 2010-06-10 at the Wayback Machine
  61. ↑ Inoue H, Nojima H, Okayama H (November 1990). "High efficiency transformation of Escherichia coli with plasmids". Gene. 96 (1): 23–8. doi:10.1016/0378-1119(90)90336-P. PMID   2265755.
  62. ↑ Donahue RA, Bloom FR (September 1998). "Transformation efficiency of E. coli electroporated with large plasmid DNA" (PDF). Focus. 20 (3): 77–78. Archived from the original on September 3, 2011.{{cite journal}}: CS1 maint: unfit URL (link)
  63. ↑ Birnboim HC, Doly J (November 1979). "A rapid alkaline extraction procedure for screening recombinant plasmid DNA". Nucleic Acids Research. 7 (6): 1513–23. doi:10.1093/nar/7.6.1513. PMC   342324 . PMID   388356.
This page is based on this Wikipedia article
Text is available under the CC BY-SA 4.0 license; additional terms may apply.
Images, videos and audio are available under their respective licenses.