Cellular Transformation
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In
molecular biology Molecular biology is a branch of biology that seeks to understand the molecule, molecular basis of biological activity in and between Cell (biology), cells, including biomolecule, biomolecular synthesis, modification, mechanisms, and interactio ...
and
genetics Genetics is the study of genes, genetic variation, and heredity in organisms.Hartl D, Jones E (2005) It is an important branch in biology because heredity is vital to organisms' evolution. Gregor Mendel, a Moravian Augustinians, Augustinian ...
, transformation is the genetic alteration of a
cell Cell most often refers to: * Cell (biology), the functional basic unit of life * Cellphone, a phone connected to a cellular network * Clandestine cell, a penetration-resistant form of a secret or outlawed organization * Electrochemical cell, a de ...
resulting from the direct uptake and incorporation of exogenous genetic material from its surroundings through the
cell membrane The cell membrane (also known as the plasma membrane or cytoplasmic membrane, and historically referred to as the plasmalemma) is a biological membrane that separates and protects the interior of a cell from the outside environment (the extr ...
(s). For transformation to take place, the recipient bacterium must be in a state of
competence Broad concept article: *Competence (polyseme), capacity or ability to perform effectively Competence or competency may also refer to: *Competence (human resources), ability of a person to do a job properly **Competence-based management, performa ...
, 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. Transformation is one of three processes that lead to
horizontal gene transfer 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 e ...
, in which exogenous genetic material passes from one bacterium to another, the other two being
conjugation Conjugation or conjugate may refer to: Linguistics *Grammatical conjugation, the modification of a verb from its basic form *Emotive conjugation or Russell's conjugation, the use of loaded language Mathematics *Complex conjugation, the change o ...
(transfer of
genetic material Nucleic acids are large biomolecules that are crucial in all cells and viruses. They are composed of nucleotides, which are the monomer components: a 5-carbon sugar, a phosphate group and a nitrogenous base. The two main classes of nucleic aci ...
between two bacterial cells in direct contact) and transduction (injection of foreign DNA by a
bacteriophage A bacteriophage (), also known informally as a phage (), is a virus that infects and replicates within bacteria. The term is derived . Bacteriophages are composed of proteins that Capsid, encapsulate a DNA or RNA genome, and may have structu ...
virus into the host bacterium). In transformation, the genetic material passes through the intervening medium, and uptake is completely dependent on the recipient bacterium. As of 2014 about 80 species of bacteria were known to be capable of transformation, about evenly divided between
Gram-positive In bacteriology, gram-positive bacteria are bacteria that give a positive result in the Gram stain test, which is traditionally used to quickly classify bacteria into two broad categories according to their type of cell wall. The Gram stain is ...
and
Gram-negative bacteria Gram-negative bacteria are bacteria that, unlike gram-positive bacteria, do not retain the Crystal violet, crystal violet stain used in the Gram staining method of bacterial differentiation. Their defining characteristic is that their cell envelo ...
; the number might be an overestimate since several of the reports are supported by single papers. "Transformation" may also be used to describe the insertion of new genetic material into nonbacterial cells, including animal and plant cells; however, because "
transformation Transformation may refer to: Science and mathematics In biology and medicine * Metamorphosis, the biological process of changing physical form after birth or hatching * Malignant transformation, the process of cells becoming cancerous * Trans ...
" has a special meaning in relation to animal cells, indicating progression to a cancerous state, the process is usually called "
transfection Transfection is the process of deliberately introducing naked or purified nucleic acids into eukaryotic cells. It may also refer to other methods and cell types, although other terms are often preferred: " transformation" is typically used to des ...
".


History

Transformation in bacteria was first demonstrated in 1928 by the British bacteriologist
Frederick Griffith Frederick Griffith (1877–1941) was a British bacteriologist whose focus was the epidemiology and pathology of bacterial pneumonia. In January 1928 he reported what is now known as Griffith's experiment, the first widely accepted demonstrat ...
. 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 ''Streptococcus pneumoniae'', or pneumococcus, is a Gram-positive, spherical bacteria, hemolysis (microbiology), alpha-hemolytic member of the genus ''Streptococcus''. ''S. pneumoniae'' cells are usually found in pairs (diplococci) and do not f ...
'' could be made
virulent Virulence is a pathogen's or microorganism's ability to cause damage to a host. In most cases, especially in animal systems, virulence refers to the degree of damage caused by a microbe to its host. The pathogenicity of an organism—its abilit ...
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 Oswald Theodore Avery Jr. (October 21, 1877 – February 20, 1955) was a Canadian-American physician and medical researcher. The major part of his career was spent at the Rockefeller Hospital in New York City. Avery was one of the first molecu ...
, Colin MacLeod, and
Maclyn McCarty Maclyn McCarty (June 9, 1911 – January 2, 2005) was an American geneticist, a research scientist described in 2005 as "the last surviving member of a Manhattan scientific team that overturned medical dogma in the 1940s and became the first to ...
. 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) 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 A genetic marker is a gene or DNA sequence with a known location on a chromosome that can be used to identify individuals or species. It can be described as a variation (which may arise due to mutation or alteration in the genomic loci) that can be ...
and the discovery of other methods of genetic transfer (
conjugation Conjugation or conjugate may refer to: Linguistics *Grammatical conjugation, the modification of a verb from its basic form *Emotive conjugation or Russell's conjugation, the use of loaded language Mathematics *Complex conjugation, the change o ...
in 1947 and transduction in 1953) by
Joshua Lederberg Joshua Lederberg (May 23, 1925 – February 2, 2008) was an American molecular biology, molecular biologist known for his work in microbial genetics, artificial intelligence, and the United States space program. He was 33 years old when he won t ...
that Avery's experiments were accepted. It was originally thought that ''
Escherichia coli ''Escherichia coli'' ( )Wells, J. C. (2000) Longman Pronunciation Dictionary. Harlow ngland Pearson Education Ltd. is a gram-negative, facultative anaerobic, rod-shaped, coliform bacterium of the genus '' Escherichia'' that is commonly fo ...
'', 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. Two years later in 1972,
Stanley Norman Cohen Stanley Norman Cohen (born February 17, 1935) is an American geneticist and the Kwoh-Ting Li Professor in the Stanford University School of Medicine. Stanley Cohen and Herbert Boyer were the first scientists to transplant genes from one living o ...
, Annie Chang and Leslie Hsu showed that treatment is also effective for transformation of plasmid DNA. The method of transformation by Mandel and Higa was later improved upon by
Douglas Hanahan Douglas Hanahan (born 1951) is an American biologist, professor, and Director Emeritus of the Swiss Institute for Experimental Cancer Research at EPFL (École polytechnique Fédérale de Lausanne) in Lausanne, Switzerland. He is a Distinguishe ...
. The discovery of artificially induced competence in ''E. coli'' created an efficient and convenient procedure for transforming bacteria which allows for simpler
molecular cloning Molecular cloning is a set of experimental methods in molecular biology that are used to assemble recombinant DNA molecules and to direct their DNA replication, replication within Host (biology), host organisms. The use of the word ''cloning'' re ...
methods in
biotechnology Biotechnology is a multidisciplinary field that involves the integration of natural sciences and Engineering Science, engineering sciences in order to achieve the application of organisms and parts thereof for products and services. Specialists ...
and
research Research is creative and systematic work undertaken to increase the stock of knowledge. It involves the collection, organization, and analysis of evidence to increase understanding of a topic, characterized by a particular attentiveness to ...
, and it is now a routinely used laboratory procedure. Transformation using
electroporation Electroporation, also known as electropermeabilization, is a microbiological and biotechnological technique in which an electric field is applied to cells to briefly increase the permeability of the cell membrane. The application of a high-vo ...
was developed in the late 1980s, increasing the efficiency of in-vitro transformation and increasing the number of
bacterial strain Bacteria (; : bacterium) are ubiquitous, mostly free-living organisms often consisting of one biological cell. They constitute a large domain of prokaryotic microorganisms. Typically a few micrometres in length, bacteria were among the fi ...
s that could be transformed. 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. In 1897 a bacterium that caused plant tumors, ''
Agrobacterium tumefaciens ''Agrobacterium tumefaciens'' (also known as ''Rhizobium radiobacter'') is the causal agent of crown gall disease (the formation of tumours) in over 140 species of eudicots. It is a rod-shaped, Gram-negative soil bacterium. Symptoms are cause ...
'', was discovered and in the early 1970s the tumor-inducing agent was found to be a DNA
plasmid 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 and ...
called the
Ti plasmid A tumour inducing (Ti) plasmid is a plasmid found in pathogenic species of ''Agrobacterium'', including Agrobacterium tumefaciens, ''A. tumefaciens'', Rhizobium rhizogenes, ''A. rhizogenes'', ''A. rubi'' and Allorhizobium vitis, ''A. vitis''. Ev ...
. 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. Not all plant cells are susceptible to infection by ''A. tumefaciens'', so other methods were developed, including
electroporation Electroporation, also known as electropermeabilization, is a microbiological and biotechnological technique in which an electric field is applied to cells to briefly increase the permeability of the cell membrane. The application of a high-vo ...
and micro-injection. Particle bombardment was made possible with the invention of the Biolistic Particle Delivery System (gene gun) by John Sanford in the 1980s.


Definitions

Transformation is one of three forms of
horizontal gene transfer 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 e ...
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 Homologous recombination is a type of genetic recombination in which genetic information is exchanged between two similar or identical molecules of double-stranded or single-stranded nucleic acids (usually DNA as in Cell (biology), cellular organi ...
; the other two are transduction, carried out by means of a
bacteriophage A bacteriophage (), also known informally as a phage (), is a virus that infects and replicates within bacteria. The term is derived . Bacteriophages are composed of proteins that Capsid, encapsulate a DNA or RNA genome, and may have structu ...
, and
conjugation Conjugation or conjugate may refer to: Linguistics *Grammatical conjugation, the modification of a verb from its basic form *Emotive conjugation or Russell's conjugation, the use of loaded language Mathematics *Complex conjugation, the change o ...
, 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.
Competence Broad concept article: *Competence (polyseme), capacity or ability to perform effectively Competence or competency may also refer to: *Competence (human resources), ability of a person to do a job properly **Competence-based management, performa ...
refers to a temporary state of being able to take up exogenous DNA from the environment; it may be induced in a laboratory. 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 Genetic diversity is the total number of genetic characteristics in the genetic makeup of a species. It ranges widely, from the number of species to differences within species, and can be correlated to the span of survival for a species. It is d ...
. Transformation has been studied in medically important
Gram-negative bacteria Gram-negative bacteria are bacteria that, unlike gram-positive bacteria, do not retain the Crystal violet, crystal violet stain used in the Gram staining method of bacterial differentiation. Their defining characteristic is that their cell envelo ...
species such as ''
Helicobacter pylori ''Helicobacter pylori'', previously known as ''Campylobacter pylori'', is a gram-negative, Flagellum#bacterial, flagellated, Bacterial cellular morphologies#Helical, helical bacterium. Mutants can have a rod or curved rod shape that exhibits l ...
'', ''
Legionella pneumophila ''Legionella pneumophila'', the primary causative agent for Legionnaires' disease, Legionnaire's disease, is an Aerobic organism, aerobic, pleomorphic, Flagellum, flagellated, non-spore-forming, Gram-negative bacteria, Gram-negative bacterium. ' ...
'', ''
Neisseria meningitidis ''Neisseria meningitidis'', often referred to as the meningococcus, is a Gram-negative bacterium that can cause meningitis and other forms of meningococcal disease such as meningococcemia, a life-threatening sepsis. The bacterium is referred to a ...
'', ''
Neisseria gonorrhoeae ''Neisseria gonorrhoeae'', also known as ''gonococcus'' (singular) or ''gonococci'' (plural), is a species of Gram-negative diplococci bacteria first isolated by Albert Ludwig Sigesmund Neisser, Albert Neisser in 1879. An obligate human pathog ...
'', ''
Haemophilus influenzae ''Haemophilus influenzae'' (formerly called Pfeiffer's bacillus or ''Bacillus influenzae'') is a Gram-negative, Motility, non-motile, Coccobacillus, coccobacillary, facultative anaerobic organism, facultatively anaerobic, Capnophile, capnophili ...
'' and ''
Vibrio cholerae ''Vibrio cholerae'' is a species of Gram-negative bacteria, Gram-negative, Facultative anaerobic organism, facultative anaerobe and Vibrio, comma-shaped bacteria. The bacteria naturally live in Brackish water, brackish or saltwater where they att ...
''. It has also been studied in Gram-negative species found in soil such as '' Pseudomonas stutzeri'', ''Acinetobacter baylyi'', and Gram-negative
plant pathogens Plant diseases are diseases in plants caused by pathogens (infectious organisms) and environmental conditions (physiological factors). Organisms that cause infectious disease include fungi, oomycetes, bacteria, viruses, viroids, virus-like orga ...
such as ''
Ralstonia solanacearum ''Ralstonia solanacearum'' is an aerobic non-spore-forming, Gram-negative, plant pathogenic bacterium. ''R. solanacearum'' is soil-borne and motile with a polar flagellar tuft. It colonises the xylem, causing bacterial wilt in a very wide ran ...
'' and ''
Xylella fastidiosa ''Xylella fastidiosa'' is an aerobic, Gram-negative bacterium of the genus ''Xylella''. It is a plant pathogen, that grows in the water transport tissues of plants ( xylem vessels) and is transmitted exclusively by xylem sap-feeding insects suc ...
''. Transformation among
Gram-positive bacteria In bacteriology, gram-positive bacteria are bacteria that give a positive result in the Gram stain test, which is traditionally used to quickly classify bacteria into two broad categories according to their type of cell wall. The Gram stain ...
has been studied in medically important species such as ''
Streptococcus pneumoniae ''Streptococcus pneumoniae'', or pneumococcus, is a Gram-positive, spherical bacteria, hemolysis (microbiology), alpha-hemolytic member of the genus ''Streptococcus''. ''S. pneumoniae'' cells are usually found in pairs (diplococci) and do not f ...
'', ''
Streptococcus mutans ''Streptococcus mutans'' is a Facultative anaerobic organism, facultatively anaerobic, gram-positive coccus (round bacteria, bacterium) commonly found in the human oral cavity and is a significant contributor to dental caries, tooth decay. The m ...
'', ''
Staphylococcus aureus ''Staphylococcus aureus'' is a Gram-positive spherically shaped bacterium, a member of the Bacillota, and is a usual member of the microbiota of the body, frequently found in the upper respiratory tract and on the skin. It is often posi ...
'' and ''
Streptococcus sanguinis ''Streptococcus sanguinis'', formerly known as ''Streptococcus sanguis'', is a Gram-positive facultative anaerobic coccus species of bacteria and a member of the Viridans Streptococcus group. ''S. sanguinis'' is a normal inhabitant of the healt ...
'' and in Gram-positive soil bacterium ''
Bacillus subtilis ''Bacillus subtilis'' (), known also as the hay bacillus or grass bacillus, is a gram-positive, catalase-positive bacterium, found in soil and the gastrointestinal tract of ruminants, humans and marine sponges. As a member of the genus ''Bacill ...
''. It has also been reported in at least 30 species of ''
Pseudomonadota Pseudomonadota (synonym "Proteobacteria") is a major phylum of gram-negative bacteria. Currently, they are considered the predominant phylum within the domain of bacteria. They are naturally found as pathogenic and free-living (non- parasitic) ...
'' distributed in several different classes. The best studied ''Pseudomonadota'' with respect to transformation are the medically important human pathogens ''
Neisseria gonorrhoeae ''Neisseria gonorrhoeae'', also known as ''gonococcus'' (singular) or ''gonococci'' (plural), is a species of Gram-negative diplococci bacteria first isolated by Albert Ludwig Sigesmund Neisser, Albert Neisser in 1879. An obligate human pathog ...
'', ''
Haemophilus influenzae ''Haemophilus influenzae'' (formerly called Pfeiffer's bacillus or ''Bacillus influenzae'') is a Gram-negative, Motility, non-motile, Coccobacillus, coccobacillary, facultative anaerobic organism, facultatively anaerobic, Capnophile, capnophili ...
'', and ''
Helicobacter pylori ''Helicobacter pylori'', previously known as ''Campylobacter pylori'', is a gram-negative, Flagellum#bacterial, flagellated, Bacterial cellular morphologies#Helical, helical bacterium. Mutants can have a rod or curved rod shape that exhibits l ...
''. "Transformation" may also be used to describe the insertion of new genetic material into nonbacterial cells, including animal and plant cells; however, because "
transformation Transformation may refer to: Science and mathematics In biology and medicine * Metamorphosis, the biological process of changing physical form after birth or hatching * Malignant transformation, the process of cells becoming cancerous * Trans ...
" has a special meaning in relation to animal cells, indicating progression to a cancerous state, the process is usually called "
transfection Transfection is the process of deliberately introducing naked or purified nucleic acids into eukaryotic cells. It may also refer to other methods and cell types, although other terms are often preferred: " transformation" is typically used to des ...
".


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 The type 2 secretion system (often referred to as the type II secretion system or by the initials T2SS) is a type of protein secretion machinery found in various species of Gram-negative bacteria, including many human pathogens such as ''Pseudomo ...
, as well as DNA
translocase Translocase is a general term for a protein that assists in moving another molecule, usually across a cell membrane. These Enzyme, enzymes catalyze the movement of ions or molecules across membranes or their separation within membranes. The reactio ...
complex at the cytoplasmic membrane. 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 The cell membrane (also known as the plasma membrane or cytoplasmic membrane, and historically referred to as the plasmalemma) is a biological membrane that separates and protects the cytoplasm, interior of a Cell (biology), cell from the extrac ...
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
RecA RecA is a 38 kilodalton protein essential for the repair and maintenance of DNA in bacteria. Structural and functional homologs to RecA have been found in all kingdoms of life. RecA serves as an archetype for this class of homologous DNA repair p ...
-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 Pilin refers to a class of fibrous proteins that are found in pilus structures in bacteria. These structures can be used for the exchange of genetic material, or as a cell adhesion mechanism. Although not all bacteria have pili or fimbriae, bact ...
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.


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. 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 ''Bacillus subtilis'' (), known also as the hay bacillus or grass bacillus, is a gram-positive, catalase-positive bacterium, found in soil and the gastrointestinal tract of ruminants, humans and marine sponges. As a member of the genus ''Bacill ...
'' 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. In ''B. subtilis'' 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. 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. 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 ''Haemophilus influenzae'' (formerly called Pfeiffer's bacillus or ''Bacillus influenzae'') is a Gram-negative, Motility, non-motile, Coccobacillus, coccobacillary, facultative anaerobic organism, facultatively anaerobic, Capnophile, capnophili ...
'' occurs most efficiently at the end of exponential growth as bacterial growth approaches stationary phase. Transformation in ''
Streptococcus mutans ''Streptococcus mutans'' is a Facultative anaerobic organism, facultatively anaerobic, gram-positive coccus (round bacteria, bacterium) commonly found in the human oral cavity and is a significant contributor to dental caries, tooth decay. The m ...
'', as well as in many other streptococci, occurs at high cell density and is associated with
biofilm A biofilm is a Syntrophy, syntrophic Microbial consortium, community of microorganisms in which cell (biology), cells cell adhesion, stick to each other and often also to a surface. These adherent cells become embedded within a slimy ext ...
formation. Competence in ''B. subtilis'' is induced toward the end of logarithmic growth, especially under conditions of amino acid limitation. Similarly, in ''
Micrococcus luteus ''Micrococcus luteus'' is a Gram-positive to Gram-variable, nonmotile, tetrad-arranging, pigmented, saprotrophic coccus bacterium in the family Micrococcaceae. It is urease and catalase positive. An obligate aerobe, ''M. luteus'' is found in soi ...
'' (a representative of the less well studied ''
Actinomycetota The Actinomycetota (or Actinobacteria) are a diverse phylum of Gram-positive bacteria with high GC content. They can be terrestrial or aquatic. They are of great importance to land flora because of their contributions to soil systems. In soil t ...
'' phylum), competence develops during the mid-late exponential growth phase and is also triggered by amino acids starvation. By releasing intact host and plasmid DNA, certain
bacteriophage A bacteriophage (), also known informally as a phage (), is a virus that infects and replicates within bacteria. The term is derived . Bacteriophages are composed of proteins that Capsid, encapsulate a DNA or RNA genome, and may have structu ...
s are thought to contribute to transformation.


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). In ''B. subtilis'', transformation is increased by UV light, a DNA damaging agent. 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 Using ''Legionella pneumophila'', 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 ''L. pneumophila''. Charpentier et al. suggested that competence for transformation probably evolved as a DNA damage response. Natural transformation in the extraordinarily radiation resistant bacterium ''
Deinococcus radiodurans ''Deinococcus radiodurans'' is a bacterium, an extremophile and one of the most radiation-resistant organisms known. It can survive cold, dehydration, vacuum, and acid, and therefore is known as a polyextremophile. ''The Guinness Book Of World ...
'' is associated with the repair of DNA damage under stressful conditions. 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 DNA repair is a collection of processes by which a cell (biology), cell identifies and corrects damage to the DNA molecules that encode its genome. A weakened capacity for DNA repair is a risk factor for the development of cancer. DNA is cons ...
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. 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. 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 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 Sexually reproducing animals, plants, fungi and protists are thought to have evolved from a common ancestor that was a single-celled eukaryotic species. Sexual reproduction is widespread in eukaryotes, though a few eukaryotic species have ...
;
Meiosis Meiosis () is a special type of cell division of germ cells in sexually-reproducing organisms that produces the gametes, the sperm or egg cells. It involves two rounds of division that ultimately result in four cells, each with only one c ...
.)


Methods and mechanisms of transformation in laboratory


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. Typically the cells are incubated in a solution containing
divalent In chemistry, the valence (US spelling) or valency (British spelling) of an atom is a measure of its combining capacity with other atoms when it forms chemical compounds or molecules. Valence is generally understood to be the number of chemica ...
cation An ion () is an atom or molecule with a net electrical charge. The charge of an electron is considered to be negative by convention and this charge is equal and opposite to the charge of a proton, which is considered to be positive by convent ...
s (often
calcium chloride Calcium chloride is an inorganic compound, a Salt (chemistry), salt with the chemical formula . It is a white crystalline solid at room temperature, and it is highly soluble in water. It can be created by neutralising hydrochloric acid with cal ...
) 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 Lipopolysaccharide (LPS), now more commonly known as endotoxin, is a collective term for components of the outermost membrane of the cell envelope of gram-negative bacteria, such as '' E. coli'' and ''Salmonella'' with a common structural archit ...
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. The surface of bacteria such as ''E. coli'' is negatively charged due to
phospholipids Phospholipids are a class of lipids whose molecule has a hydrophilic "head" containing a phosphate group and two hydrophobic "tails" derived from fatty acids, joined by an alcohol residue (usually a glycerol molecule). Marine phospholipids typi ...
and
lipopolysaccharide Lipopolysaccharide (LPS), now more commonly known as endotoxin, is a collective term for components of the outermost membrane of the cell envelope of gram-negative bacteria, such as '' E. coli'' and ''Salmonella'' with a common structural archit ...
s 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. 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 Electroporation, also known as electropermeabilization, is a microbiological and biotechnological technique in which an electric field is applied to cells to briefly increase the permeability of the cell membrane. The application of a high-vo ...
is another method of promoting competence. In this method the cells are briefly shocked with an
electric field An electric field (sometimes called E-field) is a field (physics), physical field that surrounds electrically charged particles such as electrons. In classical electromagnetism, the electric field of a single charge (or group of charges) descri ...
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 Yeasts are eukaryotic, single-celled microorganisms classified as members of the fungus kingdom (biology), kingdom. The first yeast originated hundreds of millions of years ago, and at least 1,500 species are currently recognized. They are est ...
, including ''
Saccharomyces cerevisiae ''Saccharomyces cerevisiae'' () (brewer's yeast or baker's yeast) is a species of yeast (single-celled fungal microorganisms). The species has been instrumental in winemaking, baking, and brewing since ancient times. It is believed to have be ...
'', may be transformed by exogenous DNA in the environment. Several methods have been developed to facilitate this transformation at high frequency in the lab. * Yeast cells may be treated with enzymes to degrade their cell walls, yielding
spheroplast A spheroplast (or sphaeroplast in British usage) is a microbial cell from which the cell wall has been almost completely removed, as by the action of penicillin or lysozyme. According to some definitions, the term is used to describe Gram-negative ...
s. These cells are very fragile but take up foreign DNA at a high rate. * Exposing intact yeast cells to
alkali In chemistry, an alkali (; from the Arabic word , ) is a basic salt of an alkali metal or an alkaline earth metal. An alkali can also be defined as a base that dissolves in water. A solution of a soluble base has a pH greater than 7.0. The a ...
cation An ion () is an atom or molecule with a net electrical charge. The charge of an electron is considered to be negative by convention and this charge is equal and opposite to the charge of a proton, which is considered to be positive by convent ...
s such as those of
caesium Caesium (IUPAC spelling; also spelled cesium in American English) is a chemical element; it has Symbol (chemistry), symbol Cs and atomic number 55. It is a soft, silvery-golden alkali metal with a melting point of , which makes it one of only f ...
or
lithium Lithium (from , , ) is a chemical element; it has chemical symbol, symbol Li and atomic number 3. It is a soft, silvery-white alkali metal. Under standard temperature and pressure, standard conditions, it is the least dense metal and the ...
allows the cells to take up plasmid DNA. Later protocols adapted this transformation method, using lithium acetate,
polyethylene glycol Polyethylene glycol (PEG; ) is a polyether compound derived from petroleum with many applications, from industrial manufacturing to medicine. PEG is also known as polyethylene oxide (PEO) or polyoxyethylene (POE), depending on its molecular wei ...
, 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. *
Electroporation Electroporation, also known as electropermeabilization, is a microbiological and biotechnological technique in which an electric field is applied to cells to briefly increase the permeability of the cell membrane. The application of a high-vo ...
: Formation of transient holes in the cell membranes using electric shock; this allows DNA to enter as described above for bacteria. * Enzymatic digestion or agitation with glass beads may also be used to transform yeast cells. Efficiency – Different yeast genera and species take up foreign DNA with different efficiencies. 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.


Plants

A number of methods are available to transfer DNA into plant cells. Some
vector Vector most often refers to: * Euclidean vector, a quantity with a magnitude and a direction * Disease vector, an agent that carries and transmits an infectious pathogen into another living organism Vector may also refer to: Mathematics a ...
-mediated methods are: *''
Agrobacterium ''Agrobacterium'' is a genus of Gram-negative bacteria established by Harold J. Conn, H. J. Conn that uses horizontal gene transfer to cause tumors in plants. ''Agrobacterium tumefaciens'' is the most commonly studied species in this genus. ''Agr ...
''-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 ''Agrobacterium''. 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 herbicide resistance gene was used, could alternatively be planted in soil, then later treated with herbicide to kill wildtype segregants. Some plants species, such as ''Arabidopsis thaliana'' can be transformed by dipping the flowers or whole plant, into a suspension of ''Agrobacterium tumefaciens'', typically strain C58 (C=Cherry, 58=1958, the year in which this particular strain of ''A. tumefaciens'' 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 manner. *
Viral transformation Viral transformation is the change in growth, phenotype, or indefinite reproduction of cells caused by the introduction of inheritable material. Through this process, a virus causes harmful transformations of an in vivo cell or cell culture. The ...
( transduction): 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
RNA Ribonucleic acid (RNA) is a polymeric molecule that is essential for most biological functions, either by performing the function itself (non-coding RNA) or by forming a template for the production of proteins (messenger RNA). RNA and deoxyrib ...
which replicates in the cytoplasm of infected cell. For such genomes this method is a form of
transfection Transfection is the process of deliberately introducing naked or purified nucleic acids into eukaryotic cells. It may also refer to other methods and cell types, although other terms are often preferred: " transformation" is typically used to des ...
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 gene. Some vector-less methods include: *
Gene gun In genetic engineering, a gene gun or biolistic particle delivery system is a device used to deliver exogenous DNA (transgenes), RNA, or protein to cells. By coating particles of a heavy metal with a gene of interest and firing these micro-projec ...
: 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 transformation efficiency is lower than in ''Agrobacterium''-mediated transformation, but most plants can be transformed with this method. *
Electroporation Electroporation, also known as electropermeabilization, is a microbiological and biotechnological technique in which an electric field is applied to cells to briefly increase the permeability of the cell membrane. The application of a high-vo ...
: Formation of transient holes in cell membranes using electric pulses of high field strength; this allows DNA to enter as described above for bacteria.


Fungi

There are some methods to produce transgenic
fungi A fungus (: fungi , , , or ; or funguses) is any member of the group of eukaryotic organisms that includes microorganisms such as yeasts and mold (fungus), molds, as well as the more familiar mushrooms. These organisms are classified as one ...
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: * A major issue is the dikaryotic state 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
sporulation In biology, a spore is a unit of sexual (in fungi) or asexual reproduction that may be adapted for dispersal and for survival, often for extended periods of time, in unfavourable conditions. Spores form part of the life cycles of many plant ...
. * Fungal cell walls are quite thick hindering DNA uptake so (partial) removal is often required; complete degradation, which is sometimes necessary, yields
protoplast Protoplast (), is a biology, biological term coined by Johannes von Hanstein, Hanstein in 1880 to refer to the entire cell, excluding the cell wall. Protoplasts can be generated by stripping the cell wall from plant, bacterium, bacterial, or f ...
s. * Mycelial fungi consist of filamentous
hypha A hypha (; ) is a long, branching, filamentous structure of a fungus, oomycete, or actinobacterium. In most fungi, hyphae are the main mode of vegetative growth, and are collectively called a mycelium. Structure A hypha consists of one o ...
e, 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. * Additionally, growth (and thereby mitosis) of these fungi exclusively occurs at the tip of their hyphae which can also deliver issues. As stated earlier, an array of methods used for plant transformation do also work in fungi: * 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
acetosyringone Acetosyringone is a phenolic natural product and a chemical compound related to acetophenone and 2,6-dimethoxyphenol. It was first described in relation to lignan/phenylpropanoid-type phytochemicals, with isolation from a variety of plant sour ...
. * Thanks to development of an expression system for small RNAs in fungi the introduction of a CRISPR/CAS9-system 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 market. * Physical methods like electroporation, biolistics ("gene gun"), sonoporation that uses cavitation of gas bubbles produced by ultrasound to penetrate the cell membrane, etc. are also applicable to fungi.


Animals

Introduction of DNA into animal cells is usually called
transfection Transfection is the process of deliberately introducing naked or purified nucleic acids into eukaryotic cells. It may also refer to other methods and cell types, although other terms are often preferred: " transformation" is typically used to des ...
, 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 ''Escherichia coli'' ( )Wells, J. C. (2000) Longman Pronunciation Dictionary. Harlow ngland Pearson Education Ltd. is a gram-negative, facultative anaerobic, rod-shaped, coliform bacterium of the genus '' Escherichia'' that is commonly fo ...
'' 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 The origin of replication (also called the replication origin) is a particular sequence in a genome at which replication is initiated. Propagation of the genetic material between generations requires timely and accurate duplication of DNA by semi ...
, 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 (in 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. Protocols, however, exist for making supercompetent cells that may yield a transformation efficiency of over 109. 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 Exonucleases are enzymes that work by cleaving nucleotides one at a time from the end (exo) of a polynucleotide chain. A hydrolyzing reaction that breaks phosphodiester bonds at either the 3′ or the 5′ end occurs. Its close relative is th ...
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 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 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. The plasmid therefore requires a
selectable marker A selectable marker is a gene introduced into cell (biology), cells, especially bacteria or cells in cell culture, culture, which confers one or more traits suitable for artificial selection. They are a type of reporter gene used in laboratory micr ...
such that those cells without the plasmid may be killed or have their growth arrested.
Antibiotic resistance Antimicrobial resistance (AMR or AR) occurs when microbes evolve mechanisms that protect them from antimicrobials, which are drugs used to treat infections. This resistance affects all classes of microbes, including bacteria (antibiotic resis ...
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 Auxotrophy ( "to increase"; ''τροφή'' "nourishment") is the inability of an organism to synthesize a particular organic compound required for its growth (as defined by IUPAC). An auxotroph is an organism that displays this characteristic; ''a ...
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 gene Reporter genes are molecular tools widely used in molecular biology, genetics, and biotechnology to study gene function, expression patterns, and regulatory mechanisms. These genes encode proteins that produce easily detectable signals, such as ...
s can be used as markers, such as the '' lacZ'' gene which codes for
β-galactosidase β-Galactosidase (EC 3.2.1.23, beta-gal or β-gal; systematic name β-D-galactoside galactohydrolase) is a glycoside hydrolase enzyme that catalyzes hydrolysis of terminal non-reducing β-D-galactose residues in β-D-galactosides. (This enzym ...
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 A multiple cloning site (MCS), also called a polylinker, is a short segment of DNA which contains many (up to ~20) restriction sites—a standard feature of engineered plasmids. Restriction sites within an MCS are typically unique, occurring onl ...
, where a gene of interest may be ligated into the plasmid
vector Vector most often refers to: * Euclidean vector, a quantity with a magnitude and a direction * Disease vector, an agent that carries and transmits an infectious pathogen into another living organism Vector may also refer to: Mathematics a ...
, 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 The green fluorescent protein (GFP) is a protein that exhibits green fluorescence when exposed to light in the blue to ultraviolet range. The label ''GFP'' traditionally refers to the protein first isolated from the jellyfish ''Aequorea victo ...
(GFP), which produces cells that glow green under blue light, and the enzyme
luciferase Luciferase is a generic term for the class of oxidative enzymes that produce bioluminescence, and is usually distinguished from a photoprotein. The name was first used by Raphaël Dubois who invented the words ''luciferin'' and ''luciferase'' ...
, which catalyzes a reaction with
luciferin Luciferin () is a generic term for the light-emitting chemical compound, compound found in organisms that generate bioluminescence. Luciferins typically undergo an enzyme-catalyzed reaction with Oxygen, molecular oxygen. The resulting transforma ...
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.


References


External links


Bacterial Transformation
(a Flash Animation)

At the Max Planck Institute for Molecular Plant Physiology in Potsdam-Golm plant cells are 'bombarded' using a particle gun {{Portal bar, Biology Gene delivery Modification of genetic information Molecular biology