Transformation (genetics)
Encyclopedia
In molecular biology
transformation is the genetic
alteration of a cell
resulting from the direct uptake, incorporation and expression
of exogenous gene
tic material (exogenous DNA
) from its surroundings and taken up through the cell membrane(s). Transformation occurs naturally in some species of bacteria, but it can also be effected by artificial means in other cells. Bacteria
that are capable of being transformed, whether naturally or artificially, are called competent
. Transformation is one of three processes by which exogenous genetic material may be introduced into a bacterial cell, 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). 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 term should be avoided for animal cells when describing introduction of exogenous genetic material. Introduction of foreign DNA into eukaryotic cell
s is usually called "transfection
".
. Griffith discovered that a harmless 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
). 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. .
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. Two years later in 1972, Stanley Cohen, Annie Chang and Leslie Hsu showed that CaCl2 treatment is also effective for transformation of plasmid DNA. The method of transformation by Mandel and Higa was later improved upon by Douglas Hanahan
. 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. 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 1907 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
. 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
and micro-injection
. Particle bombardment was made possible with the invention of the Biolistic Particle Delivery System (gene gun) by John Sanford
in 1990.
(DNA without associated cells or proteins) and competence
refers to the state of being able to take up exogenous DNA from the environment. There are two forms of competence: natural and artificial.
. Some species upon cell death release their DNA to be taken up by other cells, however transformation works best with DNA from closely related species. These naturally competent bacteria carry sets of genes that provide the protein machinery to bring DNA across the cell membrane(s). The transport of the exogeneous 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.
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. Only single-stranded DNA may pass through, one strand is therefore degraded by nucleases in the process, and 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 however 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.
cations, most commonly calcium chloride
solution under cold condition, and then exposed to a pulse of heat shock. However, the mechanism of the uptake of DNA via chemically-induced competence in this calcium chloride transformation
is unclear. The surface of bacteria such as E. coli is negatively charged due to phospholipids and lipopolysaccharide
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. It is suggested that exposing the cells to divalent cations in cold condition may also change or weaken the cell surface structure of the cells making it more permeable to DNA. The heat-pulse is thought to create a thermal imbalance on either side of the cell membrane, which forces the DNA to enter the cells either through 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.
, and is discussed in the corresponding article.
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 1x108 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 CaCl2 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
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 CaCl2 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.
such that those cells without the plasmid may be killed or 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 and sugars. This method requires the use of suitably mutated strains that are deficient in the synthesis of a particular biomolecule.
The transformed cells with plasmid however need not contain the desired recombinant DNA inserted in cloning experiment. Various techniques may be employed to screen for those containing 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.
Molecular biology
Molecular biology is the branch of biology that deals with the molecular basis of biological activity. This field overlaps with other areas of biology and chemistry, particularly genetics and biochemistry...
transformation is the genetic
Introduction to genetics
Genetics is the study of genes, and tries to explain what they are and how they work. Genes are how living organisms inherit features from their ancestors; for example, children usually look like their parents because they have inherited their parents' genes...
alteration of a cell
Cell (biology)
The cell is the basic structural and functional unit of all known living organisms. It is the smallest unit of life that is classified as a living thing, and is often called the building block of life. The Alberts text discusses how the "cellular building blocks" move to shape developing embryos....
resulting from the direct uptake, incorporation and expression
Gene expression
Gene expression is the process by which information from a gene is used in the synthesis of a functional gene product. These products are often proteins, but in non-protein coding genes such as ribosomal RNA , transfer RNA or small nuclear RNA genes, the product is a functional RNA...
of exogenous gene
Gene
A gene is a molecular unit of heredity of a living organism. It is a name given to some stretches of DNA and RNA that code for a type of protein or for an RNA chain that has a function in the organism. Living beings depend on genes, as they specify all proteins and functional RNA chains...
tic material (exogenous DNA
Exogenous DNA
Exogenous DNA refers to any deoxyribonucleic acid that originates outside of the organism of concern or study.The introduction of exogenous DNA into a cell is called...
) from its surroundings and taken up through the cell membrane(s). Transformation occurs naturally in some species of bacteria, but it can also be effected by artificial means in other cells. Bacteria
Bacteria
Bacteria are a large domain of prokaryotic microorganisms. Typically a few micrometres in length, bacteria have a wide range of shapes, ranging from spheres to rods and spirals...
that are capable of being transformed, whether naturally or artificially, are called competent
Competence (biology)
In microbiology, genetics, cell biology and molecular biology, competence is the ability of a cell to take up extracellular DNA from its environment...
. Transformation is one of three processes by which exogenous genetic material may be introduced into a bacterial cell, the other two being conjugation
Bacterial conjugation
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...
(transfer of genetic material between two bacterial cells in direct contact), and transduction
Transduction (genetics)
Transduction is the process by which DNA is transferred from one bacterium to another by a virus. It also refers to the process whereby foreign DNA is introduced into another cell via a viral vector. Transduction does not require cell-to-cell contact , and it is DNAase resistant...
(injection of foreign DNA by a bacteriophage virus into the host bacterium). 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 term should be avoided for animal cells when describing introduction of exogenous genetic material. Introduction of foreign DNA into eukaryotic cell
Eukaryotic Cell
Eukaryotic Cell is an academic journal published by the American Society for Microbiology. The title is commonly abbreviated EC and the ISSN is 1535-9778 for the print version, and 1535-9786 for the electronic version....
s is usually called "transfection
Transfection
Transfection is the process of deliberately introducing nucleic acids into cells. The term is used notably for non-viral methods in eukaryotic cells...
".
History
Transformation was first demonstrated in 1928 by British bacteriologist Frederick GriffithFrederick Griffith
Frederick Griffith 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 demonstrations of bacterial transformation, whereby a bacterium distinctly...
. Griffith discovered that a harmless strain of Streptococcus pneumoniae
Streptococcus pneumoniae
Streptococcus pneumoniae, or pneumococcus, is Gram-positive, alpha-hemolytic, aerotolerant anaerobic member of the genus Streptococcus. A significant human pathogenic bacterium, S...
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
Oswald Avery
Oswald Theodore Avery ForMemRS was a Canadian-born American physician and medical researcher. The major part of his career was spent at the Rockefeller University Hospital in New York City...
, Colin MacLeod
Colin MacLeod
Colin Munro MacLeod was a Canadian-American geneticist.- Biography :Born in Port Hastings, Nova Scotia, Canada MacLeod entered McGill University at the age of 16 , and completed his medical studies by age 23.In his early years as a research scientist, MacLeod, together with Oswald Avery and...
, and Maclyn McCarty
Maclyn McCarty
Maclyn McCarty was an American geneticist.Maclyn McCarty, who devoted his life as a physician-scientist to studying infectious disease organisms, was best known for his part in the monumental discovery that DNA, rather than protein, constituted the chemical nature of a gene...
. 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
Avery-MacLeod-McCarty experiment
The Avery–MacLeod–McCarty experiment was an experimental demonstration, reported in 1944 by Oswald Avery, Colin MacLeod, and Maclyn McCarty, that DNA is the substance that causes bacterial transformation...
). 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
Bacterial conjugation
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...
in 1947 and transduction
Transduction (genetics)
Transduction is the process by which DNA is transferred from one bacterium to another by a virus. It also refers to the process whereby foreign DNA is introduced into another cell via a viral vector. Transduction does not require cell-to-cell contact , and it is DNAase resistant...
in 1953) by Joshua Lederberg
Joshua Lederberg
Joshua Lederberg ForMemRS was an American molecular biologist known for his work in microbial genetics, artificial intelligence, and the United States space program. He was just 33 years old when he won the 1958 Nobel Prize in Physiology or Medicine for discovering that bacteria can mate and...
that Avery's experiments were accepted. .
It was originally thought that Escherichia coli
Escherichia coli
Escherichia coli is a Gram-negative, rod-shaped bacterium that is commonly found in the lower intestine of warm-blooded organisms . Most E. coli strains are harmless, but some serotypes can cause serious food poisoning in humans, and are occasionally responsible for product recalls...
, 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
Lambda phage
Enterobacteria phage λ is a temperate bacteriophage that infects Escherichia coli.Lambda phage is a virus particle consisting of a head, containing double-stranded linear DNA as its genetic material, and a tail that can have tail fibers. The phage particle recognizes and binds to its host, E...
without the use of helper phage
Helper virus
A helper virus is a virus used when producing copies of a helper dependent viral vector which does not have the ability to replicate on its own. The helper virus is used to coinfect cells alongside the viral vector and provides the necessary enzymes for replication of the genome of the viral vector....
after treatment with calcium chloride solution. Two years later in 1972, Stanley Cohen, Annie Chang and Leslie Hsu showed that CaCl2 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
Professor Douglas Hanahan an American biologist, and director of the Swiss Institute for Experimental Cancer Research.In 1983 he developed Super Optimal Broth, a microbiological growth medium....
. 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
Molecular cloning refers to a set of experimental methods in molecular biology that are used to assemble recombinant DNA molecules and to direct their replication within host organisms...
methods in biotechnology
Biotechnology
Biotechnology is a field of applied biology that involves the use of living organisms and bioprocesses in engineering, technology, medicine and other fields requiring bioproducts. Biotechnology also utilizes these products for manufacturing purpose...
and research
Research
Research can be defined as the scientific search for knowledge, or as any systematic investigation, to establish novel facts, solve new or existing problems, prove new ideas, or develop new theories, usually using a scientific method...
, and it is now a routinely-used laboratory procedure.
Transformation using electroporation
Electroporation
Electroporation, or electropermeabilization, is a significant increase in the electrical conductivity and permeability of the cell plasma membrane caused by an externally applied electrical field...
was developed in the late 1980s, increasing the efficiency of in-vitro transformation and increasing the number of bacterial strains 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 1907 a bacterium that caused plant tumors, Agrobacterium tumefaciens
Agrobacterium tumefaciens
Agrobacterium tumefaciens is the causal agent of crown gall disease in over 140 species of dicot. It is a rod shaped, Gram negative soil bacterium...
, was discovered and in the early 1970s the tumor inducing agent was found to be a DNA plasmid
Plasmid
In microbiology and genetics, a plasmid is a DNA molecule that is separate from, and can replicate independently of, the chromosomal DNA. They are double-stranded and, in many cases, circular...
called the Ti plasmid
Ti plasmid
Ti plasmid is a circular plasmid that often, but not always, is a part of the genetic equipment that Agrobacterium tumefaciens and Agrobacterium rhizogenes use to transduce its genetic material to plants. Ti stands for tumor inducing. The Ti plasmid is lost when Agrobacterium is grown above 28°C....
. 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
Electroporation, or electropermeabilization, is a significant increase in the electrical conductivity and permeability of the cell plasma membrane caused by an externally applied electrical field...
and micro-injection
Microinjection
Microinjection refers to the process of using a glass micropipette to insert substances at a microscopic or borderline macroscopic level into a single living cell. It is a simple mechanical process in which a needle roughly 0.5 to 5 micrometers in diameter penetrates the cell membrane and/or the...
. Particle bombardment was made possible with the invention of the Biolistic Particle Delivery System (gene gun) by John Sanford
John C. Sanford
-Academic career:Sanford graduated in 1976 from the University of Minnesota with a BSc in horticulture. He then went to the University of Wisconsin–Madison where he received an MSc in 1978 and a PhD in 1980 in plant breeding and genetics. Between 1980 and 1986 Sanford was an assistant professor at...
in 1990.
Bacteria
Bacterial transformation may be referred to as a stable genetic change brought about by the uptake of naked DNANaked DNA
Naked DNA is histone-free DNA that is passed from cell to cell during a gene transfer process called transformation or transfection. In transformation , purified or naked DNA is taken up by the recipient cell which will give the recipient cell a new characteristic or phenotype...
(DNA without associated cells or proteins) and competence
Competence (biology)
In microbiology, genetics, cell biology and molecular biology, competence is the ability of a cell to take up extracellular DNA from its environment...
refers to the state of being able to take up exogenous DNA from the environment. There are two forms of competence: natural and artificial.
Natural competence
About 1% of bacterial species are capable of naturally taking up DNA under laboratory conditions; more may be able to take it up in their natural environments. DNA material can be transferred between different strains of bacteria, in a process that is called horizontal gene transferHorizontal gene transfer
Horizontal gene transfer , also lateral gene transfer , is any process in which an organism incorporates genetic material from another organism without being the offspring of that organism...
. Some species upon cell death release their DNA to be taken up by other cells, however transformation works best with DNA from closely related species. These naturally competent bacteria carry sets of genes that provide the protein machinery to bring DNA across the cell membrane(s). The transport of the exogeneous 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
Translocase
Translocase is a general term for an enzyme that assists in moving another molecule, usually across a membrane.Translocases are most common secretion system in Gram positive bacteria.Examples include:...
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
Cell membrane
The cell membrane or plasma membrane is a biological membrane that separates the interior of all cells from the outside environment. The cell membrane is selectively permeable to ions and organic molecules and controls the movement of substances in and out of cells. It basically protects the cell...
via DNA translocase. Only single-stranded DNA may pass through, one strand is therefore degraded by nucleases in the process, and the translocated single-stranded DNA may then be integrated into the bacterial chromosomes by a RecA
RecA
RecA is a 38 kilodalton Escherichia coli protein essential for the repair and maintenance of DNA. RecA has a structural and functional homolog in every species in which it has been seriously sought and serves as an archetype for this class of homologous DNA repair proteins...
-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
Pilin refers to a class of fibrous proteins that are found in pilus structures in bacteria. Bacterial pili are used in the exchange of genetic material during bacterial conjugation, and a short pilus called a fimbrium is used as a cell adhesion mechanism. Although not all bacteria have pili or...
may be required for competence however 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.
Artificial competence
Artificial competence can be induced in laboratory procedures that involves 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 divalentDivalent
In chemistry, a divalent ion or molecule has a valence of two and thus can form two bonds with other ions or molecules. An older term for divalent is bivalent....
cations, most commonly calcium chloride
Calcium chloride
Calcium chloride, CaCl2, is a salt of calcium and chlorine. It behaves as a typical ionic halide, and is solid at room temperature. Common applications include brine for refrigeration plants, ice and dust control on roads, and desiccation...
solution under cold condition, and then exposed to a pulse of heat shock. However, the mechanism of the uptake of DNA via chemically-induced competence in this calcium chloride transformation
Calcium Chloride Transformation
Calcium chloride transformation is a laboratory technique in prokaryotic cell biology. It increases the ability of a prokaryotic cell to incorporate plasmid DNA allowing them to be genetically transformed. The addition of calcium chloride to a cell suspension promotes the binding of plasmid DNA...
is unclear. The surface of bacteria such as E. coli is negatively charged due to phospholipids and lipopolysaccharide
Lipopolysaccharide
Lipopolysaccharides , also known as lipoglycans, are large molecules consisting of a lipid and a polysaccharide joined by a covalent bond; they are found in the outer membrane of Gram-negative bacteria, act as endotoxins and elicit strong immune responses in animals.-Functions:LPS is the major...
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. It is suggested that exposing the cells to divalent cations in cold condition may also change or weaken the cell surface structure of the cells making it more permeable to DNA. The heat-pulse is thought to create a thermal imbalance on either side of the cell membrane, which forces the DNA to enter the cells either through cell pores or the damaged cell wall.
Electroporation
Electroporation
Electroporation, or electropermeabilization, is a significant increase in the electrical conductivity and permeability of the cell plasma membrane caused by an externally applied electrical field...
is another method of promoting competence. In this method the cells are briefly shocked with an electric field
Electric field
In physics, an electric field surrounds electrically charged particles and time-varying magnetic fields. The electric field depicts the force exerted on other electrically charged objects by the electrically charged particle the field is surrounding...
of 10-20 kV
Volt
The volt is the SI derived unit for electric potential, electric potential difference, and electromotive force. The volt is named in honor of the Italian physicist Alessandro Volta , who invented the voltaic pile, possibly the first chemical battery.- Definition :A single volt is defined as the...
/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.
Plants
A number of mechanisms are available to transfer DNA into plant cells:- AgrobacteriumAgrobacteriumAgrobacterium 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...
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 10 minutes in a fluid containing suspended Agrobacterium. Some cells along the cut will be transformed by the bacterium, that inserts its DNA into the cell. Placed on selectable rooting and shooting media, the plants will regrow. Some plants species can be transformed just by dipping the flowers into suspension of Agrobacterium and then planting the seeds in a selective medium. Unfortunately, many plants are not transformable by this method. - Particle bombardmentGene gunA gene gun or a biolistic particle delivery system, originally designed for plant transformation, is a device for injecting cells with genetic information. The payload is an elemental particle of a heavy metal coated with plasmid DNA...
: 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 efficiencyTransformation efficiencyTransformation efficiency is the efficiency by which cells can take up extracellular DNA and express genes encoded by it. This is based on the competence of the cells. It can be calculated by dividing the number of successful transformants by the amount of DNA used during a transformation procedure...
is lower than in Agrobacterium mediated transformation, but most plants can be transformed with this method. - ElectroporationElectroporationElectroporation, or electropermeabilization, is a significant increase in the electrical conductivity and permeability of the cell plasma membrane caused by an externally applied electrical field...
: make transient holes in cell membranes using electric shock; this allows DNA to enter as described above for Bacteria. - Viral transformationViral transformationViral transformation most commonly refers to the virus-induced malignant transformation of an animal cell in a body or cell culture. In molecular biology, the term may also refer to the transfection of DNA into a host cell using a viral vector....
(transductionTransduction (genetics)Transduction is the process by which DNA is transferred from one bacterium to another by a virus. It also refers to the process whereby foreign DNA is introduced into another cell via a viral vector. Transduction does not require cell-to-cell contact , and it is DNAase resistant...
): 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 RNARNARibonucleic acid , or RNA, is one of the three major macromolecules that are essential for all known forms of life....
which replicates in the cytoplasm of infected cell. For such genomes this method is a form of transfectionTransfectionTransfection is the process of deliberately introducing nucleic acids into cells. The term is used notably for non-viral methods in eukaryotic cells...
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.
Animals
Introduction of DNA into animal cells is usually called transfectionTransfection
Transfection is the process of deliberately introducing nucleic acids into cells. The term is used notably for non-viral methods in eukaryotic cells...
, 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 coliEscherichia coli
Escherichia coli is a Gram-negative, rod-shaped bacterium that is commonly found in the lower intestine of warm-blooded organisms . Most E. coli strains are harmless, but some serotypes can cause serious food poisoning in humans, and are occasionally responsible for product recalls...
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
Origin of replication
The origin of replication is a particular sequence in a genome at which replication is initiated. This can either be DNA replication in living organisms such as prokaryotes and eukaryotes, or RNA replication in RNA viruses, such as double-stranded RNA viruses...
, 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
Transformation efficiency
Transformation efficiency is the efficiency by which cells can take up extracellular DNA and express genes encoded by it. This is based on the competence of the cells. It can be calculated by dividing the number of successful transformants by the amount of DNA used during a transformation procedure...
and is measured in colony forming unit (cfu) per μg DNA used. A transformation efficiency of 1x108 cfu/μg for a small plasmid like pUC19
PUC19
pUC19 is one of a series of plasmid cloning vectors created by Messing and co-workers in the University of California. The p in its name stands for plasmid and UC represents the University in which it was created. It is a circular double stranded DNA and has 2686 base pairs...
is roughly equivalent to 1 in 2000 molecules of the plasmid used being transformed.
In calcium chloride transformation
Calcium Chloride Transformation
Calcium chloride transformation is a laboratory technique in prokaryotic cell biology. It increases the ability of a prokaryotic cell to incorporate plasmid DNA allowing them to be genetically transformed. The addition of calcium chloride to a cell suspension promotes the binding of plasmid DNA...
, the cells are prepared by chilling cells in the presence of Ca2+ (in CaCl2 solution
Calcium chloride
Calcium chloride, CaCl2, is a salt of calcium and chlorine. It behaves as a typical ionic halide, and is solid at room temperature. Common applications include brine for refrigeration plants, ice and dust control on roads, and desiccation...
) making the cell become permeable to plasmid DNA
Plasmid
In microbiology and genetics, a plasmid is a DNA molecule that is separate from, and can replicate independently of, the chromosomal DNA. They are double-stranded and, in many cases, circular...
. 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
Exonuclease
Exonucleases are enzymes that work by cleaving nucleotides one at a time from the end of a polynucleotide chain. A hydrolyzing reaction that breaks phosphodiester bonds at either the 3’ or the 5’ end occurs. Its close relative is the endonuclease, which cleaves phosphodiester bonds in the middle ...
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 CaCl2 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 markerSelectable marker
A selectable marker is a gene introduced into a cell, especially a bacterium or to cells in culture, that confers a trait suitable for artificial selection. They are a type of reporter gene used in laboratory microbiology, molecular biology, and genetic engineering to indicate the success of a...
such that those cells without the plasmid may be killed or 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
Auxotrophy
Auxotrophy is most commonly defined as the inability of an organism to synthesize a particular organic compound required for its growth . An auxotroph is an organism that displays this characteristic; auxotrophic is the corresponding adjective...
markers that can compensate for an inability to metabolise certain amino acids and sugars. This method requires the use of suitably mutated strains that are deficient in the synthesis of a particular biomolecule.
The transformed cells with plasmid however need not contain the desired recombinant DNA inserted in cloning experiment. Various techniques may be employed to screen for those containing the insert. Reporter genes
Reporter gene
In molecular biology, a reporter gene is a gene that researchers attach to a regulatory sequence of another gene of interest in cell culture, animals or plants. Certain genes are chosen as reporters because the characteristics they confer on organisms expressing them are easily identified and...
can be used as markers
Marker gene
A marker gene is a gene used in molecular biology to determine if a nucleic acid sequence has been successfully inserted into an organism's DNA. There are two types of marker genes: a selectable marker and a marker for screening.-Selectable marker:...
, such as the lacZ
Lac operon
The lac operon is an operon required for the transport and metabolism of lactose in Escherichia coli and some other enteric bacteria. It consists of three adjacent structural genes, lacZ, lacY and lacA. The lac operon is regulated by several factors including the availability of glucose and of...
gene which codes for β-galactosidase
Beta-galactosidase
β-galactosidase, also called beta-gal or β-gal, is a hydrolase enzyme that catalyzes the hydrolysis of β-galactosides into monosaccharides. Substrates of different β-galactosidases include ganglioside GM1, lactosylceramides, lactose, and various glycoproteins...
used in blue-white screening. This method of screening relies on the principle of α-complementation
Complementation (genetics)
In genetics, complementation refers to a relationship between two different strains of an organism which both have homozygous recessive mutations that produce the same phenotype . These strains are true breeding for their mutation...
, 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
X-gal
X-gal is an organic compound consisting of galactose linked to a substituted indole. The compound was synthesized by Jerome Horwitz and collaborators in Detroit, MI, in 1964. The formal chemical name is often shortened to less accurate but also less cumbersome phrases such as bromochloroindoxyl...
, forming characteristic blue colonies. However, the multiple cloning site
Multiple cloning site
A multiple cloning site , also called a polylinker, is a short segment of DNA which contains many restriction sites - a standard feature of engineered plasmids. Restriction sites within an MCS are typically unique, occurring only once within a given plasmid. MCSs are commonly used during...
, where a gene of interest may be ligated
DNA ligase
In molecular biology, DNA ligase is a specific type of enzyme, a ligase, that repairs single-stranded discontinuities in double stranded DNA molecules, in simple words strands that have double-strand break . Purified DNA ligase is used in gene cloning to join DNA molecules together...
into the plasmid vector
Vector (molecular biology)
In molecular biology, a vector is a DNA molecule used as a vehicle to transfer foreign genetic material into another cell. The four major types of vectors are plasmids, viruses, cosmids, and artificial chromosomes...
, 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
Green fluorescent protein
The green fluorescent protein is a protein composed of 238 amino acid residues that exhibits bright green fluorescence when exposed to blue light. Although many other marine organisms have similar green fluorescent proteins, GFP traditionally refers to the protein first isolated from the...
(GFP), which produces cells that glow green under blue light, and the enzyme luciferase
Luciferase
Luciferase is a generic term for the class of oxidative enzymes used in bioluminescence and is distinct from a photoprotein. One famous example is the firefly luciferase from the firefly Photinus pyralis. "Firefly luciferase" as a laboratory reagent usually refers to P...
, which catalyzes a reaction with luciferin
Luciferin
Luciferins are a class of light-emitting biological pigments found in organisms that cause bioluminescence...
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.
External links
- Bacterial Transformation (a Flash Animation)
- “Ready, aim, fire!” At the Max Planck Institute for Molecular Plant Physiology in Potsdam-Golm plant cells are ‘bombarded’ using a particle gun