experiment for transformation

Transformation in Bacteria

Transformation in bacteria was first studied by a scientist Frederick Griffith in 1928 . According to Griffith, the DNA or gene transfer can occur either naturally or artificially from one type of bacteria to another. For example, Transformation of non-virulent strain to a virulent cell or vice versa.

To explain the transformation principle, Griffith performed certain experiments on the mice by taking pathogenic bacteria Streptococcus pneumoniae . Transformation results in gene alteration in the recipient cell, due to the incorporation of free DNA from its surrounding through the cell membrane.

The transformation process is widely used in gene cloning, DNA linkage, generation of cDNA libraries and protein expression. Here, we will discuss the definition, stages, competence in the transformation of bacteria. You will also get to know the transformation principle of the bacteria through the Griffith experiment (transformation experiment).

Content: Transformation in Bacteria

Definition of transformation, streptococcus pneumoniae, strains used in griffith experiment, transformation experiment, transforming factor, stages of transformation.

Transformation can define as the process of taking up of an extracellular or free DNA strand of one bacterial cell ( donor’s cell ) by the competent bacterial cell ( recipient’s cell ). The taking up of the DNA strand occurs either by natural or artificial means. The transformation occurs mostly in the closely related species. Therefore, transformation merely refers to the direct insertion , incorporation and expression of the exogenous DNA in the competent bacterial cell (gets transformed by the inclusion of free DNA).

Classification :

Kingdom : Bacteria
Phylum : Firmicutes
Class : Bacilli
Order : Lactobacillales
Family : Streptococcaceae
Genus : Streptococcus
Species : pneumoniae

To demonstrate the transformation principle, Frederick Griffith had taken the pathogenic bacteria Streptococcus pneumoniae . Further, he observed two different strains of Streptococcus pneumoniae and named it as S-III and R-II strain.

S-III strain : It is the smooth strain of Streptococcus pneumonia , which is encapsulated with the polysaccharide. S-III strain will act as virulent or wild strain , as the polysaccharide is a virulent factor.
R-II strain : It is the rough strain of Streptococcus pneumonia , which lacks the polysaccharide covering. The R-II strain will act as mutant or avirulent strain , as the polysaccharide is absent.

To explain the theory of transformation principle, Frederick Griffith performed a series of experiments where he injected two different strains of Streptococcus pneumoniae into the mice and reported the particular strain’s effect into the mice.

In his first experiment, Griffith used a rough strain of Streptococcus pneumonia ( R-II ) and injected it into the mice. After doing this, he observed that the R-II strain of bacteria did not affect the mice and the mice lived . Therefore, Griffith named R-II strain as an “ Avirulent strain ”.
In his second experiment, Griffith used a smooth strain of Streptococcus pneumonia ( S-III ) and injected it into the mice. After doing this, he observed that the S-III strain of bacteria killed the mice. Therefore, Griffith named S-III strain as a “ Virulent strain ”.
In the third experiment, Griffith used smooth or virulent S-III and subjected it to the heat to destroy the virulence. Then, he injected the heat-killed S-III strain into the mice. After doing this, he observed that the heat-killed S-III strain did not affect the mice and the mice lived . Therefore, Griffith concluded that the virulence of the S-III strain becomes ineffective the heat exposure.
In the fourth experiment, Griffith used rough R-II strain plus dead or heat-killed S-III strain and injected into the mice. After that, he observed the death of mice. Then, he concluded that the S-III strain had transferred something which transformed the R-II strain into the virulent strain (R-III) and caused the death of mice.

To explain the transformation factor (whether it was a protein or some other component), Avery , Macleod and McCarty performed a series of experiments. Their experiment to identify the transformation of R-II to virulent type can be summarized into the following sequential steps:

First, they extracted different components like protein, polysaccharide, lipid, RNA and DNA from the heat-killed S-III strain.
After that, they added R-II strain individually into each test tubes.
In the third step, they used specific enzymes for the digestion of specific components.
Then, they injected it to the mice.

After doing this experiment, they observed the death of four mice except for the last one. They concluded that the DNA is the transformation factor which has transformed the R-II strain to the virulent type. Therefore, the DNA is the heritable material that has transferred the virulence from the dead or heat-killed S-III strain to the R-II strain.

As the DNA of S-III or virulent strain is destroyed by the enzyme DNase , there will not be any transformation between the heat-killed S-III strain and the R-II strain, and thus there will be no effect on the mice. They also concluded that even though the polysaccharide is a virulent factor, but still it is not involved in the transformation as it is not heritable.

There are three stages of transformation which include :

Competence is the first stage where a cell must be competent to take up the DNA. To develop competence, the cell responds to the environmental signal, allowing the binding and penetration of the free DNA .
The DNA binding is the second stage of transformation in which the exogenous or free DNA binds to the recipient’s cell wall due to developed competence. This stage occurs at the time of incubation of bacterial cell culture on ice. The DNA will bind to the recipient cell wall of bacteria by forming calcium chloride plus a DNA complex.
DNA integration is the incorporation of the exogenous DNA that has entered to the recipient cell cytoplasm. Therefore, the insertion of foreign DNA into the chromosome of the recipient cell will cause transformation.

To carry out the transformation process, the bacteria should be competent to take up the free DNA. Competence can define as the physiological state , where a recipient cell is in a state where it can respond to the environmental conditions such as starvation and cell density. Therefore, when a cell becomes competent, it can take up the exogenous DNA from the donor’s cell.

In the process of transformation, competence can be of two types :

Natural competence

Artificial competence.

It is a type where a transformation occurs naturally in response to environmental signals and extreme conditions. About 1% of bacteria can develop competence naturally. A set of genes are carried by the naturally competent bacteria. The genes (DNA) then migrate across the cell membrane naturally and infuse within the recipient’s cell.

In this type, a transformation is induced artificially by some chemical or physical methods. Thus, the transformation process is forced or do not occur naturally. Artificial competence can be achieved by both chemical and physical methods. The artificial competence can be achieved by the chemical method through the divalent cation method and physical method through the electroporation.

Divalent cation method : It was first introduced by the two scientists Mandel and Higa in 1970 . In the divalent cation method, the E.coli in the log phase of growth are taken from the culture. Then, E.coli culture is centrifuged. From the E.coli culture, the pellet of bacteria is resuspended in the divalent ion solution like calcium chloride . After that, the culture is kept under cold conditions that result in the weakening of bacteria’s cell surface and allow the binding of free DNA molecule.

Then, the bacterial suspension is suddenly subjected to the high temperature (42 Degrees Celsius) for 30 seconds in the boiling water bath, and the process called heat shock . It results in the thermal imbalance within the bacterial cell and forces the binding of free DNA into the cell.

Electroporation : It is an alternative method of chemical transformation. In electroporation, the bacterial cell is subjected to a high voltage of 15 kV/cm for a 5 µ sec under the influence of an electric field, and the process is called electroshock . The electric shock enhances the ability to take up the free DNA strand. In 1982, a technique of introducing free DNA into the mice was carried out by a scientist Neumann where he treated it with the short pulses at high voltage.

Neumann concluded that the electric shock increases the cell’s membrane potential and thereby increases the cell permeability to take up the charged molecule like DNA.

We can conclude that the non-competent cell has to be competent to carry out the transformation. The competence is developed by the environmental signals like temperature, pH, heat etc., making the cell competent by enhancing the ability to take up the free DNA.

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Molecular Biology

Griffith Experiment - Transformation in Bacteria, DNA as Genetic Material

Griffith's Experiment in 1928 demonstrated bacterial transformation, where non-virulent bacteria turned virulent upon exposure to heat-killed virulent strains. Avery, MacLeod, and McCarty experiment later confirmed in 1944 that DNA, not proteins, was the genetic material responsible for this transformation. Griffith Experiment in conclusion recognized DNA's significant role in heredity. In this article, we will study the Frederick Griffith Experiment - steps, strain of bacteria, and Griffith Experiment summary.

Table of Content

Griffith Experiment & Transforming Principle

Griffith experiment diagram, r strain and s strain bacteria.

Griffith’s Experiment - Transformation in Bacteria

Impact of the Griffith Experiment

Dna as genetic material.

Frederick Griffith conducted an experiment that demonstrated the transfer of genetic information between bacteria. The experiment showed that a heat-killed virulent strain could transform a non-lethal strain of bacteria . Griffith called the material that was transferred the "transforming principle". Griffith's experiment involved mixing living non-virulent bacteria with a heat-inactivated virulent form. The bacteria used in the experiment were Streptococcus pneumoniae, which showed two growth patterns. One culture plate had s mooth, shiny colonies (S), while the other had rough colonies (R) .

Griffith's experiment proved that some organisms can acquire new properties from their environment and from one another. However, it took almost 20 years for Avery, McLeod, and McCarty to confirm that nucleic acids, not proteins , are the molecules of heredity

Also Read : Mendel's Laws of Inheritance

The diagram of griffith experiment is shown below:

The R strain and S strain bacteria are two variants of the bacterium Streptococcus pneumonia, used by Frederick Griffith in his experiment. S strains are pathogenic, meaning they can cause disease. R strains are non-pathogenic, meaning they do not cause disease. Some other differences between R and S strains are:

Appearance: S strains have a smooth capsule , or outer coat, made of polysaccharides. R strains lack a capsule and have a rough appearance.
Colonies: S strains produce rough colonies, while R strains produce smooth colonies.
Virulence: S strains are virulent, while R strains are non-virulent.
Immune responses: The capsule of S strains allows the cell to escape the immune responses of the host mouse.
Mice: Mice injected with S strains die within a few days, while mice injected with R strains do not die.

In Griffith's experiment, when he injected mice with the heat-killed S strain and live R strain , the mice unexpectedly died. This revealed a transformation process where the R strain had taken up genetic material from the heat-killed S strain and become virulent. This observation helped in understanding bacterial transformation and the role of DNA as genetic material.

Also Read: Genetic Code - Molecular Basis of Inheritance

Griffith Experiment of Transformation in Bacteria

In 1928, English bacteriologist Frederick Griffith conducted an experiment that demonstrated how bacteria can change their function and form through transformation. The experiment was the first to suggest that bacteria can transfer genetic information through transformation. The experiment involved two strains of the bacterium Streptococcus pneumoniae: a virulent (disease-causing) strain (S) and a non-virulent (non-disease-causing) strain (R).

Transformation is the process of one thing changing into another. In molecular biology and genetics, transformation is the genetic alteration of a cell. It's one of three processes that lead to horizontal gene transfer , along with conjugation and transduction. The detail description of the Griffith’s Experiment - Transformation in Bacteria is as follows:

Griffith Experiment Steps

In the experiment, Griffith injected two types of Streptococcus pneumoniae into mice.

Griffith then subjected the virulent, smooth strain (S) to heat that killed the bacteria. This heat-killed strain (S) was no longer capable of causing disease.
Griffith injected mice with the heat-killed virulent strain (S). Surprisingly, the mice survived, indicating that the heat-killed bacteria alone were not harmful.
Griffith mixed the heat-killed virulent strain (S) with the live non-virulent, rough strain (R) and injected this mixture into mice.
The mice developed pneumonia and died, even though the strain injected was previously non-virulent.

Observations and Conclusion

Griffith concluded that some factor or biomolecule in the heat-killed virulent bacteria (S) had transformed the live non-virulent bacteria (R) into a virulent form. This phenomenon was termed "transformation," though Griffith could not identify the nature of the transforming substance.

Significance

Griffith's experiment laid the groundwork for understanding genetic transformation and proved that DNA , rather than proteins, carried genetic information. This discovery was fundamental to the development of molecular genetics and is also used in a variety of genetic engineering applications.

Conclusion - Griffith Experiment

Frederick Griffith's 1928 experiment on Streptococcus pneumoniae demonstrated bacterial transformation through a transfer of hereditary traits between strains. In Griffith experiment conclusion, the result showed that the harmless R strain could be transformed into a virulent form when exposed to the heat-killed S strain. Subsequent work by Avery, MacLeod, and McCarty in 1944 identified DNA as the transforming substance, establishing it as the genetic material. The discovery laid the foundation for molecular genetics, confirming the role of DNA in transmitting hereditary information.

Also Read: Inherited Traits Lethal Allele - Examples, & its Types Difference Between Phenotype and Genotype Ratio Importance of Variation

FAQs on Frederick Griffith Experiment

What was griffith’s experiment and why was it important.

Frederick Griffith conducted an experiment that suggested bacteria can transfer genetic information through transformation. The experiment was important because it showed that bacteria can change their function and form through transformation.

What is the Griffith Experiment Conclusion?

Frederick Griffith experiment concluded that bacteria can transfer genetic information through a process called transformation.

What was the Most Significant Conclusion of Griffith’s Experiments with Pneumonia in Mice?

Griffith conducted experiments with mice and Streptococcus pneumonia bacteria. He concluded that heat-killed bacteria can convert live avirulent cells to virulent cells. Griffith called this phenomenon transformation.

What did Frederick Griffith Want to Learn about Bacteria?

Frederick Griffith, a British bacteriologist, wanted to learn how bacteria could acquire new traits and how certain types of bacteria produce pneumonia.

How did the Two Types of Bacteria Used by Griffith Differ?

The two types of bacteria used by Griffith were the R strain, lacking a virulent capsule and non-pathogenic, and the S strain, possessing a smooth capsule and causing pneumonia in mice, making it pathogenic.

What was Oswald Avery's Experiment?

The experiment demonstrated that DNA was the only molecule that transformed from one bacterial strain to another.

What is Griffith's Transforming Principle?

Griffith performed an experiment with bacteria and mice and discovered that bacteria can incorporate foreign genetic material from their environment, which he called the transforming principle.

Why is Chapter Griffith Experiment Class 12 Important?

The Griffith Experiment in Class 12 biology is important as it describes bacterial transformation, highlighting the role of genetic material in heredity and laying the foundation for modern molecular biology and genetics research.

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Griffith Experiment
The Transforming Principle
Griffith's Experiment: Bacterial Transformation
DNA Experiments (Griffith & Avery, McCarty, MacLeod & Hershey, Chase)
Transformation in Bacteria
Lab 10-3: Transformation Experiment

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