Transformation, Practice problems and FAQs

How did spiderman get his powers? You must have learnt from the stories that he got bitten by a spider which caused the spider DNA to be incorporated in his DNA, thus giving hime spider like powers. Do you think it is possible in real life? Well, maybe not to this extent but our scientists have made discoveries in biotechnology where we can manipulate the DNA of an organism and mix the DNAs of two different organisms. You must be wondering how is that possible? Right? Biotechnology provides access to this method. It can make a genetically modified organism or GMO through Recombinant DNA (rDNA) technology.

Did you know that the human insulin gene has been cloned into bacterial cells for mass production of insulin? For this, scientists have used bacterial plasmids as vectors that can carry the human insulin gene into the bacterial cells. A recombinant DNA is formed by ligating the human insulin gene into a specific site of the plasmid vector DNA and then this recombinant DNA is introduced into the bacterial cells, such that it multiplies by and amplifies the insulin gene, while also expressing the gene to produce insulin.

The focus of our discussion in this article will be the techniques that are used to introduce foreign DNAs into cells.

Transformation

Transformation is the process by which a cell takes up foreign DNA from its surrounding. In the field of biotechnology, transformation techniques are used to genetically alter a host cell by introducing a foreign recombinant DNA (rDNA), consisting of a vector DNA and a gene of interest (GOI), into the host cell. So transformation can be defined as the process of transfer of exogenous naked DNA into a desired host cell.

Fig: Process of transformation

Prerequisites for transformation

Vector

We cannot simply insert the GOI into the host. There are some prerequisites for that. First, is the insertion of the GOI into a vector. Vectors are self replicating small-sized plasmid, cosmid or phage DNA molecules that can be manipulated to form a rDNA by inserting the gene of interest in them. Being self replicating, the vector can replicate within the host cell and hence form multiple copies of the GOI. A good vector should have the following characteristics -

An origin of replication such that it can self-replicate
Recognition sites for restriction endonucleases such that these enzymes can identify these specific recognition sites and cut the DNA at these specific sites (unique to each restriction endonuclease) to create gaps where the GOI can be inserted using the ligase enzyme.
Selectable markers such as antibiotic resistance genes which would confer antibiotic resistance to cells that would be successfully transformed with the vector carrying the GOI. Non-transformants will not be able to grow on antibiotic containing media.
Small size

The most commonly used vectors are bacterial plasmids. They are the extrachromosomal circular DNA found in bacteria.

Fig: Bacterial plasmid

Competent host cells

The next step is to insert the rDNA into a host cell. The most commonly used host cells are bacterial cells. This is because bacteria have a very short generation time of a few minutes and multiply very fast. Hence it creates multiple copies of the gene of interest in a short time.

Host cells do not readily take in the foreign DNA molecules and have to be made competent for the process of transformation. Competence refers to the ability of a cell to take up foreign DNA from its environment. This is because DNA is hydrophilic in nature and hence cannot readily pass through the hydrophobic cell membrane.

The cell membrane is composed of a phospholipid bilayer in which the hydrophilic heads of the phospholipids are arranged on either side whereas, the hydrophobic tails are arranged towards the inner side, to protect them from the surrounding environment. Thus the DNA molecule finds it difficult to pass through the hydrophobic lipid bilayer of the host cell membrane.

Both DNA and the phospholipids in the cell membrane are negatively charged and repel each other. This is another reason why DNA cannot readily pass through the cell membrane of the host cell, unless the host cells are treated with chemical agents to make them competent.

Fig: Like charges (negative) on rDNA and bacterial membrane

Thus to make the host cells readily uptake the DNA, the cells are made competent by the addition of chemical agents like CaCl₂, MgCl₂,etc which dissociate to release divalent cations (Mg²⁺, Ca²⁺, etc). These positive ions attract the negative charges on the DNA phosphate backbone and the phosphate heads of the phospholipids. This allows the DNA to bind to the phospholipid bilayer. Thus it becomes easier for the DNA to pass through the cell membrane, once the cells are subjected to different transformation techniques.

Fig: Positively charged divalent calcium ions neutralise the negative

Fig: Calcium rich environment helping the rDNA to enter the bacteria

Now that the competent host cells have the ability to take up the DNA, how do we introduce the DNA into the competent cells?

Methods of transformation

There are different methods of transformation used to introduce the rDNA into the host cells. Some of them are heat shock method, microinjection, biolistic gun, disarmed pathogen and electroporation. We will check them out one by one.

Fig: Techniques of transformation

Heat Shock Method

In this method a solution containing rDNA molecules is added to a vial containing competent bacterial culture.

FIg: Competent Bacterial Cells in the vial and rDNA with GOI in the pipette

Fig: rDNA added into the bacterial culture using a micropipette.

The mixture is then incubated in ice for a few minutes to bring down the cell’s temperature.

Fig: Incubation of mixture of host cells and rDNA molecule in ice

This is followed by putting the mixture in a water bath at 42^oC fors some time before placing it back into ice. The sudden and brief exposure to high temperature creates transient pores in the cell membrane which allows the competent cells to take up the rDNA. Placing the cells back into the ice makes sure that the pores are sealed and the DNA or other cell contents do not leak out.

Fig: Mixture of rDNA and competent host cells being subjected to heat shock

Now the transformed cells are plated and then incubated at room temperature. All the daughter cells in the bacterial colonies arising from the host cells transformed with the rDNA will now have a copy of the rDNA in themselves.

Microinjection

This is a physical method which requires the use of the microscope. In this method the rDNA molecules are injected directly into the nucleus of the animal cells using a microsyringe.

Fig: Method of microinjection

Fig: Microinjection under microscope

Biolistic or gene gun

Biolistic method is also called the gene gun method because a gun-like instrument is used to insert rDNA molecules inside host cells. Generally this method is used for introducing rDNA into plant cells. Microparticles of gold or tungsten (about 1-2 micron in size) are coated with the foreign DNA and bombarded on to the cell at high velocity. This occurs in a vacuum. As the high velocity particles travel down the instrument, they transfer the rDNA into host cells. Only the rDNA is transferred into the target host cells and the particles are held back.

Fig: Steps of biolistic method of gene transfer

Electroporation

Electroporation is the technique of introducing DNA into plant cells by exposing them to a very high voltage electric pulse. This creates transient pores in the cell membrane and allows the foreign DNA to enter the cells. Removal of the pulse makes the cell membrane stable again and the cell is restored to its original state.

Fig: Electric pulse creating transient pores in the cell membrane during electroporation

Disarmed Pathogen

Agrobacterium tumefaciens is a bacterial plant pathogen which carries the Ti (tumour inducing) Plasmid. The T-DNA segment of the Ti plasmid integrates itself into the plant cell’s genome to induce tumour formation and cause crown gall disease. This characteristic of the plasmid is used to transfer the gene of interest in the plant cell.

Fig: Agrobacterium tumefaciens causing the crown gall disease

But what are disarmed pathogens? Disarmed pathogens are organisms which have lost the pathogenicity, i.e. they cannot harm the host anymore but they have the ability to infect the host.

The T-DNA segment of the Ti Plasmid can be genetically manipulated to carry foreign DNA. A restriction enzyme can cut the T- DNA segment at a specific site and insert the DNA fragment carrying the gene of interest into The T-DNA segment using ligase. Once modified, the T-DNA becomes incapable of inducing tumours but the Agrobacterium is still capable of infecting the host plant cell..

Fig: Agrobacterium with GOI

So, if we incorporate the GOI into the T-DNA segment of the Ti plasmid and introduce the recombinant Ti plasmid into Agrobacterium, and then let it infect the plant cell, then the GOI will enter the plant cell without causing infection.

Fig: Agrobacterium with GOI inside infected host plant cell

During Agrobacterium infection, the recombinant T-DNA segment enters the plant host cell and gets integrated into its genome. This finally helps in integration of the gene of interest into the plant genome and thus genetically modify the plant. Thus Ti plasmids are considered to be excellent plant cell vectors.

Confirmation of transformants

The host cells which have successfully undergone transformation and have taken up the rDNA are called transformed hosts, transformants or recombinant cells. The cells which have not undergone successful transformation, do not have the rDNA and are hence known as non-transformants or non-recombinants. But how do we differentiate the transformants from the non-transformants? For that we need to go for the next step which is the confirmation of transformants.

This step involves culturing the transformed host cell culture onto petri plates and the selection of host cell colonies that have arisen from the transformed cells or recombinant cells. The selection of recombinant colonies is done by two methods. The first method is antibiotic resistance and the second one is insertional inactivation.

Fig: Methods of confirmation of transformants

Antibiotic resistance

In normal conditions the antibiotics kill bacteria. So if we culture the bacteria in a medium containing the antibiotics, they are unable to grow and die.

The vector DNA used to carry the GOI in the rDNA usually has antibiotic resistance genes and hence transformants will be able to grow on antibiotic containing media whereas non-transformants cannot. This helps in the selection of transformed cells which can then be selected and further cultured to amplify the GOI or obtain its product at a larger scale.

Fig: Bacterial colonies transformed with pBR322 plasmid vector survive in presence of antibiotic ampicillin

Insertional inactivation

The insertion of a foreign DNA within the coding sequence of a functional gene results in its inactivation. This is known as insertional inactivation. This phenomenon helps in the selection of recombinants.

The 𝛃-galactosidase enzyme, coded by the LacZ gene, converts a chromogenic substance, X-Gal present in the culture medium into galactose and a blue coloured byproduct. Thus, the bacterial cells which have a functional LacZ gene, produce blue coloured colonies when the growth medium contains X-Gal.

Fig: Bacterial plasmid with functional LacZ gene

If transformants get the foreign DNA incorporated within the coding sequence of the LacZ gene, then it causes insertional inactivation of the gene which is why the transformants no longer produce a functional 𝛃-galactosidase enzyme. Hence, they are unable to break down X-Gal to produce the blue coloured byproduct.

Fig: GOI inserted in the LacZ gene

Thus, the transformants produce non-coloured colonies when grown in an X-Gal containing medium. This makes selection of recombinants over non-recombinants becomes very easy as recombinants do not produce coloured colonies whereas non-recombinants give blue-coloured colonies.

Fig: Recombinant colonies show white colonies while non-recombinants show blue.

Practice Problems

1. Assertion: Gold or tungsten microparticles coated with DNA are used in biolistics.

Reason: Gold and Tungsten are inert.

Both the assertion and reason are true and the reason is the correct explanation of assertion
Both the assertion and reason are true and reason is not the correct explanation of assertion
Assertion is true but the reason is false
Both the assertion and reason are false

Solution: The process of transfer of exogenous DNA into host cells is known as transformation.

Biolistics is one of the methods of direct gene delivery into host cells by bombarding plant cells with high velocity micro-particles of gold or tungsten coated with DNA using a gene gun.. Gold or tungsten is used as they are inert in nature. Hence, they will not be toxic to the target cells.

Hence the correct option is a.

2. Read the following statements and choose the correct option.

I. High velocity micro-particles of ___A____ coated with DNA are bombarded into a ____B____ with the help of a gene gun.

II. ___C______ is the direct injection of rDNA into the nucleus of an animal cell in the method called as microinjection.

A - Plant cells, B - tungsten, C - gene gun
A - Plant cells, B - gold/ aluminium , C - electroporation
A - Animal cells, B - aluminium/ tungsten, C - electroporation
A - Gold/ tungsten, B - plant cells, C - microinjection

Solution: Biolistics, microinjection and electroporation are methods of direct gene delivery into the host cells. In the biolistics or gene gun method, the plant cells are bombarded with high velocity micro-particles of gold or tungsten coated with DNA.

In microinjection, recombinant DNA is directly injected into the nucleus of an animal cell using a microsyringe.

Hence the correct option is d.

3. Assertion: The bacterial cells must first be made ‘competent’ to take up foreign DNA.

Reason: DNA is hydrophilic and cannot pass through the hydrophobic cell membrane.

Select the correct option.

Both the assertion and reason are true and the reason is the correct explanation of the assertion.
Both the assertion and reason are true and reason is not the correct explanation of assertion.
Assertion is true but the reason is false.
Both the assertion and reason are false.

Solution: Cell competence refers to a cell's ability to take up foreign (extracellular) DNA from its surrounding environment. Bacterial cells are made competent to make them ready to take up foreign DNA. This is necessary because the cell membrane is composed of a bilayer of phospholipids. The hydrophilic heads of the phospholipids are arranged on either side and interact with the external environment. DNA being a hydrophilic molecule finds it difficult to pass through the hydrophobic lipid bilayer of the host cell membrane.

Hence the correct option is a.

4. The Ti plasmid is chosen as an effective plant cell vector because

it induces tumours in all organisms.
it is readily available.
a specific segment of this plasmid integrates itself into the host genome during infection.
it can induce tumours even after modification

Solution: The T-DNA segment of the Ti plasmid integrates itself into the plant cell’s genome to induce tumour formation in plants. The T-DNA segment of the Ti Plasmid can be genetically modified to carry foreign DNA. A restriction enzyme is used to cut the T- DNA segment at a specific site and the DNA fragment carrying the gene of interest is ligated into this segment with the help of a ligase enzyme. Once modified, the T-DNA becomes incapable of inducing tumours. This property of the plasmid makes it an effective vector for transferring foreign genes into plant cells.

Hence the correct option is c.

FAQs

1. What are the advantages of transformation for bacteria?
Answer: Bacteria may be able to adapt fast to shifting environments by transforming environmental DNA. While this can benefit bacteria by aiding the spread of antibiotic resistance, it can also be harmful if it leads the population to lose favourable alleles.

2. What is the definition of cell transformation?
Answer: Cell transformation refers to the changes that occur when normal homeostatic control, particularly of cell division, is lost, resulting in the emergence of a neoplastic phenotype (a sort of tissue growth that is abnormal and excessive).

3. Who was the first to discover transformation?
Answer: Fred Griffith discovered transformation in London in 1928, and reported that a pneumococcal strain could take on the shape of another strain even if the donor strain was dead.

4. How does transformation improve bacterial genetic diversity?
Answer: Competent cells can be transformed by absorbing naked DNA released by dying cells into their cytoplasm, where it can recombine with the host genome. When this happens, the transformed cell attains the genetic characteristics of the dead cell whose DNA it has taken up during transformation. As seen in Griffith’s experiment, the living non-virulent S. pneumoniae with rough glycocalyx become virulent in nature and produce a smooth glycocalyx when grown along with heat-killed virulent bacteria. This is because the DNA of the heat-killed virulent bacteria, that had leaked into the growth medium was taken up by the non-virulent ones during transformation.