5 Steps of Genetic Engineering Used in rDNA technology

Genetic engineering is the process of transferring the desired gene from an organism of interest to an organism of choice to obtain the desired product by applying the principle of biotechnology. The process occurs in  basic steps as

1. Isolation of the desired gene (gene cloning technology)
   
2. Selection of vector and insertion of a gene
   
3. Transfer of rDNA vector into host cells.
   
4. Multiplication, Identification, and isolation of recombinant gene cells
   
5. Expression of cloned genes inside the host cell to get the product.

The procedure followed is called rDNA technology.

In short, the desired substance like insulin for diabetic people is produced by the transfer of the desired gene (DNA) from a parent organism (here human) to a different organism (E-coli a bacteria).

In most cases, the desired organism is human or other organisms of human interest. While the organism of choice is mostly bacteria or yeast.

But why only bacteria and yeast? Because they can be quickly grown and their life cycle completes in a few hours to days. Due to this, we get the desired product formed in a short time

Because of such a short lifespan, they express the transferred gene to the fullest and we obtain the product very fast.

Steps of Genetic Engineering

To isolate the desired gene, the entire gene or DNA from the organism of interest has to be extracted.

This can be done by homogenization of tissue (breaking the cells) or by the use of surfactants to break up the cell membrane of the cell of choice.

Once the homogenate is obtained, the entire gene is separated by differential centrifugation (density-based).

This whole genome is now taken up to isolate the desired gene.

1. Isolation of the desired gene

Here the DNA coding for the desired protein is isolated. This is a critical task and can be done by any of the following four methods like

• Mechanical shearing.
• Chemical synthesis.
• By the use of restriction endonucleases.
• Complimentary DNA method.

Mechanical shearing

Here the required gene is cut off from the whole gene by the use of mechanical force. This can be done by methods like sonication, nebulization, point shink shearing, needle shear, etc. This method leads to the formation of random DNA fragments.

Chemical synthesis

As the name indicates, here the desired gene is synthesized by the use of free nucleotides. For this, the target protein is isolated and from it, the required nucleotide sequence is deduced.

Using restriction endonuclease enzymes

In this method, the whole genome is taken and subjected to the enzyme restriction endonucleases. This enzyme cuts the DNA at specific points like scissors. The gene obtained by this is quite perfect and hence widely used.

Complimentary DNA method

Here the desired DNA sequence is synthesized from the messenger RNA which codes the specific protein of choice. For this, the enzyme reverse transcriptase is used to synthesize the double-stranded DNA sequence.

The isolated genes are purified and taken for the next step to fix to a vector.

2. Selection of vector and insertion of the desired gene.

A vector is a vehicle to carry the desired gene into the genome of another organism. This helps us to see that the gene is not destroyed during transfer. Also, the gene will be operational inside the new organism due to the vector. These vectors have some specific properties like

1. It should be capable of independent multiplication. This is possible if the gene has the “Ori gene”
2. It should have a restriction site i.e. a site where the isolated gene can be fixed using restriction endonuclease. This is also called multiple cloning sites.
3. The vector should have a gene promoter sequence like a β-galactosidase gene.
4. Should have a Marker gene that helps to identify transgenic cells.

There are many types of vectors like

• Plasmids
• Cosmids
• Phasmid
• Transposons
• Bacteriophage (virus)
• Yeast cloning vector
• Shuttle vectors
• Human artificial vectors.

These vectors are large pieces of DNA molecules mostly.

Plasmids

These are naturally occurring DNA moieties from bacteria. The plasmid is a circular, single-stranded, and self-replicable DNA molecule present inside bacteria. They help in the sexual reproduction of bacteria by transfer of genetic matter from one to another. Here we use them to transfer the desired gene.

Cosmids

Cosmid is similar to plasmid DNA but can accommodate large DNA pieces.

Bacteriophage

It is a virus that attacks bacteria and inserts its gene into the bacterial cell for multiplication. 

Transposons

These are movable genes or jumping genes that move from one cell to another or plasmid to the nucleus. The size is very small like 1kb to 2kb (1kb =1000nucleotide). This transposon has no “marker gene” or “ori gene.”

Yeast vector

These are plasmids capable of replication in the yeast. They are used to transfer the desired gene into fungi. This is similar to plasmid with little modifications.

Shuttle vectors

This vector can propagate in two different host cells. They are manipulated in E. coli and then used in yeast. Plasmids and yeast vectors are also shuttle vectors.

Human artificial vectors.

Human artificial chromosome vectors are more advanced than the ones described above. They are considered ideal gene delivery vectors due to stable episomal (desired gene) maintenance and the ability to carry large genes.

Essential Steps of Genetic Engineering

Shuttle vector: These vectors have ori-gene, promoter genes for both bacteria and fungi. So it is two in one type of process.

Insertion of Gene into the vector.

The vector loaded with the desired gene is now transferred into the host cell. a is done by any of the four techniques viz.

Cohesive technique

Here cohesive ends are formed for joining with the vector. Restriction endonuclease enzyme is used to cut the desired gene and also plasmid. By this cohesive ends are formed. These cohesive ends in both the plasmid and the desired genes are easily attachable.

Homopolymer chain

Here polymers are formed at the ends of the gene to fix with the vector.

Blunt end joining.

Here the genes with blunt ends are joined to the vector by use of a DNA ligase enzyme.

Use of Cos sites.

The Cos site is one that has 12 nucleotide chains. The vector with the gene is transferred into a bacteriophage. As we know, the bacteriophage is a virus that attacks bacteria and multiplies. So bacteriophages transfer the desired gene-loaded vectors.

3. Transfer of r-DNA into host cells.

Now the vector with the desired gene is transferred into the organism of interest. i.e., bacteria or fungi in most cases.

The host cells suitable for this purpose are

• Prokaryotes: Bacteria like E.coli & Bacillus subtilis
• Eukaryotes: They can be whole plant cells, animal cells
• Fungi cells like saccharomyces cerevisiae.

This transfer of the recombinant vector (i.e. vector + Desired gene) is done such that the entire recombinant vector gets incorporated into the host cell genome. This is done by methods like

Transformation

This is homologous gene recombination into bacteria. The gene is passed into the cell. For this, we use two methods

By use of CaCl2

Here calcium chloride is added into bacterial suspension taken in a Petri dish and cooled to 0-4 degrees.

Then rDNA is added and the temperature is suddenly raised to 42°C for a short time to generate heat shock. The loaded vector enters the cell through cell wall pores and gets incorporated into the genome of the host.

This method is not suitable for heat-sensitive bacteria.

By use of lysosomal enzymes

This lysosomal enzyme destroys bacterial cell walls. So this catalytic enzyme is taken in low concentration along with plasmids (vector) and added to the bacterial culture. The cell wall cracks and plasmids enter. Then the enzyme is removed by centrifugation and supernatant discarding.

By Transduction

Here the desired gene is loaded into cosmid and inserted into an empty capsule of the virus. The transformed virus is introduced into a beaker of E. coli. This virus enters into E. coli by transduction methods leading to the incorporation of rDNA into the E.coli genome.

Conjugation

This is a natural sexual process of bacteria where the exchange of their plasmid occurs through the formation of inter-cell cytoplasmic bridges.

Other methods include the use of liposomes, particle bombardment, etc.

4. Multiplication, Identification & isolation of transgenic cells

Once the transfer of genes is done, the cells are allowed to multiply profusely.

Then they are identified and isolated from culture media.

For this isolation, a few methods are followed like

Antibiotic sensitivity technique

This is based on the replica plating method. Here the bacteria with the desired gene are isolated into another media. For this, the solution of bacteria is taken and added to antibiotic ampicillin. Those with ampicillin resistance genes multiply. While all those without vectors do not grow and are inhibited. The remaining ones grow into visible colonies.

A cylindrical vessel with a flat bottom with a muslin cloth wound is pressed over those colonies. E colonies get fixed to the cloth which is again touched to the surface of fresh media. Thus the bacteria with r-DNA are isolated. These are grown in culture media in the presence of the promoter genes to get the desired product.

The above method is not suitable for yeast and virus. So other immunological techniques like nucleic acid hybridization, and polymer chain reaction are used.

Direct phenotypic identification

Here transgenic bacteria are identified based on the newly developed characters. For example, bacteria with β-lactamase producing genes survive the culture media when ampicillin is added while remaining die.

5. Expression of the desired gene inside the host cell:

After isolation, the host cells are cultured by the fermentation process to produce the desired product. The culture broth has all the required nutrients.

However, the host cell may not express the rDNA and synthesize the desired product. Because the host cell doesn’t require the product for itself.

Further, all the genes in the genome do not activate at all times. So the transgenic gene needs and external stimuli to produce the mRNA by transcription. This mRNA which is coded for the desired substance is translated into the protein 

For this purpose, gene promoters or expression agents like Lac operon or Tryptophan operon are used

When these agents are added to cultural media, they express the rDNA to produce the desired product.

For example, in the presence of lactose in culture media, the lac-operon gene is active and there is the production of insulin by E.Coli and in the absence of lactose, there is no production of insulin.

This is how we manufacture many vaccines like hepatitis B, vitamins like B12, hormones like Insulin, etc.

Without this technique, we needed to extract them from animals which could be insufficient to meet the market demands.

Also, the product obtained has compatibility problems with the human body as it was from another source. 

See more applications of rDNA technology.

References: 

1. Basic steps of genetic engineering 
2. Human artificial chromosome (HAC) vector
3. Cacl2 induced bacterial transformation.
4. Gene promoters overview.