5 Steps of Genetic Engineering Used in rDNA technology
Genetic engineering is the process of transfer of 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 procedure followed is called the r-DNA technology.
In short, the desired substance is produced by transfer of desired gene (DNA) from a parent organism to a different organism.
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 also their life cycle completes in few hours to days. Due to this, we get the desired product formed in a short time
Because of such short lifespan, they express the transferred gene to the fullest and we obtain the product very fast.
The process occurs in 5 steps as
- Isolation of desired gene (gene cloning technology)
- Selection of vector and insertion of a gene
- Transfer of r-DNA vector into host cells.
- Identification, isolation of recombinant gene cells
- Expression of cloned genes
Before isolation of the desired gene, firstly one has to remove the entire gene or DNA from the organism of interest. This can be done by homogenization of tissue or by 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 or density based centrifugation.
This gene is now subjected to a different method to isolate the desired gene.
1.Isolation of the desired gene: Here the DNA or gene from an organism is isolated. That is from the whole genome obtained in the previous step, the part of DNA coding for the desired protein is isolated. This is a critical task and can be done by four methods like
i) Mechanical shearing.
ii) Chemical synthesis.
iii) By use of restriction endonucleases.
iv) Complimentary DNA method.
The isolated genes are purified and taken for next step to fix to a vector.
2. Selection of vector: 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 vector. These vectors have some specific properties like
- It should be capable of independent multiplication. This is possible if the gene has “Ori gene”
- 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.
- The vector should have a gene promoter sequence like a β-galactosidase gene.
- Should have Marker gene which helps to identify transgenic cells.
There are many types of vectors like
a) Plasmids: These are naturally occurring proteins from bacteria.
e) Bacteriophage (virus)
g) Shuttle vectors:
These vectors are large pieces of DNA molecules mostly.
The plasmid is a circular, single-stranded and self-replicable DNA molecule present inside bacteria. They help in sexual reproduction of bacteria by transfer of genetic matter from one to another. Here we use them to transfer the desired gene.
A bacteriophage is a virus which attacks bacteria and inserts its gene into the bacterial cell for multiplication. Cosmid is similar to plasmid DNA but can accommodate large DNA pieces.
Transposons: These are movable genes or jumping genes which 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” and “ori gene.”
Yeast cloning vector: These are used to transfer the desired gene into fungi. This is similar plasmid with little modification.
Shuttle vector: These vectors have ori-gene, promoter gene for both bacteria and fungi. So it is two in one type of the process.
3.Transfer of r-DNA:
The isolated gene is now transferred into the vector in this step. 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 plasmid and desired gene 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 vector by use of DNA ligase enzyme.
- Use of Cos sites. Cos site is one which has 12 nucleotide chains. The vector with the gene is transferred into a bacteriophage. As we know, the bacteriophage is a virus which attacks bacteria and multiplies. So bacteriophages transfer the desired gene loaded vectors.
4. Transformation of r-DNA:
Here the vector with the tagged desired gene is transferred into the organism of interest, i.e., bacteria or fungi in most cases. This is done by creating holes in the bacterial cell wall. For this, we use two methods
By use of CaCl2: Here bacteria and calcium chloride are taken in a Petri dish and cooled to 0-4 degrees. Then rDNA is added and the temperature is suddenly raised to 42degree. When cooled the bacterial wall shrinks and when heated instantly, it expands abnormally creating pores in the wall. The loaded vector enters the cell through these pores.
For those bacteria which do not tolerate this temperature, this method is not used.
By use of lysosomal enzymes: This lysosomal enzyme destroys bacterial cell wall. 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 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. The modified virus enters into E-coli by transduction methods.
5. Identification, isolation & culture of transgenic bacteria:
Once the transformation is done, now we need to identify and isolate those bacteria from culture media which have the vector within.
For this few methods are followed like
Antibiotic sensitivity technique: This is based on replica plating method. Here the bacteria with the desired gene are isolated on to another media. For this, the solution of bacteria is taken and added with antibiotic ampicillin. Those with ampicillin resistance gene multiply. While all those without vector do not grow and are inhibited. The remaining ones grow into visible colonies.
A cylindrical vessel with a flat bottom with 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 promoter gene to get the desired product.
The above method is not suitable for yeast and virus. So other immunological techniques like nucleic acid hybridization, 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 gene survive the culture media when added with ampicillin while remaining die.
After isolation, the bacteria are cultured by fermentation process to produce the desired product. The culture broth has all the required nutrients. Also, it has gene promoters which encourage the transgenic gene in the bacteria to get activated and produce the product. But why do we need a promoter sequence?
Because all the genes in the genetic material do not activate at all the times. So the transgenic gene needs and external stimuli to produce the m-RNA by transcription. This m-RNA which is coded for desired substance is translated into the protein.
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 or by other means which was insufficient to market demand. Also, the product obtained has compatibility problems with the human body as it was from another source. But the product obtained by this method is an exact copy of the one produced in the body, so it is compatible.