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 principles 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 brief, the desired substance, like insulin for diabetes, is produced by transferring the insulin gene (DNA) from a healthy human to a bacteria (E-coli).“

In most cases, the desired organism is human or plant, while the organism of choice is mostly a bacteria or yeast.

Steps of Genetic Engineering

• Initially, the whole genome, or DNA from the organism (of interest), is extracted.

• This can be done by homogenizing tissue (breaking the cells) or using surfactants to break up the cell membrane.

• The entire gene is separated from this homogenate 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 using physical or 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, the desired gene is synthesized by using free nucleotides.

• For this, the target protein is isolated, and the required nucleotide sequence is deduced.

Using restriction endonuclease enzymes (precise genetic cutting)

• The whole genome is taken and exposed to the restriction endonuclease enzymes in this method.

• 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 (DNA synthesized from mRNA)

• Here, the desired DNA sequence is synthesized from the messenger RNA, which codes the specific protein of choice.

• For this, the enzyme reverse transcriptase synthesizes the double-stranded DNA sequence.

• The isolated genes are purified and taken to the next step, which is to fix them in a vector.

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

• A vector is a vehicle meant 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. A marker gene should be used to help 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 mostly large pieces of DNA molecules.

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 but with few modifications.

Shuttle vectors

• These vectors have ori-gene and promoter genes for both bacteria and fungi.

• So, 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.

Then Insertion of Gene into the vector.

• After selecting the suitable vector, the desired gene has to be inserted into it.

• This can be done using any of the four techniques, viz.

Cohesive technique

• Here, cohesive ends are formed to join with the vector using Restriction endonuclease enzymes.

• The Restriction endonuclease enzyme cuts the desired gene and also the plasmid.

• Through this, cohesive ends are formed 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 using a DNA ligase enzyme.

Use of Cos sites.

• The Cos site are12 base single-stranded cohesive ends found in lambda (λ) phage DNA of bacteriophage lambda.
• These are suitable for the transfer of large DNA fragments.

• This vector with the gene is transferred into a bacteriophage.

• A bacteriophage is a virus that penetrates bacterial cells and multiplies.

• Indirectly, bacteriophages transfer the desired gene-loaded vectors.

3. Transfer of rDNA into host cells.

The vector with the desired gene is now 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 to incorporate the entire recombinant vector into the host cell genome.

This is done by methods like

1. Transformation

• This is homologous gene recombination into bacteria.

• The gene is passed into the cell. For this, we use two methods

By use of Calcium Chloride (CaCl₂)

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

• 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 host genome.

⚠This method is not suitable for heat-sensitive bacteria.

By use of lysosomal enzymes

• This lysosomal enzyme can destroy bacterial cell walls.

• So, a low dose of this catalytic enzyme is taken along with plasmids (vector) and added to the bacterial culture.

• This leads to cracks in the cell wall of bacteria, facilitating the plasmid entry.

• Then, the enzyme is removed by centrifugation, and the supernatant is discarded.

2. By Transduction

• The desired gene is loaded into a cosmid and inserted into an empty virus capsule.

• The transformed virus is introduced into a beaker of E. coli.

• The introduced virus enters into E. coli by transduction method, leading to the incorporation of rDNA into the E.coli genome.

3. Conjugation

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

4. Alternative methods

• 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.

• After multiplication, 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 medium.

• For this, the bacteria solution is taken and added to the 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.

• 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 their newly developed characteristics.

• For example, bacteria with β-lactamase producing genes survive the culture media when ampicillin is added while remaining dead.

5. Expression of the desired gene inside the host cell

• After isolation, the recombinant cells are subjected to fermentation with the required nutrients in the culture broth.

• However, despite sufficient nutrition, the recombinant cell may not express the rDNA or synthesize the desired product.

• This is because the host cell doesn’t require the product for itself.

• So, the desired gene has to be stimulated through an external messenger to get the desired product.

• Further, all the genes in the genome do not always activate.

• So, the transgenic gene needs external stimuli to produce the mRNA by transcription.

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

• When these agents are added to cultural media, the rDNA gets activated and produces 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 insulin production.

• This is how many vaccines, such as hepatitis B, vitamin B12, and hormones like insulin, are manufactured.

• Without this technique, we would have needed to extract them from animals, which would have been insufficient to meet the market demands.

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

CRISPR for gene therapy and research

• This is an advancement in genetic engineering where CRISPR-Cas9 allows direct and precise gene editing without needing a vector.

• CRISPR, like molecular scissors, helps cut and edit DNA at specific locations with remarkable accuracy.

See more applications of rDNA technology.

But why only bacteria and yeast? Because they can be quickly grown, and their life cycle is completed 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 quickly.

Genetic Engineering Quiz

References: 

1. Basic steps of genetic engineering 
2. Human artificial chromosome (HAC) vector
3. Cacl2 induced bacterial transformation.
4. Gene promoters overview.
5. Bacteriophage Lambda as a Cloning Vector