Biotechnology in Microbiology: Genetic Modification of Microorganism

Biotechnology in Microbiology: Genetic Modification of Microorganisms

Biotechnology in microbiology involves using microorganisms to produce valuable products or to address environmental challenges. This involves the genetic modification of microorganisms whereby the genetic makeup of microbes is modified to enhance their metabolic capabilities or to produce desired substances. Genetically modified microorganisms have applications in medicine, agriculture, industry, environmental management, etc.

Recombinant DNA technology or gene cloning

The term gene cloning can be defined as the isolation and amplification of an individual gene sequence by insertion of that sequence into a bacterium where it can be replicated.  Cloning a fragment of DNA allows indefinite amounts to be produced from a single original molecule.  Recombinant DNA technology has important applications in gene mapping, detection of inherited diseases, cancer research, immunology, etc. 

Basic steps in gene cloning experiments are

3. The DNA (cloned DNA, insert DNA, target DNA, foreign DNA) from the donor organism is extracted and enzymatically cleaved.
4. This fragment of DNA is then inserted (joined) into a circular DNA molecule called vector (vehicle) to form a new recombinant DNA (rDNA) molecule. 
5. This recombinant DNA (rDNA) molecule is then transferred into a host cell.  This process is known as transformation.
6. Within the host cell the vector multiplies, producing numerous identical copies not only of itself but also of the gene that it carries. 
7. When the host cell divides, copies of the rDNA molecules are passed to the progeny and further replication takes place.
8. After a large number of cell division a clone or colony of identical host cell is produced.  This clone contains copies of rDNA molecules. 
9. These rDNA molecules is then screened and isolated.

The DNA segment to be cloned is called foreign, passenger or target DNA or DNA insert. Vectors or cloning vehicles are self-replicating DNA molecules and most commonly used vectors are bacterial plasmids, bacteriophages or DNA viruses. Recombinant DNAs are introduced into a suitable organism, usually a bacterium; this organism is called host, while the process by which the rDNA is introduced into the host is called transformation. 

Recombinant DNA is called DNA chimera because of their analogy with the Chimera of mythology – a creature with the head of a lion, body of a goat and tail of a serpent.

The construction of such composite or artificial recombinant molecules is termed as genetic engineering or gene manipulation because of the potential for creating novel genetic combinations.

The process has also been termed molecular cloning or gene cloning because a line of genetically identical organisms, all of which contain the composite molecule or r DNA, can be propagated and grown in bulk, hence amplifying the composite molecule and the gene product whose synthesis it directs.

DNA cloning procedure has 5 essential parts

1. Preparation of DNA sample
2. Cutting of DNA molecules
3. Joining of desired DNA to the vector (ligation)
4. Transformation or transfer of rDNA to bacterial cells
5. Screening and identification

Preparation of DNA sample

Total cell DNA is required as a source of material from which genes for cloning is obtained.  This may be DNA from a culture of bacteria, plants, animals, etc.  Steps involved are

1. A culture of bacteria is grown and then harvested
2. The cells are broken open to release their contents
3. This extract is treated to remove all components except the DNA
4. The resulting DNA solution is concentrated

 

Cutting of DNA molecules

For molecular cloning, both the source DNA that contains the target sequence and the cloning vector must be consistently cut into discrete and reproducible fragments.

Restriction endonucleases are enzymes that make site specific cuts in the DNA.  There are three types of restriction endonucleases, but relevant enzymes for gene cloning are type II. 

Eg. EcoRI, PvuII, AluI

Cloning vehicles or vectors

One of the most important elements in rDNA technology is the vector.  Vectors are the carrier DNA into which foreign DNA or genes of interest are inserted to make a recombinant DNA.  Vectors along with these foreign DNA are then introduced into appropriate host cell and are maintained for study or expression. 

There are different types of cloning vectors.  Important among them are plasmid vectors.  Others include bacteriophage, cosmid, viruses, yeast cloning vectors, etc.

Joining of desired DNA to the vector (ligation)

The final step in the construction of rDNA molecule is the joining together of the vector molecule and the DNA to be cloned.   This process is called as ligation and the enzyme that catalyses the reaction is called DNA ligase. 

DNA ligase seals single stranded nicks between adjacent nucleotides in a duplex DNA chain. The enzyme catalyses the formation of a phosphodiester bond between adjacent 3’OH and 5’P termini in DNA.

Transformation or transfer of rDNA to bacterial cells

Once a mixture of rDNA is obtained, it is allowed to be taken up by the suitable bacterial cells.  The event of entering the plasmid containing foreign DNA fragment into a bacterial cell is known as transformation.  Chemical transformation, electroporation, Microinjuction, etc are the commonly used methods. 

Screening and identification

The bacteria with the vector are grown in a culture. Then the cells are lysed and the cloned gene or gene product is harvested in an efficient manner.

Applications of Genetic Modification in Microorganisms

1. Medicine and Pharmaceuticals

•    Genetically modified microorganisms are used to produce therapeutic proteins like insulin, growth hormones, clotting factors, etc. For example, Escherichia coli and yeast are engineered to produce recombinant insulin for diabetes treatment.
•   Microorganisms are genetically modified to produce antigens or weakened strains to be used as vaccines. For example, genetically engineered yeast is used to produce hepatitis B vaccine.
•   Genetic modification is used to enhance the Antibiotic Production by modifying the metabolic pathways.
•   Gene Therapy is the approach where Viruses are used to carry therapeutic genes into human cells to treat genetic disorders such as cystic fibrosis.

2. Agriculture

•     Plants could be Genetically modified to resist specific pests without harming beneficial insects or the environment. For example, Bacillus thuringiensis (Bt) toxin is engineered in plants to produce proteins toxic to insect larvae, Bt cotton, Bt Brinjal are examples
•   Genetic modification of nitrogen-fixing bacteria, such as Rhizobium species, can improve their efficiency.
•    Microorganisms are engineered to enhance plant growth by producing growth-promoting hormones or solubilizing essential nutrients like phosphorus.

3. Industrial Biotechnology

•     Genetically modified microorganisms are used to produce biofuels, such as ethanol and biodiesel, from renewable resources.
•     Microorganisms are modified to produce improved levels of biodegradable plastics like polyhydroxyalkanoates (PHAs), enzymes, aminoacids, vitamins, etc

4. Environmental Biotechnology

•    Genetically modified microorganisms are used to clean up environmental pollutants, such as oil spills, heavy metals, and toxic chemicals. Pseudomonas putida is a genetically engineered bacterium, also known as a "superbug", developed by Professor Ananda Mohan Chakrabarty.  This organism could be used to clean up oil spills. 
•   Genetically modified microorganisms are developed as biosensors to detect environmental pollutants like arsenic.

Human insulin is produced in Escherichia coli (E. coli) using recombinant DNA technology:

Steps involved 

1. Cloning synthetic genes for the A and B chains of human insulin separately into plasmid pBR322. 
2. The cloned genes are fused to an E. coli β-galactosidase gene. 
3. The insulin peptides are cleaved from the β-galactosidase. 
4. The insulin peptides are detected by radioimmunoassay and purified. 
5. The inclusion bodies of insulin precursors are solubilized and refolded to create active insulin. 

The process of producing insulin in E. coli involves:

• Producing insulin precursors (IPs) as inclusion bodies by using genetically modified E coli
• Solubilizing and refolding the insulin precursors to create fully functional polypeptides

(https://images.app.goo.gl/bjxRZVGqsUHCaus16)

Genentech was the first company which produced recombinant human insulin in E. coli in 1978. The successful production of human insulin in bacteria led to the approval of human insulin for treating diabetes in 1982.