GENETIC ENGINEERING (Recombinant DNA
Technology)
It is defined as the experimental
arrangement of genetic material for
scientific medical or industrial purposes.
Genetic engineering studies and biological materials to be used in biotechnological production are created.
Hosts Used in Molecular Cloning Host should have the following
characteristics:
Be able to grow quickly on an inexpensive medium.
It should not be a pathogen.
It should be capable of receiving DNA.
Genetic structure should remain stable in culture.
Genetic structure should be well known.
Must have appropriate enzymes for vector
replication.
Prokaryotic hosts
Gram negative E. coli;
Some strains are pathogenic,
Non-pathogen strains to produce endotoxin, Periplasmic proteins make it difficult to
isolate and purify recombinant proteins, Their ability to contaminate the resulting
product limits their use as a host.
These problems are tried to be solved by
using modified E. coli strains in cloning
studies.
Prokaryotic hosts
Gram-positive B. subtilis Not pathogen
Does not produce endotoxin Sporiferous
Lack of periplasms and
Naturally capable of secreting protein out of the cell Transformation is the most appropriate method for
cloning B. subtilis.
Disadvantage
Their plasmids are unstable and cannot retain foreign DNA in their cells.
Eukaryotic hosts
Yeast Saccharomyces cerevisiae
Prokaryotes cannot replicate most of the plasmids in their cells
Plasmid vectors such as yeast artificial chromosome (YAC) are used in cloning studies with this yeast.
Posttranslational systems
Mammalian cell culture systems (human genetics, cancer, infective diseases and physiological
studies)
The disadvantage is that it is expensive and difficult to produce on a large scale
Transfection of eukaryotic cells
Eukaryotic microorganisms, animal and plant cells, can receive foreign DNA into their cells, similar to transformation in prokaryotes.
In order to avoid confusion with the
transformation involved in cancer formation
in mammalian cells, this event in eukaryotic
cells is called transfection.
Genetic Engineering Applications in Biotechnology
Genetically modified organisms (GMOs) were obtained in vitro.
Production of many proteins such as insulin, interferon, growth hormones and viral antigens has been achieved with some moieties.
Nowadays, studies on the use of GMOs in
the fields of bioremediation and other
environmental biotechnology are
continuing in the development of industrial
fermentations in medicine and agriculture.
Genetic Engineering
Applications in Biotechnology
GM GM B.thuringiensis B.thuringiensis toxin is produced in toxin is produced in plants.
plants.
1. Insects may develop resistance to this 1. Insects may develop resistance to this toxin.
toxin.
2. Allergic reactions can occur in people 2. Allergic reactions can occur in people who consume toxins.
who consume toxins.
A new protein produced by GMO can affect A new protein produced by GMO can affect human health.
human health.
Production of mammalian products by GMOs
The production of human proteins used as pharmaceuticals is the most important field of biotechnology.
These proteins are found in very small
amounts in normal tissue. Therefore, it is very difficult to obtain from normal tissues.
Their production in cell cultures is very expensive.
These products are produced in genetically
modified microorganisms with high yield and
inexpensive.
Insulin production
It produces a long polypeptide called preproinsulin which contains a signal sequence necessary for protein secretion before the insulin gene.
Proinsulin is produced from preproinsulin.
The proinsulin has an A chain containing 21 amino acids, a sequence of 35 amino acids, and a B chain containing 30 amino acids.
By separating the A and B polypeptides
before the proinsulin and then recombining
them, enzymatic insulin is produced.
Insulin production
In insulin production using genetic
engineering techniques, polynucleotides are first synthesized, then cut with a suitable
restriction enzyme and transferred to a plasmid vector.
Proinsulin is isolated from the host cells,
the fragment is removed, the A and B chains
are chemically linked together by disulfide
bonds to produce insulin.
Vaccine production
The genes responsible for virulence are excised from the pathogen.
This vaccine, which stimulates the immune system
when administered to the body but is not pathogen, is called a recombinant live attenuated vaccine.
Flower vaccine is used in vector vaccine production.
The gene encoding an immunogenic envelope protein of a pathogen virus is transferred to the smallpox virus.
When the recombinant pox virus infects the animal cell, the sheath protein of the pathogen virus is produced
and the immune system is stimulated.
Vaccine production
Recombinant vaccines called subunit vaccines are produced.
The hepatitis B vaccine is produced in this way.
The gene prokaryote encoding the surface antigen of the hepatitis B virus is amplified in a host.
Subsequently, this gene is interpreted in yeast cells for posttranslational modification and the surface antigen proteins of the virus are produced in large amounts.
These purified proteins are then used as vaccines.
Vaccine production
DNA vaccines are produced.
The gene encoding the immunogen
protein of the pathogen is cloned into a plasmid or viral vector and introduced into the animal cell.
The animal produces this protein and the immune system is stimulated.
