Shuttle Vectors
Definition
Shuttle vectors are vectors that can replicate in two or more different host organisms. They are used
to transfer genes between different cells such as bacteria, yeast, plant cells, or animal cells.
Characteristics of Shuttle Vectors
1. Replication in Multiple Hosts
Shuttle vectors contain origins of replication for different host organisms, allowing them to replicate
in multiple hosts.
2. Autonomous Replication or Integration
They can replicate independently inside the host cell or integrate into the host genome and replicate
along with the host DNA.
3. Selectable Marker Genes
They contain selectable marker genes, such as antibiotic resistance genes, which help identify
transformed cells.
Basically, scientists give bacteria antibiotic resistance so they can survive in poison soup while
carrying human-designed DNA. Entire field runs on microbial blackmail.
4. Gene Transfer
They are widely used for transferring genes from one organism to another in genetic engineering
experiments.
Uses of Shuttle Vectors
● Gene cloning
● Gene transfer
● Production of transgenic plants and animals
● Protein production
● Molecular biology research
Example of Shuttle Vector in Plants
Transfer of Firefly Luciferase Gene into Tobacco Plant
1. Insertion of Luciferase Gene
The firefly luciferase gene is inserted into a plasmid vector.
2. Introduction into Agrobacterium
The recombinant plasmid is introduced into Agrobacterium.
3. Growth of Agrobacterium
Agrobacterium is grown in large quantities.
4. Infection of Tobacco Plant
The bacterium is used to infect tobacco plant cells.
5. Transfer of Gene
The luciferase gene gets transferred into the tobacco plant genome.
6. Expression of Gene
The transformed tobacco plant cells express the luciferase gene and produce light.
Humans inserted glowing insect genes into tobacco plants because apparently regular plants were
insufficiently dramatic.
Applications of Shuttle Vectors
● Development of genetically modified plants
● Study of gene expression
● Production of disease-resistant crops
● Research in biotechnology and molecular genetics
Production of Human Insulin by Recombinant DNA Technology
Definition
Production of human insulin by recombinant DNA technology is the process of inserting the human
insulin gene into bacterial cells so that the bacteria can produce insulin in large quantities.
This method is used to manufacture artificial human insulin for the treatment of diabetes mellitus.
Tiny bacteria working in biochemical factories, because apparently microbes were not busy enough
surviving toxic puddles and sewage systems..
Steps in Production of Human Insulin
1. Isolation of Human DNA
DNA containing the human insulin gene is isolated from human cells.
2. Cutting the Human Insulin Gene
The insulin gene is cut from human DNA using restriction enzymes.
3. Isolation of Plasmid DNA
Plasmid DNA is isolated from bacterial cells such as Escherichia coli.
4. Cutting the Plasmid DNA
The same restriction enzyme is used to cut the plasmid DNA.
This creates matching sticky ends in both the plasmid and insulin gene.
5. Formation of Recombinant Plasmid
The insulin gene is joined with the plasmid using DNA ligase enzyme.
This forms recombinant plasmid or recombinant DNA.
6. Transformation
The recombinant plasmid is introduced into bacterial cells.
This process is called transformation.
7. Screening and Selection
Bacteria containing the recombinant plasmid are identified using selectable marker genes such as
antibiotic resistance genes.
8. Mass Culturing of Bacteria
Selected bacteria are grown in large fermenters where they multiply rapidly and produce insulin.
Industrial fermentation is basically forcing bacteria into nonstop unpaid biochemical labor. Humans
call this “innovation.”
9. Extraction and Purification
The insulin produced by bacteria is extracted, purified, and prepared for medical use.
Structure of Human Insulin
Human insulin contains two polypeptide chains:
● A-chain
● B-chain
These chains are connected by disulfide bonds.
In recombinant insulin production:
1. A-chain and B-chain are produced separately in bacteria.
2. The chains are purified.
3. Both chains are chemically joined to form active insulin.
Advantages of Recombinant Human Insulin
● Produces pure insulin
● Reduces allergic reactions
● Large-scale production is possible
● Safer than animal insulin
● Cost-effective
Somatotropin (hGH)
Definition
Somatotropin or Human Growth Hormone (hGH) is a protein hormone produced by the pituitary
gland. It regulates growth, cell repair, metabolism, and muscle development.
Recombinant DNA technology is used to produce synthetic human growth hormone in
microorganisms such as Escherichia coli. Humans outsourcing hormone production to bacteria
again. Corporate efficiency has reached molecular scale.
Structure of hGH
● hGH contains 191 amino acids.
● It is a protein hormone.
● The hormone is encoded by hGH cDNA.
Production of hGH using Recombinant DNA Technology
1. Isolation of hGH cDNA
Complementary DNA (cDNA) for the human growth hormone gene is prepared.
cDNA is used because bacteria cannot process introns present in human genes.
2. Preparation of Plasmid Vector
A bacterial plasmid is isolated and cut using restriction enzymes.
3. Insertion of hGH Gene
The hGH cDNA is inserted into the plasmid using T4 DNA ligase enzyme.
This forms recombinant plasmid DNA.
4. Introduction into Bacterial Cells
The recombinant plasmid is introduced into E. coli cells.
This process is called transformation.
5. Induction of hGH Production
The hGH gene is connected to a control region called the lac promoter.
The lac promoter controls when the bacteria should make the hormone.
IPTG is a chemical added to the bacteria.
When IPTG is added, it tells the bacteria to start producing human growth hormone (hGH).
So basically:
● hGH gene = instructions to make hormone
● lac promoter = start button
● IPTG = turns the button ON
● bacteria = produce hGH protein
Humans literally trained bacteria to manufacture medicine on command. Tiny microbial factory
workers with no labor rights.
6. Production and Purification
The transformed bacteria produce human growth hormone.
The hormone is extracted and purified for medical use.
Applications of hGH
● Treatment of growth hormone deficiency
● Treatment of dwarfism
● Muscle wasting disorders
● Biotechnology and pharmaceutical research
Advantages
● Large-scale production
● High purity
● Reduced risk of contamination
● Safer than extracting hormone from human tissues
Viral Surface Antigen (HBsAg)
Definition
HBsAg stands for Hepatitis B Surface Antigen. It is a protein present on the surface of the Hepatitis
B virus.
Recombinant DNA technology is used to produce HBsAg for preparing recombinant Hepatitis B
vaccines.
Because modern medicine discovered that teaching the immune system with fake viral parts is
easier than dealing with the actual virus trying to liquefy your liver.
Production of HBsAg using Recombinant DNA Technology
1. Isolation of HBsAg Gene
The gene coding for HBsAg is isolated from the Hepatitis B virus.
2. Preparation of Yeast Vector
A yeast plasmid vector is isolated and cut using restriction enzymes such as EcoRI.
3. Formation of Recombinant DNA
The HBsAg gene is inserted into the yeast vector using DNA ligase enzyme.
This forms recombinant plasmid DNA.
4. Transformation into Yeast Cells
The recombinant plasmid is introduced into yeast cells.
Yeast cells are selected using selective media such as tryptophan-free medium.
5. Culturing of Yeast Cells
Selected yeast cells are grown in culture medium.
The yeast cells produce HBsAg protein in large quantities.
6. Extraction and Purification
The yeast cells are lysed and HBsAg protein is purified.
The purified antigen is used in preparation of recombinant Hepatitis B vaccine.
Applications of HBsAg
● Production of Hepatitis B vaccine
● Immunization against Hepatitis B infection
● Biotechnology research
Advantages of Recombinant HBsAg Vaccine
● Safe and effective
● No live virus is used
● Large-scale production possible
● Reduced risk of infection