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CHAPTER 19: BIOTECHNOLOGY

Biotechnology
 Definition: Application of organism or biological processes (like reproduction, respiration) in
manufacturing process (where raw materials are converted to products)
Application of Biotechnology: 1) Food and Beverage
1) Bakery:
- Flour is added with yeast (Saccharomyces cerevisiae) to produce carbon dioxide and bread
- Saccharomyces cerevisiae produces mixture of enzymes and break down the starch. It also
encourages proving where aerobic and anaerobic produce carbon dioxide to rise the dough
2) Wine:
- Grapes + Yeast  Wine + Ethanol by alcoholic fermentation
3) Yoghurt:
- Milk + Lactate-producing bacteria  Lactic acid + Yoghurt by lactate fermentation
- Lactobacillus bulgaricus produces lactic acid, methanoic acid and ethanal
- Streptococcus thermophilus adds creamy flavour
4) Vitamin-enriched egg:
- Hen (+ Feed) to produce vitamin-enriched eggs
- Vitamin D to improve development of bones in children
- Omega 3 has double bon, can be oxidized but affect health
- Vitamin A, C and E help by acting as antioxidants
- Omega 3 helps to build cell membrane
- Vitamin B6, B12 and vitamin E increase the vitamins in eggs
- Choline is added for brain development
Application of Biotechnology: 2) Agriculture
 Organisms that have been genetically altered using genetic engineering are called as transgenic
organism
 Advantages of transgenic plants and transgenic animals:
- Increase the yields and have higher growth rate
- Improve the quality of foods
- Resistance to pest, disease and herbicide
- Resistance to environmental stress – drought, cold, heat, tolerance to wind damage, acid, salty
soils and waterlogged soils
1) Hybrid rice:
- Crossing 2 genetically different species
- F1 generation has improved quality (hybrid vigour/heterosis), higher yields, higher resistance
and more efficient in use of soil nutrient
- But F2 generation are not fertile because hybrid vigour is lost
2) Herbicide resistant plant:
- Introduce genes which confer resistance to herbicide in plants
- When weed-killer is used, only weeds are killed
- E.g. corn, wheat, sugar beet and cotton tree
3) Transgenic fish:
Process to insert new DNA into fish;
- Use micro syringe/ gene gun to insert the gene (fluorescent genes
extracted from jellyfish or firefly), or, Subject fishes’ eggs to electrical
pulse to form pores. Insert the vector carrying gene into the fish egg
- Vectors are lentivirus transferring gene into the fish
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Advantages of transgenic fish:

-Higher growth meat, more meat and higher resistance to disease
-Ornamental fish industry e.g. zebrafish with bright red, green or orange fluorescent colour
-Research in genetics, pharmaceutical application and health
-Genetically modified zebrafish can detect aquatic pollution by detecting the pollutants and
radiate green/red light
Disadvantages of transgenic fish:
- Gene for resistance to disease cause fish to absorb toxic substances like heavy metals and
mercury
- Allergies, health risks if consumed raw or uncooked
- Breed faster, lowers the genetic diversity of native population
- Short life span. If interbreed with native population, this decreases the life expectancy of native
population
- Compete with native population for food, resources and habitat
4) Temperature tolerance in plant:
- Insert gene that regulates extreme temperature
Application of Biotechnology: 3) Medicine and Forensic
1) Human growth hormone, extracted pituitary gland of dead humans to treat dwarfism
2) Human insulin, extracted from pancreas to treat diabetic patient
- Extract **mRNA** (not DNA) from pituitary gland (for human growth hormone) or pancreas
(for human insulin)
- Use enzyme reverse transcriptase to form single stand DNA by reverse transcription
- DNA polymerase is added to form second single strand of DNA and form double stranded
complementary DNA or cDNA
- Extract plasmid from a bacterial cell
- Use the same type of restriction enzyme to cut the cDNA and plasmid
Continuation for human insulin:
- Insert the gene for insulin into the plasmid to
form recombinant plasmid by using DNA
ligase and energy form ATP (Insertion)
- Insertion may take place in a medium
containing calcium chloride, CaCl2
- Recombinant plasmid introduces the gene for
insulin into the bacteria cell/ host cell
(Transformation)
- Plasmid replicates in the host bacteria
- Screen for target gene for human insulin using antibiotic screening and X-gal screening
- Amplification using industrial fermenter to produce vast amount of insulin
- Separate and purify the gene for insulin (Separation and purification of human insulin)
- Inject the insulin into diabetic patients
3) Gene therapy:
- Gene therapy works by using genetic engineering to overcome genetic disorder
- There are 2 types; Gene replacement (normal gene replace faulty gene) and Gene
supplementation (does not remove pre-existing faulty gene but add more normal genes and will
mask the effect of faulty gene)
- Gene therapy can be applied at Sperm/Ovum (germ-line gene therapy that can be inherited) or
at Body cell (somatic-cell gene therapy that cannot be inherited
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Cystic fibrosis
- Definition: Disease affect epithelial cell lining trachea
and block pancreatic duct
- Normal gene codes for cystic fibrosis transmembrane
protein (CFTP)
- CFTP transport chloride ions out of epithelial cells into
the mucus and later makes the epithelial smooth and
moist while the mucus is watery
- Mucus traps dust and microorganism to prevent lungs infection
- Effect of mutant gene on CFTP:
 Sticky mucus clogs the airway of lungs, lungs become prone to infection
 Salty sweats to remove excessive chloride ions
 Affect digestion as it may block the pancreatic duct
 Breathing difficulty and coughing
 Affect the cells secreting sweat, mucus, hormone and enzyme
- Stages of cystic fibrosis gene therapy
 Extract normal gene for CFTP and extract plasmid from bacteria
 Use the same type of restriction enzyme to cut DNA for target gene and plasmid
 Use DNA ligase to join the gene fragment with the cut plasmid to produce recombinant
plasmid
 Use recombinant plasmid to introduce gene for CFTP in bacterial host cell
 Recombinant plasmid will replicate in the host cell
 Screening for transformed host cell
 Cloning/ Amplification to increase output
 Insert recombinant plasmid into liposome (a lipid that passes through the plasma
membrane easily)
 Spray liposome into the nose
 Liposomes enter the lung tissue
 Normal genes are expressed and effect may last for few weeks. Epithelial cell will shed
off

Severe combined immunodeficiency disorder, SCID

- SCID is caused by adenosine deaminase (ADA) deficiency in infants
- The gene for ADA in SCID infants is not functioning
- ADA is used by white blood cells to produce antibodies
- Extract normal ADA gene and vector. Use the same type of restriction enzyme to cut both
- The normal gene for ADA joined to the vector by DNA ligase and ATP energy
- Vector containing ADA gene is introduced into a retrovirus that had been inactivated to become
non pathogenic
- Produce recombinant virus
- ADA gene incorporated into the DNA of the retrovirus
- A small amount of bone marrow is isolated from the SCID infant and cultured in the lab
(medium containing nutrient and carbon dioxide)
- The bone marrow cells are infected with the retrovirus that contains ADA genes
- The retrovirus integrates the normal ADA gene into the bone marrow cells
- Bone marrow cells with the normal ADA gene are reintroduced into the SCID infant
- Normal ADA gene will be transcribed, translated and produce ADA
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4) Forensic science- DNA fingerprinting

 Definition: Technique used to identify individual from the sample of DNA using non-coding
regions of DNA
 95% of the DNA do not code for any amino acid
 Unknown sample is compared against the known sample
 By comparing the non-coding parts of DNA
 The non-coding parts of DNA is repetitive. It is known as variable number tandem repeat
(VNTR)
 Polymerase chain reaction (PCR) is used to amplify the small sample
 Stages in DNA fingerprinting:
 Collect the DNA sample. If the sample is small, amplify it using PCR
 Extract the DNA from the sample
 Use restriction enzyme/endonuclease to cut the DNA into fragments (sticky end/blunt end)
 Fragments are called as restriction fragment length polymorphism, RFLP
 Separate the DNA fragment using electrophoresis (using agarose gel, electrical field and
buffer solution of pH 7)
 Add alkaline to split the double strand DNA into single stands
 Perform Southern blotting where the single strand is transferred from the gel to a
nitrocellulose membrane/ nylon membrane
 Probe with radioactive marker (process known as hybridisation) to identify the target gene
on single strand DNA with complementary base
 Autoradiography; place an x-ray film over the
nylon membrane with phosphate substrate
 Alkaline phosphatase removes the phosphate,
cause substrate to fluoresce and fogging the x-ray
film
 Develop the x-ray film and the fogging parts will
appear as dark band
 Compare the repetitive non-coding region with
another sample’s result
 Extra notes:
- Probe attach to specific base sequence in DNA fragments, process called as hybridisation
- Complementary base pairing during hybridisation occurs between probes and DNA of
sample
- During Southern blotting, DNA deposited on nylon membrane by capillarity action
- DNA fixed with the membrane by being exposed to short wavelength of light
 DNA fingerprinting is used to convict criminal suspects, establish paternity, determine the
identity of a dead body and to identify defective gene by genetic screening
Application of Biotechnology: 4) Public Health
1) Genetic screening
 Definition: Procedure to examine genetic disease
 When?
- Foetus- Prenatal diagnosis
- Carrier- Carrier diagnosis
- Future genetic disease- Predictive diagnosis
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 Screening technique:
i) Amniocentesis
- Insert a fine sterile needle into the uterus. Use ultrasound to monitor the position of the
foetus and needle
- Withdraw sample of amniotic fluid (living cell e.g. skin cell of foetus)
- Sample is centrifuged to separate cells and supernatant fluid. Cells are cultured
- Analyse karyotype for abnormality
- DNA analysis/ biochemical analysis is done to detect metabolic disorder
- When? 4th month of pregnancy/15-16 weeks of pregnancy
ii) Chorionic villus sampling
- Insert needle through abdomen or insert catheter (flexible tube) through vagina and cervix
- Along with ultrasound scanner
- Get cells from chorionic villus
- Analyse the karyogram
- When? 2-3 months of pregnancy/8-12 weeks of pregnancy
- Has higher chance causing miscarriage than amniocentesis
iii) Pre-implantation diagnosis
- In-vitro fertilization/test tube baby
- Embryo at 8 cells stage, Take sample of cell
- Carry out on the embryo before it implants in the uterus

2) Diagnostic kit
 Definition: Electronic monitoring device uses biological material like cell, enzyme or
antibody to detect or measure chemical compound
 Biological material + Substrate
- Change in heat, light, pH, mass, flow of electron, new chemical
- Transducer converts change into electrical signal
- Amplify electrical signal to give digital display
 Example: Diagnostic kit to detect blood glucose
- Glucose oxidase in immobilized form + glucose generate electrons
- Enzyme oxidizes glucose in blood to release electrons
- Electrons are collected and converted into electrical current
- Amount of glucose is directly proportional to the current
- Digital display to get the result in 20 seconds
 Advantages of diagnostic kit:
- Low risk of error in diagnosis
- Lower cost
- Less time
- Less expertise, need no hospital laboratories
- Less chance of sample being mishandled, lost or contaminated
3) Oil-decomposing bacteria
 Source of oil pollution:
- Collision of oil tanker
- Seepage of offshore installation
- Flashing of tanker holds (lover cavity part of ship)
 Effect of oil pollution:
- Kill seaweeds, marine invertebrates (mussels, cockles, crustacean, and fishes)
- To bird, it will mat the feathers impair flight, cause swimming difficulty and heat insulation
is lost cause bird to be under hypothermia, lower body temperature
- When taken into stomach during preening, it causes gut irritation and poison the bird
- Lower the economic value of fish as flesh is tainted with oil
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 Oil decomposing bacteria:

- Pseudomonas can break down 4 main groups of hydrocarbons in oil
- Relevant genes occur on plasmid Pseudomonas
- But no single strand contains all four plasmids
- Now, all 4 are introduced into single “super bug”
- This bacteria type is strayed onto oil-polluted surfaces
- Bacteria only able to work at oil-water interface as they need oxygen
- So, they can clean the oil better on thin surfaces rather than thick
- They function slowly at low temperature, thus, might be unsuitable for use in cold climates
- To clean oil spill, genetically engineered Pseudomonas bacteria are used
Advantages of using human insulin:
- Identical to human insulin in body, Less side effect
- Cheaper to produce
- Rapid response, Pure and uncontaminated
- Not dependent on livestock
Gene therapy
- Ex vivo (outside the body) e.g. to treat severe combined immunodeficiency disorder, SCID
- In vivo (inside the body) e.g. to treat cystic fibrosis or viral delivery system