Biotechnology Flashcards
When was the first recombinant organism produced?
E. coli in 1972
What is white biotechnology?
Industrial biotechnology- the use of living organisms or their derivatives to make industrial products Chemicals Amino acids Vitamins Enzymes
History of citric acid production
Produced in the UK from 1826
Calcium citrate produced from lemons and converted to citric acid chemically
1917 - Aspergillus niger (fungus) found to produce copious amounts of citric acid
1923 - large scale production began in New York
Over 1-2 million tonnes produced worldwide
How is acetic acid produced?
Fermentation of ethanol or methanol by microbes
200,000 tonnes produced annually
How is butanol produced?
From petroleum for fermentation of Clostridium acetobutylicum
Used in plastics, paint, resins, etc
1.2 million tonnes produced annually
Byproduct is acetone
How is lactic acid produced?
Half by microbes, the rest is chemical
Used as acidified, preservative and in plastics
Where were enzymes traditionally obtained from?
Microorganisms, plants and animals
How are enzymes produced now?
From fungi Submerged cultures (in liquid) of Aspergillus oryzae used to produce enzymes in large bioreactors
Advantages of obtaining enzymes from fungi
Cheap
High yields
Continuous
Uses of pectinases
Break down peptin in manufacture of fruit juice and baby food
Uses of proteases
Leather tanning
Many other uses
Uses of phytases
Added to animal feed to enable digestion of phosphate
What enzymes are used in detergent?
Modified subtilisin from Bacillus subtilis
Alkaline proteases
Amylase
Lipases
What are the benefits of immobilising enzymes?
Enzymes can be recycled Preserves expensive enzymes - lower production costs Absent from end product Increased stability Bound to gel or membrane
What does glucose isomerase do?
Converts glucose to fructose
High fructose corn syrup went into mass production in the 1960s
How has immobilisation changed the use of glucose isomerase?
Before the syrup only contained 15% fructose because enzyme too expensive to produce
Afterwards fructose yield was 42%
Still cheaper to produce sucrose
Technological advances in 1970s reduced glucose isomerase production costs, and sucrose prices rose, so HFCS now economically viable
How much is the production cost of glucose isomerase reduced by due to immobilisation?
40%
What is the current fructose content of high fructose corn syrup?
55%
What is red biotechnology?
Health-related biotechnology Biopharmaceuticals Recombinant proteins Vaccines Stem cells Animal models Gene therapy
Recombinant proteins
Over 100 in use, on which 50 are antibodies ($50 billion)
Main uses are replacement for missing/defective proteins and inhibition of infectious agents
Examples of recombinant proteins
Insulin
Antibodies
Blood clotting factors
Vaccines
What was the first commercially available GM hormone?
Insulin - lowers blood glucose levels
Produced by E. coli
What methods have overcome problems in insulin production?
Allergic reactions to non-human insulin
Had to tweak sequence using enzymes to modify the protein
Also tweaked DNA sequence to prevent clumping when being injected
How much are vaccines worth?
$40 billion per annum
What was the first vaccine?
Developed by Edward Jenner in 1796
Live cowpox virus infection protected against smallpox virus
First infectious disease to be eradicated worldwide (1977)
What is a subunit vaccine?
Fragments of the pathogen e.g. viral coat proteins or lipids
Developed prior to recombinant DNA technology
Hepatitis B vaccine was first example
How was the hepatitis B vaccine developed?
Blood-borne virus that causes liver disease
The virus was isolated from infected blood and the proteins purified and used in the vaccine
How are subunit vaccines produced now?
The relevant protein-encoding genes are cloned into plasmids, transformed into yeast or a cell line and the proteins would be purified
What is an inactivated vaccine?
Killed pathogen
Most common type
Vaccinate against polio (Sulk vaccine), rabies, influenza, hepatitis A
Many pathogens cannot be isolated or cultured in vitro or are too expensive to culture
Risk of infection to biotech workers
What are attenuated vaccines?
Live, weakened pathogens that no longer express the toxin gene
Can be a natural mutant or a GM mutant
Could be a related virus
Vaccinate against polio (Sabin vaccine), MMR, tuberculosis, chickenpox, cholera
Safer to produce, but needs a lot of research to identify the toxic gene
Risk that attenuated pathogens may revert to pathogenic strain
How are new vaccines developed? (first method)
- Genome sequenced to find antigens
Identify genes and clone into expression library
Determine which proteins are responsible for the immune response
How are new vaccines developed? (second method)
- Use viral genomes
Can use Vaccinia virus to make new vaccines
Clone genes of interest into a plasmid and insert into vaccine genome
Use recombinant Vaccinia virus as a vaccine against smallpox as well as other illnesses
Cheaper
How are new vaccines developed? (third method)
- DNA-based vaccines
Add the gene encoding the antigen into a plasmid
Bind the DNA to a charged particle and inject it
The DNA will bind with genomic DNA particles and the antigen will be expressed temporarily, triggering a localised immune response
Cheaper to make and easier to store
When and what was the first DNA-based vaccine?
2005
Produced against the West Nile Virus
How are new vaccines developed? (fourth method)
- Edible vaccines
Antigens can be expressed on plants, then they can be eaten
However lots must be eaten and it must be raw
What are stem cells?
Undifferentiated cells, which can differentiate into specialised cells and replicate to make more stem cells
What are the two types of stem cells?
Adult stem cells
Embryonic stem cells
What are totipotent stem cells?
They can regenerate into all cell lineages to regenerate a whole organism e.g. in plants and fungi, or embryonic stem cells within the first couple of divisions (they can generate both embryonic and extra-embryonic cells)
What are pluripotent cells?
Capable of forming all the cell lineages within an embryo, but not extra embryonic lineages
What are multipotent cells?
Have the potential to differentiate into many, but not all, cell lineages
What are hematopoietic stem cells (HSCs)
Found in bone marrow
Multipotent - replenish red and white blood cells
What are the two places that hematopoietic stem cells are found?
- Vascular niche - near blood vessels within the marrow
2. Endosteal niche - at the interface between the bone and marrow
What two cell lines do hematopoietic stem cells differentiate into?
- Lymphoid progenitor - differentiate into T and B cells
2. Myeloid progenitor - differentiate into red blood cells, macrophages and neutrophils
What are intestinal epithelial stem cells (ISCs)?
Found in the small intestine
Also called crypt base columnar cells (CBCs)
Can differentiate into four cell types
What are the four cell types that CBCs can differentiate into?
Absorptive epithelial cells (enterocytes)
Goblet cells - secretes main component of mucus
Enteroendocrine cells - control glucose levels, food intake and stomach emptying
Paneth cells - secrete anti-microbial compounds
What are embryonic stem cells (ESCs)?
Derived from the blastocyst
Pluripotent, so capable of forming all the cell lineages within an embryo, but not extraembryonic lineages
Cells can be grown in culture (since 1998)
What are induced pluripotent stem cells (iPSCs)?
Adult cells the have been undifferentiated back to stem cells
Changes in gene expression allow reversion of adult cells into stem cells
Less controversial than embryonic stem cells
Not clear if identical to ESCs
Low efficiency of production
Can cause rumours
STAP cell comtrovery
What is the controversy over STAP cells?
Research on generating pluripotent stem cells by subjecting ordinary cells to certain types of stress (e.g. bacterial toxins or physical trauma) was published in 2014 by Haruko Obokata. However, others were unable to replicate it - may be error or fraud
What stem cells are often used in stem cell therapy?
Hematopoietic stem cells most commonly used (bone marrow transplant)
HSCs also found in umbilical cord blood and some circulate in blood
What could stem cell therapy be used to treat?
Leukaemia Sickle cell anaemia Tissue regeneration Reverse ageing Parkinson’s Diabetes Spinal cord injuries Replacement for animal testing
Treatment for diabetes
Induce stem cells to differentiate into pancreatic B cells that produce insulin
Use these to treat type 1 diabetics (who cannot produce insulin)
Tested in mouse models and mice have been shown to produce insulin
More work needed to be done to prevent immune system attacking B cells
How to transform embryonic stem cells
- Make vector containing gene if interest
- Transform stem cells
- Insert stem cells into embryo
- Implant into mouse
How are knockout mice produced?
- Clone regions flanking gene into a vector
- Introduce DNA construct into mouse
- Construct replaces or disrupts gene, so no protein is made and gene function can be analysed
- Used in research of human diseases
What are knockout mice?
They have genes ‘knocked-out’ to analyse their function
How is cloning achieved?
- Remove nucleus from egg cell
- Add a different nucleus
- Allow to develop into frog
What did cloning show?
The the nucleus of a differentiated cell can regenerate an entire organism
What is forensic biotechnology?
Use of science in gathering evidence Fingerprints DNA technology Blood type DNA fingerprinting DNA databases/barcoding Biometrics (body measurements and calculations)
Features of bloodtyping
Glycolipid antigens A, B and O Different alleles of the same gene O has one fewer carbohydrate A has N-acetyl-galactosamine B has galactose
What is DNA fingerprinting?
Analysis of DNA fragments for identification
Developed in 1980s
Early method used restriction fragment length polymorphisms (RFLPs)
Process of early DNA fingerprinting
- DNA was extracted, cut with restriction enzymes and run on gel
- A Southern blot was used, where DNA was transferred to a nylon filter
- Radioactively labelled DNA probe were used, which bound to complementary DNA
- An autoradiograph is generated, where radiation-sensitive film is placed over the blot and DNA bound to the probes appear as bands on the film
- The restriction pattern is matched to DNA found on the victim
Current process of DNA fingerprinting
DNA is now amplified by PCR and sequenced
Mini satellites and microsatellites often used
Can be run on a gel or visualised by qPCR or sequencing software
What is a minisatellite?
Variable number tandem repeats (VNTRs)
10-100 base pairs repeating 5 to 50 times
Segment of repetitive DNA
What is a microsatellite?
Short tandem repeats (STRs)
2-5 base pairs repeating 5 to 50 times
Examples of phenotypic biometrics
Fingerprints
Retinal scans - look at the pattern of blood vessels behind retina
Iris recognition systems
Facial recognition
What is DNA barcoding?
Used to identify species
Uses conserved regions of genome (e.g. chloroplasts in plants, mitochondria COI in animals)
Can be used to analyse what is in food and to ID insect larvae
What is bioremediation?
The use of living organisms or their products to break down waste and pollutants in the environment
Can clean up organic waste, chemical spills, pesticides, heavy metals, etc.
Uses natural processes and reactions
How are organisms obtained for bioremediation?
Can search for organisms that degrade waste and use them directly
Can genetically modify other organisms to express the relevant genes
What are some common chemical pollutants?
Benzene Chromium Naphthalene Radioactive compounds Trace metals Trinitrotoluene (TNT)
Describe bioremediation of ground water
If drinking water is contaminated, need a bioreactor containing bacteria to separate the clean water
What microbes are used for bioremediation?
- Petroleum-eating bacteria: isolated strains of Pseudomonas found in contaminated soils contained plasmids encoding genes for degrading organic compounds such as naphthalene, octane and xylene
- E. coli used for heavy metals
- Metallothioneins (proteins on cell surface)to neutralise heavy metal pollutants such as cadmium and mercury
- Fungi Phanerochaete spp. can degrade toxic chemicals such as creosote and other pollutants that degrade bacteria
- Fusarium oxysporum and Mortierella hyaline can degrade asbestos and heavy metals
Describe the Exxon Valdez disaster
1989
Oil tanker ran aground and released 42 million litres of oil off Alaskan coast
Oil will remain for hundreds of years
What are steps for bioremediation of oil spill?
- Clear oil from surface using skimmers and vacuums
- Wash rocks with water
- Added fertiliser to encourage growth of bacteria to degrade o
Describe the Deepwater Horizon oil spill
2010
Explosion released 600 million litres of oil into Gulf of Mexico
Oil removed by various methods
Bioremediation degrades 50% of oil released
What types of bacteria are used to removed the sulphur from fossil fuels and reduce their toxicity?
Thiobacillus and Sulfolobus can be used to convert inorganic FeS2 to sulphates that can be washed away
That accounts for 30% of sulphur in fossil fuels, the rest is in thiophene rings
Rhodococcus can break down thiophene but is hard to culture
Transformed E. coli may be able to break down thiophene
Describe bioremediation of lignocellulose
It is a waste agricultural and industrial product
Microorganisms can degrade cellulose and hemicellulose, but not lignin
White rot fungi can degrade lignin using peroxidases to break the bonds between phenols in lignin
What is a white rot fungi that can degrade lignin?
Phanerochaete chrysosporium
Using bioremediation to recover valuable metals
Cooler, nickel, boron, gold
Many microbes convert metal products into metal oxides or ores
Useful for recovery of metals from waste solutions from industrial manufacturing processes
Bioremediation of radioactive wastes
Most radioactive materials kill microbes, but some strains have a potential for degrading radioactive chemicals
No bacterium so far can completely metabolise radioactive elements into harmless products
What is phytoremediation?
Using plants for bioremediation of soil, water and air
Around 350 plant species naturally take up toxic materials
Poplar, juniper, grasses, Agrostis
Sunflower plants removed radioactive cesium and strontium from Chernobyl
Water hyacinths removed arsenic from water supplies in Bangladesh and India
What is biofuel?
Fuel produced through biological processes
What is the total biofuel production in billions of litres?
81 billion litres per year
Issues with biofuel production
Expensive Subsidy dependent Fuel instead of food What to do with waste products Impacts on biodiversity
Biogas
2 million people worldwide burn biogas for fuel
Could produce gas from animal dung in small bioreactors
Bioethanol
Sugarcane or maize often raw materials
Microbes convert sugars into ethanol
Production is expensive
Fuel additive
Biodiesel
Derived from plant oils and animal fats
Standalone fuel - replacement for diesel
Lignocellulose biomass as a biofuel
Agricultural waste products - woody crops, sawdust
Do not need much water or fertiliser to grow
More sustainable
Still for biodiesel and bioethanol
Less competitive due to difficulties degrading lignocellulose
Biofuels from algae and cyanobacteria
Still under development
No competition for land use
Oils easily refined into diesel
Easy to genetically manipulate algae into ethanol and butanol
Can also make biogases such as biohydrkgen and biomethane
Algae produce 10x the output of traditional biofuel feedstocks
Lots of fertiliser needed
What % of fuel in the UK is biofuel?
3%
What greenhouse saving do biofuels have compared to fossil fuels?
80%
Bioremediation of paper waste
Paper is mainly cellulose
Various microbes produce different cellulases
These cellulases have been purified and shown to degrade paper to glucose
Glucose could then be used to produce ethanol
Inefficient multi step process
Cellobiose acts as feedback inhibitor of cellulose degradation
Glucose inhibits hydrolysis of cellobiose
End products of this reaction therefore have to be removed quickly to allow continuous degradation
Fuel from microbes
Derived from plant oils and animal fats - could otherwise be used for food
Engineer E. coli to produce large quantities of fatty acids
What is green biotechnology
Use of crop plants and agricultural systems
Humans have modified plants for thousands of years
Since 1920s there has been mutational breeding, plant propagation, etc
Genetic modification
What is Agrobacterium tumefaciens?
Plant pathogen used in molecular biology
Causes crown gall (1907)
Induced plant to produce excess auxin and cytokinin
Galls contain opines synthesised by genes on the Ti plasmid
Other plant transformations
Gene gun or particle bombardment
Gold particles are coated in the DNA construct to be introduced, then they are fired at target cells
GM crops
Also called biotech or transgenic crops
First commercial GM crops in 1994
Grown in 26 countries
$18 billion annually
What 4 GM crops dominate global production?
Soybean (50%)
Maize (31%)
Cotton (13%)
Oilseed rape (4%)
Two main traits of GM crops
Herbicide tolerance
Insect resistance
Resistance to glyphosate
Herbicide
Blocks amino acid synthesis
Introduce a mutant EPSPS gene from Agrobacterium tumefaciens (aroA); glyphosate cannot bind
Plant is resistant to glyphosate
GM plants producing Bt toxin
cry genes from soil bacterium Bacillus thuringiensis
Toxic protein, which solubilises when ingested by insects
Binds to epithelial cell’s within the digestive tract and creates holes
Insects die within a few days
Spray plants with it in organic agriculture
Other GM traits
Herbicide tolerance
Virus resistance - mosaic viruses
Quality traits - drought tolerance, golden rice, photosynthetic genes
