L. 22 - Cell Factories & Biotechnology Flashcards
L.O.
- Explain why microbes are useful for biotechnology, using examples of specific fungi (cerevisiae) and bacteria (E.coli)
- Explain what a plasmid is, and define the roles of different kinds of plasmids in nature and in biotechnology
- Define the terms “DNA cloning”, “recombinant DNA”, “GMO”
- Explain how recombinant DNA and GMOs are made, and especially which enzymes do which jobs in this process
- Discuss why vaccines are important, and how recombinant DNA methods can be used to make them
Fermentation (traditional biochemistry)
- Fermented alcohol and to preserve food
- Mixed cultures of naturally occuring bacteria
- Yeast fermentation is ancient ~7000BC China
Types of Biotechnology
Molecular biotechnology:
- Cellular machinery inside cells
Cellular biotechnology:
- Whole cells and microbes
Cellular biotechnology
- Early/ ancient methods
- Fermentation
Molecular biotechnology
- Modern methods
- Looking at manipulating microbes and enzymes
Microbes for biotechnology
- Virus
- Archaea
- Algae
- Fungi
- Protists
Viruses for biotechnology
Viruses are used as vectors
Vectors = carry gene in DNA
- Source of enzymes (T4 Liagse, for ligating)
Archaea for biotechnology
- Diverse living conditions
- Source of enzymes (PCR, thermostable polymerase, to copy DNA)
Bacteria for biotechnology
- Host for DNA cloning
- Express proteins
- Quick and easy to work with
Algae for biotechnology
- Eukaryotes
- Green biotechnology
- Biofuels, (convert CO2 and light into biofuels)
- Carbon negative
- Low maintenance
Fungi for biotechnology
Yeast = Cloning and expression hosts, similar to bacteria
Moulds = antibiotic synthesis, penecillin
Host cell examples
Bacteria: (e-coli)
- Fast growing
- Easy to extract/ add plasmid DNA
- Model organisms
Yeasts: cerevisiae
- Bread and beer
- Express Eukayrotic genes (body recognise as eukaryotic)
- Generally safely recognised (food grade)
Biosynthesis of high-value products from simple raw materials. All starting from DNA
Vectors
- Deliver DNA (usually foreign)
Plasmids:
- Circular DNA
- Replicate independently of chromosomes
- Most common vector
Viruses:
- ‘hijacking’ host cells
Plasmids
Naturally found and allow genes to be swapped (Horizontal gene transfer)
Non-essential genes for growth and division of bacteria (can help in certain conditions - antibiotic resistance)
Key factors of plasmids
Selectable (gene) marker:
- Forces bacteria to takeup and sue the plasmid for anitibiotic resistance/ metabolic activity
Replication function:
- Persistance in host
- Help bacteria keep the plasmid
Cloning site:
- Manipulate site
- Insert foreign genes
Molecular cloning
- Make many copies of entity
3 Steps:
1. Recombinant DNA preperation (to be modified)
2. Transformation and screening
3. Copying and expression
Molecular cloning step 1:
Recombinant DNA preperation
Recombinant DNA:
- Foreign DNA and plasmid vector mixed
Identify gene of interest:
- Digest DNA (cut with enzyme)
- Ligate DNA (join foreign DNA with vector)
Molecular cloning step 2:
Transformation and screening:
Transformation:
- Host cell picks up recombinant DNA under stressful conditions
Screening:
- Selectrable (gene) marker to see it
- Transformation is NOT 100% efficient
eg.
antibiotic resistance
metabolic activity
PCR (thermostable polymerase)
Molecular cloning step 3:
Copying and expressing:
Copies of organisms/ DNA:
- express molecules/ proteins
Cloning Site:
- induce growth/ gene expression
- lacZα gene
PCR:
- copying DNA
Express foreign protein:
- End product
- Used to make another product
Molecular cloning end result
Genetically modified Organism (GMO):
- Foreign gene inside
Risks:
- depends on gene added
- antibiotic resistance gene transfer into pathogens
- commercial risks
Vaccines
- Recombinant products
- Some disease have no cure, prevention is only option
- Vaccines lead to ‘herd immunity’
- Adaptive immune response to recognise antigens associated with the pathogen
- live or dead microbes
Smallpox
- Variola virus
- Smallpox is erradicated due to vaccines
Preparing a recombinant vaccine
1. Recombinant DNA preperation:
- identify gene of interest
- Digest DNA
- Ligate DNA
2. Transformation and screening:
- microbe takes up recombinant DNA
- select those that did
3. Copying and expressing:
- Express foreign gene
- purification
- Antigen product
[heft]
(O = Plasmid) .(≠ = DNA)
O + ≠≠≠≠
C + .≠ ≠ ≠ ≠ .<=Digest
C≠ .<=Ligate
