L. 22 - Cell Factories & Biotechnology

Question 1

L.O.

Answer 1

• 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

Question 2

Fermentation (traditional biochemistry)

Answer 2

• Fermented alcohol and to preserve food
• Mixed cultures of naturally occuring bacteria
• Yeast fermentation is ancient ~7000BC China

Question 3

Types of Biotechnology

Answer 3

Molecular biotechnology:
- Cellular machinery inside cells

Cellular biotechnology:
- Whole cells and microbes

Question 4

Cellular biotechnology

Answer 4

• Early/ ancient methods
• Fermentation

Question 5

Molecular biotechnology

Answer 5

• Modern methods
• Looking at manipulating microbes and enzymes

Question 6

Microbes for biotechnology

Answer 6

• Virus
• Archaea
• Algae
• Fungi
• Protists

Question 7

Viruses for biotechnology

Answer 7

Viruses are used as vectors
Vectors = carry gene in DNA
- Source of enzymes (T4 Liagse, for ligating)

Question 8

Archaea for biotechnology

Answer 8

• Diverse living conditions
• Source of enzymes (PCR, thermostable polymerase, to copy DNA)

Question 9

Bacteria for biotechnology

Answer 9

• Host for DNA cloning
• Express proteins
• Quick and easy to work with

Question 10

Algae for biotechnology

Answer 10

• Eukaryotes
• Green biotechnology
• Biofuels, (convert CO2 and light into biofuels)
• Carbon negative
• Low maintenance

Question 11

Fungi for biotechnology

Answer 11

Yeast = Cloning and expression hosts, similar to bacteria
Moulds = antibiotic synthesis, penecillin

Question 12

Host cell examples

Answer 12

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

Question 13

Vectors

Answer 13

• Deliver DNA (usually foreign)

Plasmids:
- Circular DNA
- Replicate independently of chromosomes
- Most common vector

Viruses:
- ‘hijacking’ host cells

Question 14

Plasmids

Answer 14

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)

Question 15

Key factors of plasmids

Answer 15

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

Question 16

Molecular cloning

Answer 16

• Make many copies of entity

3 Steps:
1. Recombinant DNA preperation (to be modified)
2. Transformation and screening
3. Copying and expression

Question 17

Molecular cloning step 1:

Answer 17

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)

Question 18

Molecular cloning step 2:

Answer 18

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)

Question 19

Molecular cloning step 3:

Answer 19

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

Question 20

Molecular cloning end result

Answer 20

Genetically modified Organism (GMO):

• Foreign gene inside

Risks:
- depends on gene added
- antibiotic resistance gene transfer into pathogens
- commercial risks

Question 21

Vaccines

Answer 21

• 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

Question 22

Smallpox

Answer 22

• Variola virus
• Smallpox is erradicated due to vaccines

Question 23

Preparing a recombinant vaccine

Answer 23

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

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(O = Plasmid) .(≠ = DNA)
O + ≠≠≠≠

C + .≠ ≠ ≠ ≠ .<=Digest

C≠ .<=Ligate