LECTURE 21 - Cell Factories and Biotechnology

Question 1

Why should we care about biotechnology?

Answer 1

• food
• drink
• medicine
• agriculture
• fuel
• cleaning

Question 2

Traditional biotechnology - Fermentation

Answer 2

• “Biotechnology” has been done by humans for >9,000 years
  i.e. fermentation to preserve foods, or make alcohol
• Early fermentations used mixed cultures of naturally-occurring bacteria & fungi

intentionally introduce specific beneficial microorganisms (often lactic acid bacteria, yeast, or other desirable cultures) to the food. These microorganisms help preserve the food by outcompeting spoilage microorganisms, producing antimicrobial compounds, and creating an environment that is inhospitable to spoilage agents. The process is carefully controlled to ensure safety and quality.

Question 3

Cellular vs. molecular biotechnology

Answer 3

• Fermentation could be defined as “cellular biotechnology”
  –> need some biology skills (esp. microbiology)
  –> don’t need understanding of DNA, RNA, proteins
• Modern methods are “molecular biotechnology”
  –> need high-level biology skills (esp. microbiology)
  –> AND need understanding of DNA, RNA, proteins

Question 4

Viruses in molecular biotechnology

Answer 4

• Vectors to carry genes into new hosts
• Source of enzymes eg. T4 ligase

Question 5

arhaea in molecular biotechnology

Answer 5

• Source of thermostable
  polymerase enzymes for
  copying DNA sequences

Question 6

bacteria in molecular biotechnology

Answer 6

• Excellent hosts for
  cloning DNA and
  expressing proteins

Question 7

algae in molecular biotechnology

Answer 7

• Conversion of CO2 + light
  into biofuels (ethanol, H2)

Question 8

fungi [yeast] in molecular biotechnology

Answer 8

• Excellent cloning and
  expression hosts

Question 9

fungi [moulds] in molecular biotechnology

Answer 9

• Antibiotic
  synthesis

Question 10

Host cells for biotechnology - the ‘workhorses’

Answer 10

Bacteria e.g. E.coli
* Fastest growth
* Very easy to extract or add plasmid DNA
Yeast e.g. Saccharomyces
* Better for expressing eukaryote genes
* Generally recognized as safe (GRAS)

Question 11

Host cells for biotechnology - what do they provide?

Answer 11

• The host cell contains machinery for biosynthesis of high-
  value products from simple raw materials

USING : Sugars/Ammonia/Phosphate/Trace metals
with: Ribosomes/Amino acids/ Enzymes/ Metabolic pathways
INTO : food/drink/medicine/fuel

Question 12

Biotech processes - what do we need to provide?

Answer 12

• The host cell factory needs instructions or a
  blueprint to tell it which products to make
  Instructions = add DNA !

Question 13

How to deliver the instructions? … plasmids

Answer 13

• Plasmids: circular DNA elements found in microbes; replicate
  independently of the chromosome(s)
• Plasmids are the most commonly used vector for delivery of
  foreign DNA into a target host cell
• Viruses can also be used
  as vectors

Question 14

plasmids def

Answer 14

• Plasmids: circular DNA elements found in microbes; replicate independently of chromosomes

Question 15

Key features of plasmids used for biotech

Answer 15

Cloning site [has restriction site(s) / can be cut by restriction enzymes, so (new) gene can be added ]
–>Add foreign genes here

Replication functions [replicate, independently & fast, / have origin of replication so, high copy number / large number of plasmids]
–> Ensures persistence in host

Selectable marker [have marker genes so, recombinants / transformed bacteria ,
can be recognised]
–> Enables us to force cells to take up plasmid

Question 16

Cloning def

Answer 16

o make many copies of a biological entity
1. Creating identical organisms
OR
2. Making copies of a piece of DNA by adding it into a plasmid, then replicating the plasmid

Question 17

Tools needed for DNA cloning – Enzymes !

Answer 17

Copying DNA : thermostabe polymerase
Cutting DNA : restriction enzyme
Joining DNA : T4 ligase

thermostable polymerase : archaea
t4 ligase : virus

Question 18

DNA Cloning - Part 1. Digestion and ligation

Answer 18

Organism of interest [This organism contains the DNA fragment you want to clone.]
1. Extract DNA [Extract the DNA from this organism: DNA can be found in the nucleus of eukaryotic cells or in the nucleoid region of prokaryotic cells (like bacteria).]

2. Cut DNA [This is done using enzymes known as restriction endonucleases or restriction enzymes : recognize specific DNA sequences (called restriction sites) and cleave the DNA at those sites.]
   into pieces
   ‘diestion’
3. Or copy DNA [polymerase chain reaction (PCR) to make identical copies of specific DNA regions]
   Plasmid –> cut –> ligation
   [The DNA fragment and the plasmid both have compatible ends (often created by the same restriction enzyme), which can be joined together. This joining process is called ‘ligation,’ and it’s accomplished using an enzyme called DNA ligase. The ligase covalently binds the DNA fragment and the plasmid, creating a recombinant DNA molecule.]

Question 19

DNA Cloning - Part 2. Transformation & Screening

Answer 19

1. Transformation : uptake of DNA –> the process of introducing the recombinant DNA molecule into the host organism (usually bacteria)
2. Selecting Plasmid-Containing Cells:
   use a selective marker, like an antibiotic resistance gene on the plasmid –> plate the transformed bacterial cells on agar plates containing the antibiotic –> plasmid containing cells will will survive and form colonies
3. Screening : identifying the presence of the gene of interest within the transformed cells.

• Sequence-Based Screen (Look for DNA Directly):
  *- comparing the sequence to the known sequence of the gene of interest
• Phenotypic Screen (Look for the Effect of the Gene on the Host):
  *- observing the characteristics or traits of the host cells after transformation –> identify it by observable changes.

Question 20

ligation mixture

Answer 20

This mixture contains the genetic material you want to insert into the host cells.

The ligation mixture typically contains the following components:

• DNA Fragments: The DNA fragments that you want to join together.
• Plasmid Vector
• DNA Ligase
• Buffer Solution
• Co-Factors
• Water

Question 21

DNA Cloning - Part 3. The final product… a GMO

Answer 21

A recombinant microbe refers to a microorganism (like bacteria or yeast) that has been genetically modified by inserting a specific gene of interest into its DNA
–> GMO (genetically-modified organism)

Question 22

risks with GMO

Answer 22

• Antibiotic resistance gene transfer into pathogens [these genes could potentially transfer to pathogenic bacteria, making them more resistant to antibiotics. This could complicate the treatment of infections.]
• Depends on what foreign genes were added [Some GMOs may have minimal environmental or health risks, while others may pose greater concerns. It’s important to assess each GMO on a case-by-case basis.]
• Commercial risks: legal constraints, public perceptions [Some people are worried about the potential long-term health and environmental impacts of GMOs.]

Question 23

DNA Cloning Part 4. Expression of gene(s)

Answer 23

when working with plasmids, scientists can introduce foreign genes and control their expression using promoters.
* The protein made from foreign gene may itself be the
end-product (e.g. HepB vaccine) or foreign genes could encode and
make the end-product (e.g. metabolic enzymes) from

Question 24

Examples of recombinant products - vaccines

Answer 24

• Vaccines: a primary defence against infectious disease;
  save approx. 3 million lives every year
• Protect against: smallpox, polio, rabies, diphtheria, pertussis,
  tetanus, hepatitis, measles, mumps, rubella, influenza,
  pneumonia, rotavirus, meningitis, papillomavirus (and more)
• Some diseases have no cure…
  prevention is the only option
• Vaccines lead to ‘herd immunity’

Variola virus (smallpox)
- Eradicated by vaccination

may consist of :
1. live attenuated microbes
2. killed microbes
3. antigens (proteins) produced in a GMO host
4. mRNA coding for antigens

Question 25

vaccines def

Answer 25

Vaccines: a primary defence against infectious disease;
save approx. 3 million lives every year

Question 26

Hepatitis B vaccine

Answer 26

1. Isolate gene coding for antigen by selectively
   copy DNA
   thermostable
   polymerase
   –> Surface protein
   antigen HBsAg
2. Cloning antigen gene
   yeast plasmid and HBsAg gene –> digestion [“digestion” refers to a laboratory technique in molecular biology. It involves using enzymes, known as restriction enzymes, to cut DNA at specific sites] –> ligation –> recombinant plasmid
3. Transformation into yeast
   Recombinant yeast plasmid + yeast cloning host –> GMO yeast
4. Gene Expression [the yeast cells containing the recombinant plasmidexpress the HBsAg gene, leading to the production of the HBsAg protein within the yeast cells], Protein purification
   yeast cells produce the HBsAg protein

Protein Purification: After the yeast cells have produced the HBsAg protein, it needs to be isolated and purified. This typically involves a series of lab techniques to separate the protein from other cellular components. The purified HBsAg protein can then be used for research, diagnostics, or vaccine production.