LECTURE 21 - Cell Factories and Biotechnology Flashcards
Why should we care about biotechnology?
- food
- drink
- medicine
- agriculture
- fuel
- cleaning
Traditional biotechnology - Fermentation
- “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.
Cellular vs. molecular biotechnology
- 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
Viruses in molecular biotechnology
- Vectors to carry genes into new hosts
- Source of enzymes eg. T4 ligase
arhaea in molecular biotechnology
- Source of thermostable
polymerase enzymes for
copying DNA sequences
bacteria in molecular biotechnology
- Excellent hosts for
cloning DNA and
expressing proteins
algae in molecular biotechnology
- Conversion of CO2 + light
into biofuels (ethanol, H2)
fungi [yeast] in molecular biotechnology
- Excellent cloning and
expression hosts
fungi [moulds] in molecular biotechnology
- Antibiotic
synthesis
Host cells for biotechnology - the ‘workhorses’
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)
Host cells for biotechnology - what do they provide?
- 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
Biotech processes - what do we need to provide?
- The host cell factory needs instructions or a
blueprint to tell it which products to make
Instructions = add DNA !
How to deliver the instructions? … plasmids
- 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
plasmids def
- Plasmids: circular DNA elements found in microbes; replicate independently of chromosomes
Key features of plasmids used for biotech
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
Cloning def
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
Tools needed for DNA cloning – Enzymes !
Copying DNA : thermostabe polymerase
Cutting DNA : restriction enzyme
Joining DNA : T4 ligase
thermostable polymerase : archaea
t4 ligase : virus
DNA Cloning - Part 1. Digestion and ligation
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).]
- 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’ - 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.]
DNA Cloning - Part 2. Transformation & Screening
- Transformation : uptake of DNA –> the process of introducing the recombinant DNA molecule into the host organism (usually bacteria)
-
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 - 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.
ligation mixture
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
DNA Cloning - Part 3. The final product… a GMO
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)
risks with GMO
- 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.]
DNA Cloning Part 4. Expression of gene(s)
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
Examples of recombinant products - vaccines
- 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
vaccines def
Vaccines: a primary defence against infectious disease;
save approx. 3 million lives every year
Hepatitis B vaccine
- Isolate gene coding for antigen by selectively
copy DNA
thermostable
polymerase
–> Surface protein
antigen HBsAg - 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 - Transformation into yeast
Recombinant yeast plasmid + yeast cloning host –> GMO yeast - 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.
