1
Q
Why use recombinant cells?
A
-Products of interest may be produced in very low volumes in natural cells
- Wild-type cells may be slow-growing #
- May be interested in a modified/ unique protein not found in nature
- Not ethical to use natural systems (i.e. human insulin)
2
Q
What does wild-type cell mean?
A
it describes a cell when it is in natural, non-mutated or unmodified form.
3
Q
What are the two main classifications of cells?
A
Prokaryotic (simple structure) and Eukaryotic (complex structure)
4
Q
what are the characteristics of prokaryotic cells?
A
-Typically 1-10 µm
-Outer rigid cell wall
-Inner lipid cell membrane
-Pili/flagella structures allow movement
-Embedded transport proteins in the membrane allow the passage of material into and out of the cell
-Interior cytoplasm (fluid-like) material
-DNA contained in a single closed circular loop
-Free floating ribosomes
-Free floating plasmid DNA
5
Q
What are the characteristics of eukaryotic cells?
A
- Typically 10-100 µm
- Some have an outer rigid cell wall (e.g. plant cells)
- Inner lipid cell membrane
- Cytoplasm contains cytoskeleton to give structure
- Highly organised: contain sub-cellular organelles with specific functions
- Mitochondria/ chloroplasts
- Rough / smooth endoplasmic reticulum
- Lysosomes
- Golgi body
- DNA is contained within a membrane-bound nucleus
6
Q
What are the characteristics of eukaryotic cells?
A
- Typically 10-100 µm
- Some have an outer rigid cell wall (e.g. plant cells)
- Inner lipid cell membrane
- Cytoplasm contains cytoskeleton to give structure
- Highly organised: contain sub-cellular organelles with specific functions
- Mitochondria/ chloroplasts
- Rough / smooth endoplasmic reticulum
- Lysosomes
- Golgi body
- DNA is contained within a membrane-bound nucleus
7
Q
Examples of prokaryotic cells?
A
Eubacteria, archaea
8
Q
Example of Eukaryotic cells?
A
Animal, Plant, Fungi, Protists
9
Q
What does it mean by cell differentiation?
A
As many eukaryotes are multi-cellular (multiple cells form an organism), groups of these cells become specialised to form different things or have different functions. for example, DNA is identical but only certain parts of the DNA are expressed.
10
Q
What is a host cell?
A
This is a cell you would use to host the DNA of interest
11
Q
What to consider when choosing a cell line to host a recombinant product.
A
There are a range of well charachterised cells to select from.
- Both Eukaryotic and Prokaryotic
- It depends on what product you want to synthesise
- Choose the system that is the least demanding that produces the quantity and quality of proteins needed.
12
Q
Why are prokaryotic cells - Bacteria good host cells?
A
Such as E.coli, bacterial cells :
- has fast replication (doubling time of 20 mins) however not all bacteria multiply this rapidly
- Fast metabolism, and produces a sizable biomass
- Can withstand agitation without damage due to cell walls
- Bacterial cells are also well characterised such as E.coli
- They produce a lot of cells in a small space, meaning there can be a lot of profit from using bacterial host cells.
- they’re also cheap to grow compared to mammalian cells
13
Q
What is the downside of using bacteria as host cells?
A
Bacterial cells aren’t capable of making post-translational modifications of protein.
- They arent capable of expressing mammalian characteristics, such as inclusion bodies.
- Proteases contamination in the host cells could degrade it and endotoxin could accumulate
14
Q
What are the Pros and Cons of using cytoplasmic proteins in Escherichia Coli?
A
Pros:
- Formation of inclusion bodies (insoluble granules) makes protein easier to separate
- These miss folded inclusion bodies can be easily separated and can be tinkered with using a bit of biochemistry and can be added to other systems or chemicals
Cons:
- Protein degradation by cytoplasmic proteases can occur in some proteins forms
- Inclusion bodies are incorrectly folded proteins so need further processing to fold correctly
- E.coli cytoplasm conditions prevent disulphide bonds from forming
15
Q
What Protiens are expressed in E.Coli
A
- Cytoplasmic Protein (inside the cells) (most common type)
- Secreted Proteins (Outside cell or between membranes/wall) (carry out functions)
- Inclusion bodies
16
Q
Pros and Cons of using Periplasmic Proteins?
A
Pros:
- There’s low proteolytic activity (breakdown of proteins into smaller polypeptides / Amino Acids)
- Can have disulphide bonds made there
17
Q
Pros and Cons of using Periplasmic Proteins?
A
Pros:
- There’s low proteolytic activity (breakdown of proteins into smaller polypeptides / Amino Acids)
- Can have disulphide bonds made there
Cons:
- Pushing proteins in the periplasmic area are difficult to separate, therefore it’s not desirable to manufacture proteins in this area.
18
Q
Why are disulphide bonds important?
A
They are important because:
- They induce stability
- reduces the entropy of the denatured state
- reduces flexibility
19
Q
Why is secreting the proteins from E.coli for manufacturing more desirable?
A
Pros:
This is a more preferred direction because:
- it is a more straightforward downstream process
- Can centrifuge your way down to the cells of interest, separate the desired mixture from the undesired and then you’ll get a media full of desired cells
- Due to proteins not secreting a lot of proteins, there are low contamination levels on your desired media.
Cons:
- It’s not common for E.coli to secret proteins
- Requires more genetic engineering to help the protein be transported out, i.e. carrier proteins.
20
Q
What are the intracellular factors that could negatively affect the physiology of the cell?
A
- Multicopy plasmids - although having many multicopy plasmids means you get a higher yield, this can affect the metabolic rate and cause a metabolic burden on the cells
- Proteins can misfold - can trigger a stress response, this redirects the protein production into creating something that isn’t the desired protein
- Accumulation of toxic gene products - can also trigger a stress response, this redirects the protein production into creating something that isn’t the desired protein
- High-level gene expression - having a high gene expression causes the growth to slow down and means the growth won’t be doubled, this is because of cell uses a lot of resources and energy to create the protein.
21
Q
What are the extracellular factors that could negatively affect the physiology of the cell?
A
- Accumulation of toxic metabolites - causes the desired products to be contaminated and could be difficult to separate or unusable
- Nutrient limitations - if you have some dense cells, are the nutrients spread enough that all the cells have nutrients? If cells don’t have any nutrients this could also trigger a stress response.
- Limitation of Oxygen - This is the same as nutrient limitation, if the cells can’t get enough oxygen they can trigger a stress response
22
Q
What can optimise the expression of proteins?
A
- Low proteolytic activity (proteins being broken down into smaller segments)
- Enhance disulphide bridge formation - Cysteine reduced by two enzymes in the cytoplasm called Thioredoxin and glutaredoxin, can form disulphide bridges in the cytoplasm
23
Q
Why are Eukaryotic Yeast Cells desirable in fermentation?
A
Pros:
- Share biochemical, molecular & genetic features with higher eukaryotes
- Suitable for larger-scale fermentation
- Simple, rapid reproduction
- low-cost growth
- Can secrete soluble, correctly folded and post-translational modified proteins
Cons:
- lack of a strong and strictly regulated promoter
- The secretion efficiency is low
- Not suitable for high-density cultivation
- Irregular glycosylation
24
Q
Why is glycosylation important in manufacturing biologics?
A
These chains (glycans or chains of carbohydrates) become attached to the protein via a post-translational modification process called glycosylation.
Glycosylation plays an important role in the structure, function, absorption, half-life, clearance, and safety of therapeutic proteins.
25
What are the types of Post-Translational Modification?
- Hydroxylation
- Methylation
- Lipidation
- Acetylation
- Disulfide Bond
- Phosphorylation
- Glycosylation
- Ubiquitination
- SUMOylation
26
What is Hydroxylation?
Attaches a hydroxyl group (-OH) to a side change of a protein
27
What is Methylation?
Adds a methyl group (-CH3), usually at lysine or arginine residue
28
What is Lipidation?
Attaches a lipid, such as a fatty acid, to a protein chain
29
What is Acetylation?
Adds an acetyl group to an N-terminus of a protein or at lysine residue
30
What is Disulfide Bond?
Covalently links the sulfur atoms of two different cysteine residues
31
What is SUMOylation?
Adds a small protein SUMO (small ubiquitin-like modifier) to a target protein
32
What is Ubiquitination?
Adds ubiquitin to a lysine residue of a target protein for degradation
33
What is glycosylation?
Attaches a sugar, usually to an "N" or "O" in an amino acid side chain
34
What is Phosphorylation?
Adds a phosphate to serine, threonine or tyrosine.
35
why is post-translational modification important?
Post-transcriptional modification of proteins further modulates and extends the range of possible protein functions.
This is also why it's important to know what cell line to select so there isn't a lot post-transcriptional modification after transcription.
36
What are the pros and cons of using Eukaryotic Mammalian Cells?
Pros:
- Can produce modified (folded, glycosylated) proteins
- Secretion of the product (excreted proteins are made in the endoplasmic reticulum.
- i.e. CHO cells (Chinese Hamster Ovary Cells): stable cell line
Cons:
- Expensive cell line - need complex and defined media
- Slow growth and low yield, compared to E.coli, growth could take days instead of a few hours.
- High potential for contamination from animal virus and bacteria
- More fragile as there is no cell wall
- i.e COS cells (African Green Monkey Cells): Transient expression, loses ability to synthesise cells when vectors is used - needs integration into chromosomes
37
What conditions are considered when creating a cell culture?
- A substrate or medium that supplies the essential nutrients (amino acids, carbohydrates, vitamins, minerals)
- Growth factors
- Hormones
- Gases
- A regulated physico-chemical environment (pH, Osmotic pressure, temperature)
38
Pros and cons of using Plants cell lines
Pros:
- Perform complex modifications like mammalian cells
- Products can be localised to plant organs by controlling tissue-specific regulators sequences
- Cheaper to grow - don't use fermenters for transgenic plants
Cons:
- Require for labour
- There is a chance that the propagated plants will be less resilient to disease due to the type of environment they are grown in.
- The success rate is high if correct procedures are followed, success with the tissue cultures is not a guarantee.
39
Pros and Cons of using insect cell lines?
Pros:
- Baculovirus vectors are used to insert desired genes and transfected them into cultured insect sells.
- similar to mammalian cells, it can be used to adherent and suspension cultures.
- easy purification
Cons:
- Time-consuming cloning procedure.
- Expensive media
-Glycosylation is different to the mammalian system resulting in improper maintenance of epitopes on proteins.
40
What do cells need to grow and survive?
1. Source of Carbon - from glucose or glycerol, amino acids or lipids.
2. Source of Energy to make ATP - usually glucose or glycerol
3. Many cellular processes require inorganic ions - need a source of different salts (makeup 1% of the cells)
4. Need water
5. Other nutrients - Micronutrients, vitamins, nitrogen, phosphorus, sulfur, etc.
6. Physio-chemical properties - pH, Osmotic pressure, oxygen and temperature
7. Mammalian cells need growth factors/ hormones
8. Contamination-free environment - antibiotics or ultra-clean room
41
What are the types of Bacterial Media Formulation?
There are 2 types: Complex and Defined media
Complex media - A complex (undefined) medium is one in which the exact chemical constitution of the medium is not known.
- includes lysates of other cells, e.g. yeast
- lots of batch-to-batch variation#
- commonly used in a lab than in industry
Defined media - Defined media are usually composed of pure biochemicals off the shelf; complex media usually contain complex materials of biological origin such as blood or milk or yeast extract or beef extract, the exact chemical composition of which is obviously undetermined
- Easy to control process as provides reliable ingredients
- more expensive but required for process control
42
What is a typical complex media?
Lysogeny broth (LB) - is used for feeding E.coli. It contains:
- Tryptone - milk protein that provides amino acids
- yeast extract - organic compounds, vitamins and trace elements
- NaCl
43
Example of the defined medium on insulin production in E.coli
- Tryptone - 3.5g/L
- Yeast extract - 3.5g/L
- glucose - 2.0g/L
- NaCl - 1.0g/L
- (NH4)Cl - 3.0g/L
- K2HPO4 - 4.0g/L
- (NH4)2SO4 - 2.0g/L
- K2SO4 - 3.0g/L
- MgSO4 - 1.0g/L
- Thiamine - 5mg/L
- Trace elements - 2ml/L
- Kanamycin - 30mg/L
44
What is the purpose of phenol red?
Phenol red (PR) is the standard pH indicator in various cell and tissue culture media, as it provides a quick check for the health of the culture
45
What is the basic medium of mammalian cell cultures?
Dulbecco's Modified Eagle Medium (DMEM) or Roswell Park Memorial Institute Medium (RPMI), which contains:
- Amino acids
- Vitamins,
- Inorganic salt,
- Glucose
- Phenol red
46
What is the optimal pH for a mammalian cell line?
7.4
47
Where do we get growth factors for cell culture media?
Serums from fetal and calf bovine sources are commonly used to support the growth of cell culture. This is called the Fetal bovine serum (FBS)
The fetal serum is a rich source of growth factors and is appropriate for cell cloning and for the growth of fastidious cells.
48
What factors are needed for mammalian cells to grow?
- Growth factors
- Hormones
- Adhesion factors
- Carriers (albumen)
49
What is FBS?
Foetal bovine serum
Added to DMEM to a final concentration of 10%
50
Serum-based vs Serum free
Serum based:
- Serum is blood plasma without cells
- contains lots of factors/ proteins/ molecules that are hard to define/ synthesise individually
- a cheap way of giving complex cells all their requirements
- undefined so creates batch-to-batch variation
Serum free:
- Chemically defined media available but expensive and IP controlled
- Commonly associated with specific cells (build media around specific cell lines)
- Spent media analysis: analyse what's left after the growth curve and develop a media recipe based on data
51
What is recombinant DNA technology?
DNA from one organism is recombined with DNA from another organism
52
Define Cloning
Cloning is an umbrella term meaning identical copies of DNA molecules is replicated
53
Why is recombinant DNA (rDNA) important?
rDNA is an important research tool in biology, as it allows scientists to manipulate DNA fragments in order to study them in the lab. This involves using various methods to insert a piece of DNA into a bacterial or yeast cell such as E.coli.
Synthesise in E.coli using rDNA technology means you can have an unlimited supply, it is cheap, predictable and can make a human form of insulin.
54
What is the rDNA cloning steps?
1. Isolation of genetic material (DNA)
2. Cutting of DNA at specific locations
3. Joining of DNA fragments
4. Insertion of DNA into the host cell
5. Selection and screening of transformed cells.
55
rDNA: How do you isolate the genetic material?
1. Identify the desired DNA and isolate it in its true form.
2. Since DNA exists within the cell membrane along with other macromolecules such as RNA, polysaccharides, proteins, and lipids, it must be separated and purified which involves enzymes such as lysozymes, cellulase, chitinase, ribonuclease, proteases etc.
3. Other macromolecules are removable with other enzymes or treatments. Ultimately, the addition of ethanol causes the DNA to precipitate out as fine threads. This is then spooled out to give purified DNA.
56
rDNA: What are the uses of restriction enzymes in restriction enzyme digestion?
Restriction enzymes act as molecular scissors that cut DNA at specific locations. These reactions are called the restriction enzyme digestion.
57
What is restriction enzyme digestion?
1. The most useful restriction enzymes make staggered cuts; that is, they leave a single-stranded overhang at the site of cleavage.
2. These overhangs are very useful in cloning because the unpaired nucleotides will pair with other overhangs made using the same restriction enzyme.
3. The next step in the cloning process is to cut the vector with the same restriction enzyme used to cut the donor DNA. Vectors have target sites for many different restriction enzymes, but the most convenient ones are those that occur only once in the vector molecule.
4. Cut vector DNA and donor DNA are mixed in a test tube, and the complementary ends of both types of DNA unite randomly.
58
rDNA: What happens in the Transformation phase?
1. The mixture should now contain a population of vectors each containing a different donor insert. This solution is mixed with live bacterial cells that have been specially treated to make their cells more permeable to DNA.
2. Recombinant molecules enter living cells in a process called transformation. Once inside, the recombinant DNA molecule replicates like any other plasmid DNA molecule, and many copies are subsequently produced.
3. Furthermore, when the bacterial cell divides, all of the daughter cells receive the recombinant plasmid, which again replicates in each daughter cell.
4. The original mixture of transformed bacterial cells is spread out on the surface of a growth medium in a flat dish (Petri dish) so that the cells are separated from one another.
59
What are the two transformation methods?
1. CaCl2/ heat-shock - the chemical components as well as a ligated mixture are put in a test tube and let it incubate in an ice bath for 30 mins, then it is shocked at 42C for 30 secs before returning it into the ice bath.
2. electroporation - Electrocompetent cells and purified ligated DNA are added into a centrifuge tube and put into an ice container to not denature it whilst being transported to get an electric shock. The mixture gets an electric shock and then is returned to the ice bed.
60
If bacteria were antibiotic resistant, what could go wrong?
1. Bacteria contain only empty plasmid
2. Inserted gene is mutated
3. Inserted gene may be inserted into the wrong position
4. Bacteria may acquire antibiotic resistance
5. Antibiotic plate concentration may be too low
5. Inserted gene may be toxic so is removed by the bacteria
61
If the cloning procedure was successful, what would be the desired outcome?
- Plasmid contains a single copy of your gene
- In the correct orientation
- In the correct place
- With no mutations or truncations
62
What is an expression vector?
It is a plasmid or virus that is specially designed for expressing genes in a cell. It is a vector widely used for protein production. They have basic features of a vector like ori (origin of replication), insertion site, a selectable marker, etc. Additionally, they also have regulatory elements that aid in protein synthesis. Thus, the vector DNA fragment also carries a proper sequence for protein synthesis.
Expression vectors are vectors that enable the expression of cloned genes in order to determine the successful cloning process.
Usually, cloning vectors do not allow the expression of a cloned gene which is why the use of expression vectors is required.
https://microbenotes.com/vector-molecular-biology/
63
What is a cloning vector?
It is a small DNA fragment that has the stability to act as a vector for cloning purposes. Usually, foreign DNA is inserted into the cloning vector. After reaching the target cell, they can replicate and integrate with the target or host. These vectors can be of a virus cell, bacterial cell or even plasmid of a bacterial cell.
https://microbenotes.com/vector-molecular-biology/
64
What are plasmids and vectors?
A plasmid is a genetic structure in a cell that can replicate independently.
Vectors are a type of plasmid but they are experimentally used as tools to clone, transfer and manipulate genes.
65
What are the plasmid features?
- Small circular DNA (~4-10kb) in bacteria
- Annotation starts with a ‘p’, eg. pET28a, pSK, pCMV
- Often called vectors
- Possess an origin of replication
- HIGH or LOW copy number
- Carry an antibiotic marker
- Often a multiple cloning site (MCS)
66
Pros and Cons of plasmids as vectors
Pros:
- Easy to manipulate and isolate because of the small size
- More stable because of its circular configuration.
- Replicate independent of the host.
- High copy number.
- Detection was easy because of antibiotic-resistant genes.
Cons:
- Large fragments cannot be cloned.
- The size range is only 0 to 10kb.
- Standard methods of transformation are inefficient
67
Vector feature: Origin of Replication (Ori)
- A specific set/ sequence of nucleotides where replication initiates.
- For autonomous replication inside the host cell.
- Foreign DNA attached to ori also begins to replicate.
68
Vector feature: Cloning site
- Point of entry or analysis for genetic engineering.
- Vector DNA at this site is digested and foreign DNA is inserted with the aid of restriction enzymes.
69
Vector feature: Selectable Marker
- Gene that confers resistance to particular antibiotics or selective agents which, under normal conditions, is fatal for the host organism.
- Confers the host cell the property to survive and propagate in a culture medium containing the particular antibiotics.
70
Vector feature: Marker or Reporter Gene
- Permits the screening of successful clones or recombinant cells.
- Utilised extensively in blue-white selection
71
Vector feature: Inability to transfer via conjugation
Vectors must not enable recombinant DNA to escape to the natural population of bacterial cells.
72
What are the components of the ligase reaction?
- The cut plasmid
- The cut gene
- DNA ligase (which puts the cut DNA pieces together)
- Ligase buffer
- water
73
What happens after the formation of the recombinant DNA?
Mix the plasmids with the gene of interest with bacteria that are more capable of taking up DNA through their cell membrane in a process called transformation.
The results would be on an agar plate with the bacteria with the recombinant DNA being grown in colonies.
74
What are the reasons that the colonies grew?
1. Some plasmids will only be cut only once and will re-circularise
2. Some plasmids will not be cut at all
3. Some plasmids will contain the inserted gene
4. Some bacteria may be resistant to antibiotic
75
How to control anti-biotic issues? (First plate control)
When plating the transformation mix onto an agar plate, you also plate out a set of bacteria onto a plate that doesn't contain any of the plasmids, this plate would be the negative control.
The negative control should have no plasmid containing the desired gene, and it does not have any antibiotic-resistant features.
If there are colonies that grow on the agar plate in the negative control, this shows this could suggest that the antibiotic is probably too low.
So comparing this to your agar plate with the desired gene plasmids, if your control has 20 colonies and your normal plate has 200 colonies, this suggests that 10% (20 colonies) have antibiotic-resistant properties in the mix
76
How to control for Plasmid recircularization (self-ligation)? (Second control plate)
To prevent recircularization:
- use 2 types of restriction enzyme, one for each end, so this means they can't reattach
- Dephosphorylate the 5’ ends of the vector with a phosphatase
- Use a much higher conc. of insert (molar ratio greater than 1:6)
To control this control of circularised vector:
- Transform bacteria with only the cut plasmid mixture (eg. no insert)
77
How would you control uncut parent plasmids? (Third control plate)
- You will always have uncut vector in your ligation mix
- To minimize uncut vectors, separate the cut and uncut vectors on an agarose get after digestion.
Control for uncut vector:
- Transform colonies with a "no ligase" control - only uncut vectors will have antibiotic resistance
78
Positive and negative controls in all cloning experiments
Tube 1 – bacteria with no plasmid - Negative control
Tube 2 – bacteria with uncut plasmid - Positive control
Tube 3 – bacteria with digested plasmid and insert minus ligase - Uncut vector
Tube 4 – bacteria with digested plasmid and insert minus insert - Self-annealed cut vector
Tube 5 – bacteria with DNA mix containing everything - Cloning mix
These controls are important for estimating false positives and for when things go wrong
79
What is Blue-White Selection Method?
Blue-white screening is a rapid and efficient technique for the identification of recombinant bacteria. It relies on the activity of β-galactosidase, an enzyme occurring in E. coli, which cleaves lactose into glucose and galactose.
Only white colonies will have an inserted gene.
Blue colonies if there are no inserted genes
80
What happens during the Blue-White selection?
The plasmid vector contains a lacZ gene which has the multiple cloning site in the middle (MCS).
MCS is where restriction enzymes attach to the plasmid and cut it open for the desired gene to be inserted. This results in lacZ gene being non-functional, hence why inserted gene colonies are white.
81
What is the function of lacZ gene?
lacZ gene is the gene for β-galactosidase, which breaks down lactose into glucose and galactose
82
How is lacZ gene utilized for the Blue-White selection?
- Add X-gal (5-bromo-4-chloro-3-indolyl-D-galactoside) into your agar mixture
- X-gal is a lactose analogue
- X-gal is cleaved by lacZ to give a blue product
- Non-functional lacZ = white colony containing recombinant plasmid
- Functional lacZ = blue colony containing no insert
83
What are the features of expression vectors?
- Prokaryotic or Eukaryotic promoter just upstream of the MCS
- Promoter - very strong and inducible T7 or lac-based promoter (>30% total cellular protein)
- Often include an N- or C-terminal protein tags
- Often in between the tag and protein is a protease cleavage site
- Always have an Antibiotic resistance gene
Origin of replication
84
What is T7 promoter?
- Regulates gene expression of recombinant genes
- T7 bacteriophage promoter is an extremely strong promoter
- But it will not work in E. coli unless the strain carries T7 RNA polymerase
- Strains called BL21 (DE3) carry the T7 RNA polymerase under control by lacUV5 promoter
- Complicated induction system:
(week 4a slide 26 for system)
85
What is IPTG?
- IPTG is a lactose analogue
- IPTG is not metabolised!
- We could use lactose but it would be metabolized by the bacterium
(week 4a slide 26 for system)
IPTG is the favoured inducer for E. coli expression
BUT IT IS EXPENSIVE
86
What is a promoter?
Promotes transcription of the gene.
87
What are protein tags?
- Protein tags help purify, detect or solubilise a protein.
- Fused to the N- or C- terminus of the protein
- The reason why protein is added to a product that doesn't belong there is to help with the downstream processing
88
Types of Protein Tags
- GFP - very large tags, useful for microscopy and can be used with "GFP Traps" columns
- His - Very small tags, often hexa- or deca- histidines. Purified by Nickel column purification
- GST - Large tags, help protein solubilization. Glutathione column purification. GST tag usually cleaved off with an enzyme.
89
How would you remove the tags after protein purification?
You would use a specific protease that would cleave off the tag from the enzyme at a specific place.
Once the protein has finished purifying a protease would be added and it would cleave a specific place on the tail with the tag.
These proteases are specially engineered to make sure it does not cleave parts of the protein off and would cut the desired location.
90
Types of proteases used to cleave of the tags
- Enterokinase (EKT) – an intestinal enzyme that cleaves trypsin – cleaves the site DDDDK
- Factor Xa – cleaves the sequence IEGR
- Thrombin (Thr) – targets the site LVPR/GS
- Tobacco Etch Virus protease (TEV) – cleaves ENLYFQ- (TEV has the highest specificity)
91
Which strains of E.coli are used for cloning and expression?
Almost all E. coli strains used in the lab descended from 2 strains:
- K12 = cloning strains (DH5α)
- B-strain = expression strains (BL21)
92
What is BL21?
- BL21 is the most commonly used E. coli parent expression strain
- Deleted for the Lon protease which degrades unusual proteins
Newer variants of BL21:
- BL21 (DE3) – contains T7 RNA polymerase under control of LacUV5 promoter = IPTG inducible
- BL21 (DE3) Rosetta - contain 6 tRNA genes for rare codons
93
What is the 2-tier cell bank system?
Master Cell Bank and Working Cell Bank
94
What is a Master Cell Bank?
Master Cell Bank (MCB) is an aliquot of a single pool of cells that are generally prepared from a selected clone under defined conditions.
The MCB is used to derive all working cell banks (WCB).
95
What is a Working Cell Bank?
Working Cell Bank (WCB) is a prepared aliquot of homogenous suspension of cells from a master cell bank (MCB).
Usually 1 vial of MCB to make many WCBs.
96
How do you make these cell banks?
1 Grow up your cells containing the recombinant plasmids.
2. Aliquot them under sterile conditions.
3. Freeze at -196C = master cell bank (MCB)
4. Use 1 vial of MCB to make many WCBs
97
What happens if you freeze cells?
- Freezing kills cells due to formation of ice crystals
- Freezing, causes large increase in salt/solute concentration
- Even bacteria are sensitive to freezing
You can freeze them by:
- Glycerol (10%) is used as a cryoprotectant for bacteria
- DMSO (10-20%) is used for mammalian cells (enters cells more rapidly)
- STORE INDEFINITELY AT below -196°C
- Because it is also a nutrient, it is non-toxic
- Cells are thawed rapidly at 37C to minimise damage
98
How do you thaw a cell bank?
- Cells are thawed rapidly at 37C to minimise damage, i.e. water bath.
You can inoculate media after this.
99
What is the process of "Scaling Up"?
Scaling up allows you to gradually grow bigger batches of cell cultures, to eventually have a big enough culture to inoculate to a big fermenter for a larger volume of cells. This allows the fermentation of cells to be done in a shorter time.
100
What is a growth curve?
A growth curve is a representation of the number of living cells in a population over time. This is helpful to visualise the scaling-up process.
The growth curve has different phases
- Lag phase
- Exponential (log) phase
- Deceleration and Stationary phase
- Death phase
101
Scaling up system
Select a new single colony containing the vector --> Add to 5mL = starter culture --> Grow overnight (Stationary phase) --> Add to larger culture (200ml-100ml) --> Grow to OD600 of 0.4-0.8 (Exponential phase) --> Add IPTG (0.1-1mM) --> grow bacteria to stationary phase (OD600 = over 20)
102
What is the Lag Phase?
The lag phase is the initial phase of the growth curve. A small group of cells are placed in a nutrient-rich medium that allows them to synthesize proteins and other molecules necessary for replication.
The cells increase in size but no cell division happens in this phase.
103
What is the Exponential Phase?
The exponential (Log) phase is the time when cells are dividing by binary fission and doubling in numbers after each generation time.
Metabolic activity is high as DNA, RNA, cell wall components and other substances necessary for growth are generated for division.
It is in this phase that antibiotics and disinfectants are most effective as these substances typically target bacteria cell walls or the protein synthesis processes of DNA transcription and RNA translation.
104
What is the Deceleration and Stationary Phase?
The population growth experienced in the log phase begins to decline as the available nutrients become depleted and waste products start to accumulate.
Bacterial cell growth reaches a plateau, or stationary phase, where the number of dividing cells equals the number of dying cells. This results in no overall population growth. Under less favourable conditions, competition for nutrients increases and the cells become less metabolically active. Spore forming bacteria produce endospores in this phase and pathogenic bacteria begin to generate substances (virulence factors) that help them survive harsh conditions and consequently cause disease.
105
What is the Death Phase?
As nutrients become less available and waste products increase, the number of dying cells continues to rise. In the death phase, the number of living cells increases exponentially and population growth experiences a sharp decline.
As dying cells lyse or break open, they spill their contents into the environment making these nutrients available to other bacteria. This helps spore-producing bacteria to survive long enough for spore production. Spores are able to survive the harsh conditions of the death phase and become growing bacteria when placed in an environment that supports life.
106
What is Fermentation?
It is essentially the metabolism of glucose in the absence of oxygen.
Fermentation is used by microorganisms even when oxygen is present.
107
What is limited in liquid culture?
Oxygen limitation will always occur so fermentation metabolism will always occur.
Want the bacterial cultures to be well aerated.
108
What is a problem in fermentation?
There are many waste products that are produced which lead to bacteria dying quickly
109
What is the principle of fermentation?
- The main principle of fermentation is to derive energy from carbohydrates in the absence of oxygen.
- Glucose is first partially oxidized to pyruvate by glycolysis.
- Then pyruvate is converted to alcohol or acid along with the regeneration of NAD+ which can take part in glycolysis to produce more ATP.
- Fermentation yields only about 5% of the energy obtained by aerobic respiration.
110
Can you draw a flowchart of fermentation?
https://microbenotes.com/fermentation/
111
What controls growth?
Glucose limitation
112
Can you draw and label the basic fermentation bioreactor?
Check week 6 session 5 slide 12 picture
113
What are the other conditions needed to control the fermentation?
- Temperature (typically at 18C, 25C or 37C)
- pH (typically kept ~7.0)
- Oxygen (usually kept at 30%)
- Glucose (most other nutrients will be in excess)
- Mixing
114
What are the types of Bioreactor systems?
- Batch - is a process where all the substrate and nutrients are added at zero time or soon after inoculation takes place and the vessel is allowed under a controlled environment to proceed until maximum end product concentration is achieved.
- Continuous - is a process where ingredients are added throughout the process as per the need, and the products are removed as they are formed. The exponential phase is usually prolonged in this type because of the continuous addition of nutrients.
- Fed-batch - is a modified version of batch culture, it is a semi-open system. It involves adding nutrients systematically after the batch phase. It is more productive, yields better with controlled sequential additions of nutrients, enables higher cell densities and prolongs product synthesis.
115
Draw the different growth curves of the different types of bacterial cultures
Week 6 session 5 slide 16
116
What is the difference between Batch vs. Fed-batch vs. Continuous culture?
https://microbenotes.com/batch-vs-fed-batch-vs-continuous-culture/
117
Give an example of a real-life bioreactor in the Industry
- 50L of TB medium is prepared
- Placed in a 50L Bioreactor and sterilised. Antibiotic added.
- 1L starter culture grown in 3L flask. A frozen working cell bank of E. coli culture is added.
- Grown 12h at 37°C to known OD600
- Starter culture added to 50L Bioreactor; grown at 37°C
- Dissolved oxygen is maintained at 30% and pH is maintained at 6.8
- At OD600 = 40, IPTG is added at 0.1mM
- Growth continues for 1-2 h.
- Cells then placed on ice and harvested
118
What is the UPSTREAM PROCESSING workflow?
1. Sequence retrieval from Genbank
2. Design Primers and perform PCR (or Purchase the gene)
3. Insert into a cloning vector
4. Transform cloning strain. select colonies
5. Select colonies to purify and sequence plasmids
6. Sub-clone gene into an expression vector
7. Transform an expression strain
8. Pilot expression studies
9. Larger scale fermentation/bioreactors
119
What are HeLa cells?
They were the first immortal human cells, which came from a woman called Henrietta Lack's cancer
120
Why is HeLa cells important?
this cell line has contributed to many medical breakthroughs, from research on the effects of zero gravity in outer space and the development of polio and COVID-19 vaccines, to the study of leukemia, the AIDS virus and cancer worldwide.
121
What are the disadvantages of HeLa Cell Lines?
- they were cancerous and had many errors in the genome
- Another problem with HeLa is that it doesn't have a normal human karyotype (the number and appearance of chromosomes in a cell). Henrietta Lacks (and other humans) have 46 chromosomes (diploid or a set of 23 pairs), while the HeLa genome consists of 76 to 80 chromosomes (hypertriploid, including 22 to 25 abnormal chromosomes)
122
What are CHO cells?
Chinese hamster ovary cells or in short CHO cells are derived from the ovary of a small rodent called the Chinese hamster.
CHO cells are used in biological and medical research, primarily such as genomic and chromosome studies, toxicity assays and nutrition and gene expression.
123
What are the advantages of CHO cells?
- Similar post-translational modification to humans
- Easily scalable in large-scale cultures
- High yield
- ideal for GMP protein production
- Adaptable for protein-free, animal-free production, and serum-free culture conditions
- High stability and safety profiles
- Impressive amount of approved products
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What are the disadvantages of CHO cells?
- The productivity is typically 10 to 100 times smaller than if using a microbial host and its often difficult to predict how productive the host cells will be when it's scaled up in large bioreactors
- Since it's a mammalian cells its expensive
- it grows slowly
Biotherapeutics
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