week 5 Flashcards
(30 cards)
examples of recombinant proteins: therapeutic proteins
- hormones
- blood factors
- insulin
- interferons
- tissue plasminogen activator
- vaccines
- antibodies
examples of recombinant proteins: diagnostic protein
- enzymes
- antibodies
- biosensors
examples of commercial enzymes: industrial and ‘biotech’
industrial:
- food/textile production
‘biotech’ enzymes
- restriction enzymes
- thermostable polymerases
- ligases
considerations of purifying from natural sources
- availability of starting material
- abundance of protein within cell/tissue
- contamination/ infection
- ethical considerations
- purification considerations
- cost
describe recombinant protein production
- can use non-pathogenic host that is easily cultured
- genetic engineering allows high expression of required proteins
- much reduced contamination and infection risk
- production may be more ethically acceptable procedure
describe the human growth hormone (hGH)
- naturally produced in minute quantities in pituitary gland
- required for growth and development
- purified hGH was used in treatment of growth deficiencies
- animal hormone incompatible
- hGH was isolated from human cadavers
- major HIV and prion issues - natural hGH is now banned
- Genentec started to produce it and was approved in 1985
- human growth hormone now mainly produced in E.Coli ( a very simple protein)
- single polypeptide with just 2 disulphide bonds. no cofactors required for activity, not toxic to host cell, stable and soluble
what principles do gene cloning use
- extraction of nucleic acids
- manipulation of DNA/RNA - type II restriction endonucleases, DNA ligases
- electrophoresis
- PCR
- reverse transcription
describe the ligation of DNA
- EcoR1 produces fragments with ‘sticky’ ends
- can be joined with T4 DNA ligase
how was the first recombinant DNA produced
- EcoR1 to chop up both sets of extracted DNA
- mixed the resulting fragments sets together
- used DNA ligases to join random DNA fragments together - many different combinations possible
describe the clonal growth of bacteria
- one bacteria produces two copies of itself
- all bacterial DNA is copied
- individual clones amplify into colonies of bacteria
- cloning can refer to the multiple copies of bacteria cells or sequences of DNA produced during clonal growth
- usually have specific or special functions: antibiotic resistance, nitrogen fixation, virulence
how are plasmids transferred between bacteria
- plasmids can be transferred between bacteria: horizontal gene transfer -
1. transformation - uptake of free DNA from environment
2. conjugation - direct transfer of DNA between bacteria
3. transduction - transfer of DNA through viruses (phages) - basis of transferred antibiotic resistance in bacteria - normally low (1%) transmission of plasmids between bacteria - can be increased (made competent) via heatshock, electroporation or certain chemicals (e.g. CaCl2)
what was the first ‘gene cloning’ experiment
- insertion of frog DNA into bacterial plasmids to create the first transgenic organism
1. digest both sets of DNA with EcoR1
2. combine the two DNA fragment sets and ligate
3. transfer into bacteria
4. analyse clones of bacteria (colonies) for frog DNA
describe random recombinant DNA outcomes
- plasmid without ability to replicate in bacteria
- plasmid without antibiotic resistance
- plasmid without frog DNA
describe modern plasmid cloning vector properties
- origin of replication
- resistance/selection/marker gene
- multiple cloning site - with flanking promoters/regulators
- a marker to select for insertion of fragment
what are multiple cloning (polylinker) sites
- area with many different restriction enzyme cutting sites - allow insertion of DNA
- usually one site for each RE in the MCS
- sometimes two of certain RE (one outside MCS)
what are resistance/selection/marker genes
- included in plasmid vectors to help identify and select cells that have taken up the correct plasmid
- e.g. antibiotic resistance genes. Only bacteria with the plasmid can grow on antibiotic-containing media
- e.g.lacZ gene: Encodes β-galactosidase; used in blue-white screening.
White colonies = plasmid with insert (gene disrupted).
Blue colonies = plasmid without insert (gene intact). - e.g. Fluorescent proteins
Cells expressing these proteins glow under UV light, indicating successful transformation or gene expression. - Often Combined in Vectors
describe alpha-complementation
- A mutation in the lacZ gene leads to the production of a β-galactosidase enzyme that lacks the N-terminal portion. This larger, mutant fragment is called the omega fragment (ω fragment). By itself, this fragment is inactive and cannot hydrolyze substrates like X-gal.
- The missing N-terminal portion of β-galactosidase can be supplied separately as a small α-peptide, usually encoded by a plasmid. The α-peptide alone is also inactive, but when it is expressed in a host cell that carries the omega fragment, it can bind to the omega fragment and restore enzymatic activity through complementation.
- When the α-peptide (from the plasmid) and the ω fragment (from the host genome) are co-expressed in the same cell, they associate to form an active β-galactosidase enzyme. This restoration of function allows the cell to metabolize substrates like X-gal, producing a blue color, which is a key feature of blue-white screening in cloning experiments.
describe alternative cloning strategies
Traditional Cloning (RE + Ligase)
Involves cutting both the vector and insert with restriction enzymes, then joining them with DNA ligase.
Often relies on multiple cloning sites (MCSs) containing various RE recognition sequences.
Limitations:
Difficult to control:
Insert orientation
Reading frame (risk of frameshifts)
Avoiding RE sites that occur naturally within the gene or vector.
Requires careful primer and vector design to include proper restriction sites.
Modern Cloning Kits (Patent-Based Methods)
Many kits are based on proprietary, often restriction-enzyme-free technologies, designed to make cloning faster, more accurate, and less error-prone. These include:
Gateway Cloning Based on recombination (site-specific), allows easy transfer of inserts between vectors.
TOPO Cloning Uses topoisomerase to ligate DNA with specific overhangs. Fast and efficient.
describe TA cloning
- many Taq polymerases leave a 3’ A-overhang and we can use this to clone - high-fidelity or proofreading versions do not
- basic TA cloning involves ligation of PCR products into a vector that has T-overhangs
Plasmid vector properties:
LacZ Marker: Allows blue-white screening.
Insertion of the PCR product disrupts the lacZ gene.
White colonies = successful clones (insert present).
Blue colonies = empty vector (no insert).
Antibiotic Resistance Gene: Enables selection of transformed cells.
Only bacteria with the plasmid will grow on antibiotic-containing media.
Origin of Replication (ori): Allows the plasmid to replicate independently in host cells.
- 50% of clones are anti-sense
describe TOPO cloning
The virus topoisomerase I enzyme is used to cleave and ligate DNA fragments, making it a one-step cloning process
bypasses the need for restriction enzymes or ligases
describe general procedure
Obtain and alter gene of interest:
- RE digestion of DNA
If natural RE sites are not available or suitable, design primers that include restriction sites at their 5′ ends.
This allows you to amplify the gene by PCR and then cut the product with matching REs.
c. Direct Cloning of PCR Products
Alternatively, directly clone PCR-amplified products (e.g., via TA cloning or recombinational cloning) without RE digestion.
Perform ligation with plasmid vector, transform bacteria (as in the practical!)
Select bacterial colonies via antibiotic resistance
- Extract plasmid DNA
describe obtaining and altering the gene of interest
a. Restriction Enzyme (RE) Digestion of DNA
Restriction enzymes are like molecular scissors that cut DNA at specific sequences.
You use them to cut your gene of interest and your plasmid vector in a way that both pieces will match and can be joined later.
b. RE Sites with Primers (PCR Method)
If you’re amplifying the gene by PCR, you can add restriction sites to the ends of your primers.
This way, once PCR is done, you can use those restriction enzymes to cut the PCR product — making it ready for insertion into the plasmid.
c. Direct Cloning of PCR Products (without REs)
Some modern kits let you insert PCR products directly into vectors using special enzymes (like TA cloning or Gibson Assembly).
This avoids the need for restriction digestion entirely.
describe ligation with plasmid vector
Once both the plasmid and the gene have matching ends (from RE digestion or other methods), you mix them with DNA ligase.
Ligase glues the gene into the plasmid, forming a recombinant DNA molecule.
describe transforming the bacteria
You introduce the plasmid into competent bacteria (usually E. coli) using:
Heat shock or
Electroporation
The bacteria take up the plasmid and start replicating it.
