Molecular Microbiology Flashcards
What Is Molecular Microbiology?
Branch of Microbiology devoted to studying the molecular basis of physiological processes that happen in micro-organisms
Enables manipulation of micro-organism to our means
Why do we manipulate micro-organisms to our means?
To produce commercially relevant products
e.g. Surf
Produce medicines/vaccines/therapeutics
e.g. Insulin, Hepatitis B Vaccine
Production In Large Quantities + Safe
Misused: Biological Warfare (Aum Shinrikyo)
How are micro-organisms manipulated?
- Cloning (Replicate DNA fragments in host)
- Engineer plasmids
- Transformation
- Large Scale Culture
- Harvesting
- Product Purification
- Testing (Assay)
What happens in the Cloning Stage?
Main: Gene of interest inserted into plasmid
- PCR Amplification:
- Primers designed with restriction enzyme
sites to amplify target gene from DNA.
- Primers designed with restriction enzyme
- Restriction Digestion:
- Cut PCR product + Plasmid Vector with the
same enzyme - Creates complementary “sticky ends” for
precise ligation.
- Cut PCR product + Plasmid Vector with the
- Ligation:
- DNA ligase used to glue gene into plasmid’s
Multiple Cloning Site (MCS).
- DNA ligase used to glue gene into plasmid’s
Result: Recombinant Plasmid (plasmid + gene).
What happens In the Engineer Plasmid Stage?
Aim: Design a functional plasmid in the host
- Select a Plasmid Backbone
Choose suitable plasmid vector for host (e.g.,
E. coli) + purpose (e.g., protein production) - Design Primers with Restriction Sites
Design PCR primers to amplify target geneAdd restriction enzyme sites to the primer
ends - PCR Amplify the Target Gene
Use PCR to copy insulin gene from human
DNA
** not too sure about this one **
What Happens In the Transformation Stage?
Aim: Ensure Bacterial Cells Take Up Plasmid
- Use heat shock/electric shock
- Increases membrane competency making it
porous, allowing plasmid uptake - Allow Bacteria to repair in nutrient broth
- Plate bacteria on agar with ampicillin
- Plasmid-containing bacteria survive
What Happens In The Culture Stage?
Aim: Wide-range bacterial growth to produce
protein
- Grow bacteria in giant vats (Batch/Fed-Batch
Fermenters) - Induction: Adding IPTG to activate promoter
and start protein production - Adjusting pH, Oxygen, Temperature +
nutrients for optimal growth
What Happens In The Harvesting Stage?
Goal: Collect Bacteria + Extract Protein.
- Centrifugation: Spin down bacteria into
pellet. - Break Open Bacteria using Sonication
(Sound Waves)/Lysozymes/Detergents - Remove Debris: Centrifuge to separate
cell fragments from protein.
What Happens In The Purification Stage?
Aim: Isolate protein from other bacterial
components
- Affinity Chromatography: Using a His-Tag to
bind to nickel columns - Ion exchange/Size Exclusion: Further refines
protein - Dialysis to remove salts/small molecules
What Happens In The Testing Stage?
Goal: Ensure the protein is pure, active, and
safe
- SDS-PAGE: Gel electrophoresis to check
protein size + purity. - Western Blot: To identify protein using Ab
- Bioassay: Test function in mice (e.g., Insulin’s ability to lower blood sugar)
- Sterility Tests: To ensure protein is not
contaminated
What is Botulinum Toxin?
- Spore forming Gram positive obligate
anaerobe - Disease results from absorption of toxin into
blood stream - Causes flaccid paralysis (prevents ACH
release)
Case Studies:
Aum Shinrikyo Cult
- Cultured C. botulinum from soil
- Released toxin in Tokyo + US military
installations
Iraq:
- Admitted producing 19,000L of Botulinum
- Anthrax spores: For use in aerial bombs
warheads
What Is Anthrax Toxin?
- Toxin Produced by Gram+ Bacillus
anthracis - Can produce Highly resistant Endospores
- Can be aerosolized
- Spores remain viable for years
3 forms: cutaneous, inhalation + gastrointestinal
Death caused by:
- septicemia
- hemorrhagic meningitis
Anthrax toxins:
PA (protective antigen) - B moiety (vaccine)
EF (oedema factor)
LF (lethal factor) A moiety
