There is a Group 2 organism present in production. Which containment measures must be in place in the factory before any work can be authorised?
SPEECBFS
i) Viable micro-organisms should be contained in a system which physically separates the process from the environment (closed system).
ii) Exhaust gases from the closed system should be treated so as to minimize release.
iii) Sample collection, addition of materials to a closed system and transfer of viable micro-organisms to another closed system, should be performed so as to minimize release.
iv) Bulk culture fluids should not be removed from the closed system unless the viable micro-organisms have been inactivated by validated means.
v) Seals should be designed so as to minimize release.
vi) Personnel should wear protective clothing (work clothing).
vii) Decontamination and washing facilities should be provided for personnel.
viii) The effluent before final discharge must be inactivated by validated means.
Define 4 hazard groups used to classify biological agents based on their ability to infect healthy humans.
ULCS
Group 1 – unlikely to cause human disease.
Group 2 – can cause human disease and may be a hazard to employees; it is unlikely to spread to the community and there is usually effective prophylaxis or treatment available.
Group 3 – can cause severe human disease and may be a serious hazard to employees it may spread to the community, but there is usually effective prophylaxis or treatment available.
Group 4 – causes severe human disease and is a serious hazard to employees it is likely to spread to the community and there is usually no effective prophylaxis or treatment available.
Describe 2 possible anti-biofouling strategies.
CAM
- Chemical agents - include a wide variety of antimicrobial agents, such as biocides and anti biotics and also chemical cleaners that assist in the removal of the biofilm.
- Mechanical Cleaning - Arguably the most effective anti biofouling strategy is mechanical cleaning this can be accomplished by scraping, brushing or hydraulic flushing of the biofilm.
Describe the 6 stages in biofilm formationand state their respective time frames.
RIGEAS
- Reversible adsorption of bacteria to a surface (seconds)
- Irreversible attachment of bacteria, Quorum sensing occurs (Minutes-seconds)
- Growth and division of bacteria within the slimy matrix. (Hours/days)
- Exopolymer production and biofilm formation (Hours/days)
- Attachment of other micro-organisms to the bio-film (Days/Months)
- Single cells can escape from the outer surfaces of the biofilm to start a new biofilm (Days/months)
What are the key advantages of using immobilised enzymes as opposed to free enzymes?
RSC
- Makes the recovery and reuse of the enzymes far more straight forward. This is particularly important where an expensive enzyme is in use.
- Immobilisation offers the enzymes greater stability in variable of extreme conditions i.e. temperature and PH. increased stability helps improve the efficiency of the entire process.
- Contamination is also prevented by immobilisation. The enzymes are not free to contaminate the final product. This drastically reduces the cost as purification methods are not needed.
What are the parameters of the methods of enzyme immobilisation
PCB EAR MLM
- Preparation
- Cost
- Binding force
- Enzyme leakage
- Applicability
- Running Problems
- Matrix effects
- Large diffusion barriers
- Microbial protection
enzyme immobilisation method : Adsorption
SLVY WHY NN
Immobilisation through adsorption involves the enzyme being adsorbed on to some sort of structure.
- Preparation : Simple
- Cost : Low
- Binding force : Variable
- Enzyme leakage : Yes
- Applicability : Wide
- Running Problems : High
- Matrix effects : Yes
- Large diffusion barriers : No
- Microbial protection : No
enzyme immobilisation method : Covalent coupling
DHS NSL YNN
Enzymes can be covalently attached to a solid support. This involves the forming of a stable covalent bond between the enzyme and the matrix, providing a more robust attachment.
- Preparation : Difficult
- Cost : High
- Binding force : Strong
- Enzyme leakage : No
- Applicability : Selective
- Running Problems : Low
- Matrix effects : Yes
- Large diffusion barriers : No
- Microbial protection : No
How could you exploit bacterial plasmid vectors to produce large amounts of recombinant proteins?
CSO EGR C
- Plasmids are circular, double-stranded DNA (dsDNA) molecules.
- Separate from a cell’s chromosomal DNA.
- Occur naturally in bacteria and Eukaryotic cells.
- Exist in a parasitic or symbiotic relationship with their host cell, and replicate autonomously within the host.
- Plasmids can carry genes conferring useful properties e.g. antibiotic or heavy-metal resistance.
- Plasmids contain proteins required for replication – other proteins provided by host, and other parts of the plasmid can be deleted and foreign DNA can then be inserted.
- Plasmid is cut with same restriction enzymes as foreign DNA to give sticky ends. These are joined using DNA ligase and the recombinant plasmid inserted in to E.coli (electroporation) which is then grown to amplify product.
Advantages and disadvantages of using E.coli as expression hosts.
Advantages:
- Extensive use industry
- Well understood
- Easy to grow in high densities (>100 mg/L)
- Fast growth – doubling time ~ 20min
- Simple nutritional requirements – defined simple media
Disadvantages:
- Can’t do post-translational modifications (glycosylation, acylation, isoprenylation).
- Possess pyrogens and endotoxins (gram-ve cell wall) – must be totally eliminated from proteins to be injected in animals or humans.
Advantages and disadvantages of using B.Subtillis as expression hosts.
Advantages:
- Well-known Gram +ve bacteria
- Significant industrial usage
- GRAS (Generally Regarded As Safe)
- Low conc. pyrogens.
- Can be used for production of proteins for food industry.
Disadvantages:
- Lower yields than gram –ve bacteria.
- Responds to stress by sporulating, reduces recombinant protein concentration.
Advantages and disadvantages of using Mammalian cells as expression hosts.
Advantages:
- Used extensively for production of virus vaccines, the synthesis of endogenous proteins e.g. interferon and monoclonal antibodies.
- Through increasing knowledge of cell cycle control can increasingly manipulate cell division and cell longevity.
Disadvantages:
- Complex to manipulate compared with bacteria.
- Cells are fragile.
- Complex nutritional requirements (e.g. hormones, growth factors).
What are the seven principles of HACCP?
CIC MCVR
Conduct hazard analysis
Identify critical control points
Establish critical limits
Establish monitoring procedures
Establish corrective actions
Establish verification procedures
Establish record system
What are the (6) major interactions between two organisms in a mixed culture?
Neutralism
Competition
Commensalism
Amensalism
Mutualism
Prey - Predator
What is the definition of neutralism (mixed culture)
Neutralism is an interaction where neither population is affected by the presence of the other Each species consumes different limiting substrates and neither species is affected by the end products of the other.
What is the definition of competition (mixed culture)
Competition is an indirect interaction between two populations that has negative effects on both In competition, each population competes for the same substrate.
What is the definition of commensalism (mixed culture)
Commensalism is an interaction in which one population is positively affected by the presence of the other However, the second population is not affected by the presence of the first population.
What is the definition of amensalism (mixed culture)
Amensalism is the opposite of commensalism In amensalism population A is negatively affected by the presence of the other population B However, population B is not affected by the presence of population A.
What is the definition of mutualism (mixed culture)
Mutualism is an interaction where the presence of each population has a positive effect on the other This interaction is essential to the survival of both species.
What is the definition of predation (mixed culture)
Predation involves the ingestion of prey by the predator organism In this interaction, one population benefits at the expense of the other In general, predators tend to be larger than their prey, and consume many prey during their lifetimes.
