F8 Evolution inom biotekniken, utveckling av industriella cellfabriker Flashcards
Strain improvement (stamutveckling)
The science and technology of manipulating and improving
microbial strains in order to enhance their metabolic capacitates for biotechnological application
CSI = Classical Strain Improvement
Alla traditionella, icke-GMO tekniker för att förbättra en stam
• Stamkorsning, ALE, mutagenes etc.
• Fortfarande idag den mest använda stamutvecklingstekniken!
• ”Kräver mindre kunskap, mer tid & tur”
• Effektiv t.ex. för att öka robusthet, tolerans substratspektra, ofta mindre effektiv för att öka produktspektra
New micro-organisms can be found & developed by:
- Isolation and screening
- Classical strain improvement
- Genetic engineering: introduction of targetted genetic alterations
improvement of microorganisms, the process:
isolation -> screening -> improvement -> preservation
How to get a strain?
Strain collections • Few microorganisms available • Well known • Cheap • The best strains are not available
Isolation from nature • Great variety of microorganisms • Expensive • Time consuming • Isolation locus can enhance finding strains with special properties
Enrichment (anrikning, berikning) during isolation improves…
… the likelihood of obtaining organisms with the desirable property
• Use conditions favourable for the desired organism
• Use conditions unfavourable for the non-desirable types
Enrichment on Solid media
- Used for selection of enzyme or antibiotic producers
- Selective medium that encourages growth of productive types
- May also be used to isolate organisms for bioremediation
elimination of unwanted microorganisms
antibiotics cycloheximide Pasteurisation pH temperature
classical screening protocol
Production detected by inhibition of the test organism(s).
Grow isolate in liquid culture and test cell-free broth for activity.
phenotype screening
Phenotypic screening is a type of screening used in biological research and drug discovery to identify substances (such as small molecules, peptides, or RNAi) that alter the phenotype of a cell or an organism in a desired manner.
Development of screens , ex
- Test organisms with increased sensistivities/resistances
- Genetic biosensors or reporter gene assays
- Molecular probes
- Immunologically based assays
- Robotic automation leading to high throughput screening
Classical methods of strain improvement:
• Mutagenesis • Recombination/Breeding/Mating • Protoplast fusion • Evolutionary engineering, ALE →The methods are generally used in combination and in addition to metabolic engineering to develop high producing strains.
CLASSICAL STRAIN IMPROVEMENT, + och -
+ Useful for complex, multifactorial traits
+ Useful for non-model organisms (lack of knowledge on genomics and physiology and/or genome editing tools)
+ Developed strains are not considered as GMO (no negative associations, less rigid regulations for use and disposal)
- Time consuming, labour intensive
- The microorganism must have the prerequisite genes for developing a specific trait
genetic variation can be enhanced by:
mutation
breeding/mating
interspecies hybridization/forced mating
aim of strain improvement:
- Increased production (productivity, titre) and yield
- Increased robustness and tolerance
- Rapid growth, short lag phase
- Genetic stability
- Ability to use cheap substrates & reduced need for dietary supplements
- Elimination of byproducts
- Lowered oxygen demand, higher temperature tolerance
type of mutations:
spontaneous induced - chemically - physically engineered
mutagenesis
Mutagenesis is a process by which the genetic information of an organism is changed by the production of a mutation. It may occur spontaneously in nature, or as a result of exposure to mutagens. It can also be achieved experimentally using laboratory procedures.
in vivo vs in vitro
in vivo: treating the whole organism
in vitro: directed to one gen - one protein
types of in vivo mutagenesis:
chemical
physical
insertional (disruption of random genes)
useful mutations:
- Strains not producing feedback inhibitors
- Strains not producing feedback repressors
- Auxotrophs
- Do not recognise inhibitors or repressors
- Resistant mutants
- Revertant mutants
Evolutionary engineering, ALE
ALE= adaptive laboratory evolution
Experimental improvement of cellular properties exploiting genetic variation in large microbial populations and selection through natural evolution under a controlled selection pressure for predetermined functions.
- Based on natural evolution
- No structural info required
- No understanding of mechanisms required
1) Generation of genetic diversity through a selection pressure
2) Identification of successful variants (screening)
preservation
Effective preservation is important due to high commercial value of strains isolated.
Goal is to preserve desirable characteristics.
• Eliminate possibility for genetic alteration
• Prevent contamination
• Maintain viability
