F8 Evolution inom biotekniken, utveckling av industriella cellfabriker

Question 1

Strain improvement (stamutveckling)

Answer 1

The science and technology of manipulating and improving

microbial strains in order to enhance their metabolic capacitates for biotechnological application

Question 2

CSI = Classical Strain Improvement

Answer 2

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

Question 3

New micro-organisms can be found & developed by:

Answer 3

• Isolation and screening
• Classical strain improvement
• Genetic engineering: introduction of targetted genetic alterations

Question 4

improvement of microorganisms, the process:

Answer 4

isolation -> screening -> improvement -> preservation

Question 5

How to get a strain?

Answer 5

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

Question 6

Enrichment (anrikning, berikning) during isolation improves…

Answer 6

… the likelihood of obtaining organisms with the desirable property
• Use conditions favourable for the desired organism
• Use conditions unfavourable for the non-desirable types

Question 7

Enrichment on Solid media

Answer 7

• 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

Question 8

elimination of unwanted microorganisms

Answer 8

antibiotics
cycloheximide
Pasteurisation
pH
temperature

Question 9

classical screening protocol

Answer 9

Production detected by inhibition of the test organism(s).

Grow isolate in liquid culture and test cell-free broth for activity.

Question 10

phenotype screening

Answer 10

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.

Question 11

Development of screens , ex

Answer 11

• Test organisms with increased sensistivities/resistances
• Genetic biosensors or reporter gene assays
• Molecular probes
• Immunologically based assays
• Robotic automation leading to high throughput screening

Question 12

Classical methods of strain improvement:

Answer 12

• 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.

Question 13

CLASSICAL STRAIN IMPROVEMENT, + och -

Answer 13

+ 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

Question 14

genetic variation can be enhanced by:

Answer 14

mutation
breeding/mating
interspecies hybridization/forced mating

Question 15

aim of strain improvement:

Answer 15

• 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

Question 16

type of mutations:

Answer 16

spontaneous
induced
- chemically
- physically
engineered

Question 17

mutagenesis

Answer 17

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.

Question 18

in vivo vs in vitro

Answer 18

in vivo: treating the whole organism

in vitro: directed to one gen - one protein

Question 19

types of in vivo mutagenesis:

Answer 19

chemical
physical
insertional (disruption of random genes)

Question 20

useful mutations:

Answer 20

• Strains not producing feedback inhibitors
• Strains not producing feedback repressors
• Auxotrophs
• Do not recognise inhibitors or repressors
• Resistant mutants
• Revertant mutants

Question 21

Evolutionary engineering, ALE

Answer 21

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)

Question 22

preservation

Answer 22

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