The isolation of mutants which do not produce feedback

inhibitors or repressors (Auxotrophic mutation)

Dr.Gayathri.M, VIT
3/6/2017 1
stanbury
• Demain(1972) presented several blue prints of
hypothetical mutants producing intermediates
and end products
• All hypothetical mutants are auxotrophic
mutants
• Isolation of auxotrophic mutants result in
isolation of high yielding strains provided
mutation should occur at specific site
• So design a procedure to select relevant
auxotrophs

Dr.Gayathri.M, VIT
3/6/2017 2
stanbury
Most commonly used technique to isolate
auxotrophic mutant
 Use of some enrichment culture
 Use of a technique to visually identify the mutants

Replica plating
Davis method
Sandwich technique
Dr.Gayathri.M, VIT
3/6/2017 3
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 Davis method

Survivors of mutation

Cultured on complete medium

Harvested by centrifugation

Washed and resuspended in minimal medium plus penicillin

Only growing prototrophic cells were susceptible to the penicillin-non

growing auxotrophs survive

Cells harvested by centrifugation-washed and resuspended in complete

medium

Dr.Gayathri.M, VIT
3/6/2017 4
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 Dr.Gayathri.M, VIT
3/6/2017 5
stanbury
 Filtration enrichment method
Liquid minimal medium + mutated spores

Shaken for few hours

Prototrophs will germinate

Auxotrophs will not germinate
 Replica plating

Dr.Gayathri.M, VIT
3/6/2017 7
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 Sandwich technique
Survivors of mutation

Seeded in a layer of minimal agar in a petridish

Covered with a sterile minimal agar

Plate- incubated 1-2days

Colonies developed marked on the base of the plate

Layer of supplemented agar is poured over the surface

Colonies which then appear after further incubation

period are auxotrophic
 Examples of use of auxotrophs for the
production of primary metabolites 1

Dr.Gayathri.M, VIT
3/6/2017 10
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 Examples of use of auxotrophs for the production of
primary metabolites 2

Dr.Gayathri.M, VIT
3/6/2017 11
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 Examples of use of auxotrophs for the
production of primary metabolites 3

Dr.Gayathri.M, VIT
3/6/2017 12
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 Examples of use of auxotrophs for the
production of primary metabolites 4

Dr.Gayathri.M, VIT
3/6/2017 13
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 The isolation of mutants that do not recognise
the presence of inhibitors and repressors
1) IsolationOB of analogue resistant mutants
2) The isolation of revertants.

Dr.Gayathri.M, VIT
3/6/2017 14
stanbury
• analogue resistant mutants may be expected
to overproduce the end product to which the
analogue is analogous provided that:
(i) The toxicity of the analogue is due to its
mimicing the control properties of the natural
product.
(ii) The site of resistance of the resistant mutant
is the site of control by the end product.

Dr.Gayathri.M, VIT
3/6/2017 15
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Gradient plate technique for the isolation of
analogue-resistant mutants

Dr.Gayathri.M, VIT
3/6/2017 16
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 The isolation of induced mutants producing improved yields of
secondary metabolites where directed selection is difficult to apply

Dr.Gayathri.M, VIT
3/6/2017 17
stanbury