This document discusses methods for strain development in biotechnology. There are two main methods: mutation and genetic recombination. Mutation involves inducing changes to an organism's DNA through spontaneous or induced means like radiation or chemicals. This can improve strain performance. Selection of mutants involves screening colonies for desired traits like increased product formation. Isolation techniques identify mutants with traits like antibiotic resistance or an inability to synthesize certain factors (auxotrophs). Overall, strain improvement aims to genetically modify organisms for enhanced metabolic functions in biotechnology applications.

1. Methods of strain development:
By
Dr. Harinatha Reddy A
ASSISTANT PROFESSOR
Department of Biotechnology
Bangalore

2. What is strain development?
 Improving the genetics of an organism in order to
enhance their metabolic capacities for biotechnological
applications.
 Genetic improvements made new microbial strains to
increase product formation.

3. What are the ideal features of an improved strain?
 Shorter time of fermentation
 Capable of metabolizing low-cost substrates
 Reduced O2 demand
 Decreased foam formation

4. What are the ideal features of an improved strain?
Non-production of undesirable compounds
Tolerance to high concentrations of carbon or nitrogen
sources (Resistant of changes in pH and TMP)
Resistant to infections of bacteriophages.
Improved strains can produce one metabolite as the main
product.

5. There are two different methods for improvement of strains:
 Mutation.
 Genetic recombination.

6. Mutation
 Any change that occurs in the DNA of a gene is referred to as
mutation.
 Mutation cause an improvement of genotypic and phenotypic
performances of microbes.
Mutation.
Spontaneous
mutation
Induced
mutation
Site directed
mutation

7. Spontaneous mutation:
 The spontaneous mutations occur at a very low frequency, and
usually are not suitable for industrial application.

8.  The rate of mutation can be increased by various
factors and agents called mutagens.
 Ionizing radiations (e.g. X-rays, gamma rays).
 Non-ionizing radiations (e.g. ultraviolet radiations).
 Various chemicals (e.g. Benzene, Ethidium
bromide, Nitrous oxide, Hydroxylamine,
nitrosoguanidine)
Induced mutation:

9. MUTAGEN MUTATION
INDUCED
IMPACT ON
DNA
RELATIVE
EFFECT
Ionizing
Radiations-X
Rays,gamma
rays
Single or double
strand bearkage
of DNA
Deletion/structura
l changes
high
UV
rays,chemicals
Pyrimidine
dymerisation
Trnsversion,deleti
on,frameshift
transitions from
GC AT
Medium
Hydroxylamine(NH2
OH
Deamination of
cytosine
GC AT
transitions
low
N-Methyl –N’-
Nitro N-
Nitrosoguanidine
Methylation of
bases and high
pH
GC AT
transitions
high
Nitrous
acid(HNO2)
Deamination of
A,C & G
MediumBidirectional
transitions,deletio
n,AT GC/GC
AT

10.  Among physical agents, UV has been widely used in
industry to induce mutations.
 It does not require much equipment, and is relatively
effective.
 Chemical methods other than NTG (nitrosoguanidine) are
probably best used in combination with UV.

11. Site-directed mutagenesis :
 Site-directed mutagenesis is a molecular biology method that is
used to make specific and intentional changes to the DNA sequence.
 In Site-directed mutagenesis synthetic double- stranded
oligonucleotide contain desired mutant DNA sequence is inserted
in to the plasmid vector.
 The resulting recombinant Plasmid inserted into the bacterial cells.

13. Selection of Mutants:
 Selection and isolation of the appropriate mutant strains is very
important for their industrial use.
 The best method for selection of mutants is Random screening:

14. Random screening:
In the first step
bacterial cells Exposed
to Uv rays: After incubation colonies were
formed
Colonies are randomly selected and
checked for their ability to produce the
desired industrial product.

15. Random screening:
 The mutated strains are randomly selected and checked for their
ability to produce the desired industrial product.
 The strains with maximum yield can be selected.
 Many a times, this is the only way to find the right strain of
mutants developed.

16. Disadvantage:
 Non specific.
 A very large number of colonies must be tested.
 Random screening is costly and too long procedure.

17. Isolation of antibiotic resistant strains:
 The mutated strains are grown on a selective medium
containing an antibiotic.
 The wild strains are killed while the mutant strains with
antibiotic resistance can grow.

18. Isolation of antibiotic resistant strains:
 Gradient Plate Technique:
 Replica Plating Method:

19. Gradient Plate Technique: (Preparation of Gradient plate)
media without antibiotics
(with antibiotics)

20.  An excellent way to determine the ability of organisms to
produce mutants that are resistant to antibiotic is to grow them on a
gradient plate of a particular antibiotic.
 The gradient plate consists of two wedges like layers of media: a
bottom layer of plain nutrient agar and top layer of antibiotic with
nutrient agar.
 The antibiotic in the top layer, diffuse into the bottom layer
producing a gradient of antibiotic concentration from low to high.

21.  Bacterial culture will be spread over the surface of the plate and
incubated for 24 to 72 hours.
 After incubation colonies will appear on both the gradients.
 The colonies develop in the high concentration are resistant to
antibiotic , and are considered as antibiotic resistant mutants.

22. Replica Plating Method:
Master plate
without
Antibiotic
Media without
antibiotic
Replica plates:

23. Replica Plating Method:
Master plate
without
Antibiotic
Replica plates:

24.  A simple velveteen covered colony transfer device is used to transfer
the colonies from master plate to nutrient agar medium supplemented
with or without a particular antibiotic.
 The velvet picking up the bacterial cells from the surface of this
master plate.
 The velvet with its attached microbes is then touched to the surface
of a sterile agar plate, inoculating it.


25.  In this manner, microbes can be repeatedly stamped onto media of
differing composition.
 By comparing the presence of colonies following incubation we can
indirectly determine the antibiotic resistant colonies by their presence
in antibiotic contain medium.


26. Isolation of auxotrophs from prototrophs:

27. Isolation of auxotrophs from prototrophs:
 Auxotrophs: An organism (bacteria or fungi), that has lost the ability
to synthesize growth factors required for its growth and metabolism as
the result of mutational changes (Unable to grow on Minimal media
without amino acids or growth factor: able to grow only in
complete media).
 Prototrophs an organism or cell capable of synthesizing all its
metabolites from inorganic material, requiring no organic nutrients
(Unable to grow on Minimal media).

28.  An auxotrophic mutant characterized by defect in one of the
biosynthetic pathway.
 For example His Mutants of E.coli require Histidine for their
growth.
 The isolation of such mutants can be done by growing them on
complete medium it contain Histidine.

29. Replica Plating Method:

30. Replica Plating Method:
 A simple velveteen covered colony transfer device is used to
transfer the colonies from master plate to nutrient agar medium
supplemented with or without a particular antibiotic.
 The velvet picking up the bacterial cells from the surface of this
master plate.
 The velvet with its attached microbes is then touched to the surface
of a sterile agar plate, inoculating it.

31.  A colony that develops on a complete medium fail to develop on a
minimal medium that lacks a specific growth factor.
 The microbes that do not grow on the minimal medium represent
auxotrophic strains.

32. Isolation of Antimetabolite strains:

33.  Mutants Resistant to Feedback Inhibition:
 In many microbes, the end products of metabolism, when
accumulated in the microbial cell, inhibit the enzyme activities
of many pathways.
 Mutation in the structural gene, however, can alter the
enzyme binding site and prevent these inhibitory effects.

34.  Antimetabolites are used to screening of mutant strains.
 Antimetabolites which have structural similarities with
metabolites can block the normal metabolic pathways and kill the
cells.

35.  Supplementing the screening medium with these Antimetabolites
selects only mutants with altered enzyme structure and desensitized
to inhibition effects to grow.
 The mutant strains resistant to antimetabolites can be selected
for industrial purposes.
 Mutations at the operator site or other regulatory sites on the gene
relieve such end-product repression and allow overproduction of
the biosynthetic enzyme.

36. Best example for Antimetabolites:
Natural metabolites Antimetabolites
Amino acid Proline 3,4 Dehydroproline
Phenylalanine p-Fluorophenylalanine
Vitamins Niacin 3-Acetyl pyridine
Nitrogenous
bases
Uracil 5-fluorouracil

37. For example:
 When 3,4 Dehydroproline present in medium it block the synthesis
of proline and Kill bacterial cells.
 But the mutant antimetabolic strains will grow only in this
medium.
 Antimetabolic strains resistant to feed back inhibition.
 Such Antimetabolite resistance generally used for the over
production of primary and secondary metabolites.

39. Thank you: