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Identifying essential genes
in phage
Screens for essential genes
To identify essential genes, you can screen for what types of
mutations?
Screens for essential genes
To identify essential genes, you can screen for what types of
mutations?
conditional lethal mutations
Screens for essential genes
To identify essential genes, you can screen for what types of
mutations?
conditional lethal mutations
What are the two types of conditional lethal screens that
you can do in phage?
Screens for essential genes
To identify essential genes, you can screen for what types of
mutations?
conditional lethal mutations.
What are the two types of conditional lethal screens that
you can do in phage?
•
Temperature sensitivity
•
Nonsense suppression
Screens for essential genes
To identify essential genes, you can screen for what types of
mutations?
conditional lethal mutations.
What are the two types of conditional lethal screens that
you can do in phage?
•
Temperature sensitivity
•
Nonsense suppression
Screens for essential genes
To identify essential genes, you can screen for what types of
mutations?
conditional lethal mutations.
What are the two types of conditional lethal screens that
you can do in phage?
•
Temperature sensitivity
•
Nonsense suppression
What is the observable phenotype in screens for essential
genes?
Screens for essential genes
To identify essential genes, you can screen for what types of
mutations?
conditional lethal mutations.
What are the two types of conditional lethal screens that
you can do in phage?
•
Temperature sensitivity
•
Nonsense suppression
What is the observable phenotype in screens for essential
genes?
Dead (don’t form plaques) at non-permissive conditions
Screens for essential genes
STEPS of temperature sensitivity screen:
1. Mutagenize phage particles
phage
Screens for essential genes
STEPS of temperature sensitivity screen:
2. Add mutagenized phage to E. coli at a low MOI
phage
E.coli
Screens for essential genes
STEPS of temperature sensitivity screen:
3. Plate and incubate overnight at permissive temperature
Mutagenized phage
Mutagenized
phage
E.coli
Screens for essential genes
STEPS of temperature sensitivity screen:
3. Plate and incubate overnight at permissive temperature
Why first incubate at permissive temperature?
Mutagenized phage
Mutagenized
phage
E.coli
Screens for essential genes
STEPS of temperature sensitivity screen:
3. Plate and incubate overnight at permissive temperature
Why first incubate at permissive temperature? To allow
all phage to replicate
Mutagenized phage
Mutagenized
phage
E.coli
Screens for essential genes
STEPS of temperature sensitivity screen:
3. Plate and incubate overnight at permissive temperature
Why first incubate at permissive temperature? To allow
all phage to replicate
Mutagenized phage
Mutagenized
phage
E.coli
Control: Wild type phage
Wild type E.coli
phage
Screens for essential genes
STEPS of temperature sensitivity screen:
3. Plate and incubate overnight at permissive temperature
Why first incubate at permissive temperature? To allow
all phage to replicate
Mutagenized phage
Mutagenized
phage
E.coli
Control: Wild type phage
Wild type E.coli
phage
What is the reason for wild type control?
Screens for essential genes
STEPS of temperature sensitivity screen:
3. Plate and incubate overnight at permissive temperature
Why first incubate at permissive temperature? To allow
all phage to replicate
Mutagenized phage
Mutagenized
phage
E.coli
Control: Wild type phage
Wild type E.coli
phage
What is the reason for wild type control?
to ensure that the temperature change is not
lethal to all phage
Screens for essential genes
STEPS of temperature sensitivity screen:
4.
Transfer plaques to duplicate plates and incubate overnight:
Mutagenized phage
• At permissive temperature
• At non-permissive
temperature
mutagenized
Permissive temp
Non-Permissive temp
Screens for essential genes
STEPS of temperature sensitivity screen:
4.
Transfer plaques to duplicate plates and incubate overnight:
Mutagenized phage
• At permissive temperature
• At non-permissive
temperature
Control: Wild type phage
• At permissive temperature
• At non-permissive
temperature
mutagenized
Permissive temp
Wild type
Non-Permissive temp
Permissive temp
Non-Permissive temp
Screens for essential genes
STEPS of temperature sensitivity screen:
5. Mutagenized phage that form plaques at permissive temp but not
at non-permissive temp are temperature-sensitive mutants
Mutagenized phage
• At permissive temperature
• At non-permissive
temperature
Control: Wild type phage
• At permissive temperature
• At non-permissive
temperature
mutagenized
Wild type
Temperaturesensitive mutant
Permissive temp
Non-Permissive temp
Permissive temp
Non-Permissive temp
Screens for essential genes
STEPS of nonsense-suppressor screen:
1. Mutagenize a known concentration of phage particles
phage
Screens for essential genes
STEPS of nonsense-suppressor screen:
2. Add mutagenized phage to a suppressor E. coli strain at a
low MOI; plate and incubate overnight
mutagenized
phage
suppressor
E.Coli
strain
Screens for essential genes
STEPS of nonsense-suppressor screen:
2. Add mutagenized phage to a suppressor E. coli strain at a
low MOI; plate and incubate overnight
Why incubate first on suppressor strain?
mutagenized
phage
suppressor
E.Coli
strain
Screens for essential genes
STEPS of nonsense-suppressor screen:
2. Add mutagenized phage to a suppressor E. coli strain at a
low MOI; plate and incubate overnight
Why incubate first on suppressor strain? to allow all
phage to replicate and form plaques
mutagenized
phage
suppressor
E.Coli
strain
Screens for essential genes
STEPS of nonsense-suppressor screen:
3.
Transfer plaques to duplicate plates and incubate overnight:
•
On a lawn of suppressor E.coli strain
•
On a lawn of wild type E.coli
Suppressor E.coli
Wild type E.coli
Screens for essential genes
STEPS of nonsense-suppressor screen:
4.
Those phage that are nonsense mutants produce a plaque on
the suppressor E.coli strain but not on the wild type E.coli strain
Nonsense mutant
Suppressor E.coli
Wild type E.coli
Complementation test
Complementation tests allow you to determine whether
your isolated mutations affect the same gene.
Complementation test
STEPS
1.
Infect wild type strain with two of your isolated mutants at high
MOI for 1 hour (to allow one round of infection)
am2-
am1-
Wild type E.Coli strain
Complementation test
STEPS
1.
Infect wild type strain with two of your isolated mutants at high
MOI for 1 hour (to allow one round of infection)
Why at high MOI?
am2-
am1-
Wild type E.Coli strain
Complementation test
STEPS
1.
Infect wild type strain with two of your isolated mutants at high
MOI for 1 hour (to allow one round of infection)
Why at high MOI? So each cell can be infected by both mutants
am2-
am1-
Wild type E.Coli strain
Complementation test
STEPS
2.
To determine whether the mutations complemented each
other, you must determine the amount of phage that was
successfully replicated in the co-infected wild type E. coli strain:
•
Perform a plaque assay of the phage supernatant, this time
on a suppressor E.coli strain.
am1- + am2- +
suppressor E.coli
Complementation test
STEPS
2.
To determine whether the mutations complemented each
other, you must determine the amount of phage that was
successfully replicated in the co-infected wild type E. coli strain:
•
Perform a plaque assay of the phage supernatant, this time
on a suppressor E.coli strain.
•
Positive control: wild type phage in suppressor E.coli
strain
am1- + am2- +
suppressor E.coli
wt phage +
suppressor E.coli
Complementation test
STEPS
2.
To determine whether the mutations complemented each
other, you must determine the amount of phage that was
successfully replicated in the co-infected wild type E. coli strain:
•
Perform a plaque assay of the phage supernatant, this time
on a suppressor E.coli strain.
•
Positive control: wild type phage in suppressor E.coli
strain
•
Negative control: each single mutant phage in
suppressor E.coli strain
am1- + am2- +
suppressor E.coli
wt phage +
suppressor E.coli
am1- +
suppressor E.coli
am2- + suppressor
E.coli
Complementation test
STEPS
3.
If plaques are formed in the plaque assay involving am1- + am2+ suppressor E.coli, the mutations complement each other and
are likely mutations in two different genes.
am1- + am2- +
suppressor E.coli
wt phage +
suppressor E.coli
am1- +
suppressor E.coli
am2- + suppressor
E.coli
Complementation test
STEPS
3.
If plaques are not formed in the plaque assay involving am1- +
am2- + suppressor E.coli, the mutations do not complement
each other and are likely mutations in the same gene.
am1- + am2- +
suppressor E.coli
wt phage +
suppressor E.coli
am1- +
suppressor E.coli
am2- + suppressor
E.coli
Complementation test
STEPS
3.
If plaques are not formed in the plaque assay involving am1- +
am2- + suppressor E.coli, the mutations do not complement
each other and are likely mutations in the same gene.
Why are plaques are not formed?
am1- + am2- +
suppressor E.coli
wt phage +
suppressor E.coli
am1- +
suppressor E.coli
am2- + suppressor
E.coli
Complementation test
STEPS
3.
If plaques are not formed in the plaque assay involving am1- +
am2- + suppressor E.coli, the mutations do not complement
each other and are likely mutations in the same gene.
Why are plaques are not formed?
The phage produced are unable to infect further cells.
am1- + am2- +
suppressor E.coli
wt phage +
suppressor E.coli
am1- +
suppressor E.coli
am2- + suppressor
E.coli
Mapping
Once you determine that two mutations complement each
other and thus are mutations in different genes, you can
map the genes to determine the physical distance between
them.
Mapping
STEPS
1. Infect suppressor strain with your two different mutants at high
MOI
am2-
am1-
Suppressor E.Coli strain
Mapping
STEPS
2. Allow infected cells to produce phage (1 hour)
1 hour
Suppressor E.Coli strain
Mapping
STEPS:
3. Perform a plaque assay to determine total number of phage and
number of recombinants:
- Serial dilutions of phage
- Plate on suppressor strain AND wild-type strain
100
phage
Wild type E.coli
phage
buffer
~1000 phage
Suppressor E.coli
Mapping
STEPS:
4. Count the # of plaques formed on the suppressor strain
and on the wild type strain.
Suppressor E.coli
Wild type E.coli
Mapping
STEPS:
5. Calculate the frequency of recombination:
Frequency of recombination = total recombinants/total phage
Suppressor E.coli
Wild type E.coli
Mapping
What is the genotype of the plaques formed on the wild type E.coli
strain?
Suppressor E.coli
Wild type E.coli
Mapping
What is the genotype of the plaques formed on the wild type E.coli
strain?
am1+, am2+
Suppressor E.coli
Wild type E.coli
Mapping
Recalling that we are dealing with essential genes, do the total
recombinants = # of plaques formed on the wild type E.coli strain?
Suppressor E.coli
Wild type E.coli
Mapping
Recalling that we are dealing with essential genes, do the total
recombinants = # of plaques formed on the wild type E.coli strain? No
Suppressor E.coli
Wild type E.coli
Mapping
Recalling that we are dealing with essential genes, do the total
recombinants = # of plaques formed on the wild type E.coli strain? No
What do the actual # of total recombinants equal?
Suppressor E.coli
Wild type E.coli
Mapping
Recalling that we are dealing with essential genes, do the total
recombinants = # of plaques formed on the wild type E.coli strain? No
What do the actual # of total recombinants equal?
2 X # of plaques formed on wild type E.coli
Suppressor E.coli
Wild type E.coli
Mapping
Why multiply by 2?
Suppressor E.coli
Wild type E.coli
Mapping
Why multiply by 2?
To account for the recombinants that cannot grow on wild type
E.coli: am1-, am2-
Suppressor E.coli
Wild type E.coli
Mapping
The farther the genes are from each other, the higher the
frequency of recombination is between them.
Suppressor E.coli
Wild type E.coli