Transcript Isolate Mutations Continued

Isolate Mutations Continued.
HW 2 Due Next Tue
Tue Lecture: Suppression
How to Isolate Mutants
1. Selection
2. Screens
3. Enrichment
Selection
• Only allows the growth of the mutant
Example
Resistance to Streptomycin
-Plate bacteria on plates with and without streptomycin
-On plates without = lawn
-On plates with = only resistant colonies
The mechanism of resistance is mutations in the rpsL gene which is a ribosomal protein
Mutants are rare because only changes in a few amino acids yield a resistant phenotype
without affecting its ribosomal activity
Selection
• Advantages
• Can examine large number of bacteria for rare mutants
• Very powerful
• “A selection is worth a thousand screens”
• Disadvantages
• Usually hard to develop experiments with direct selections
• Can not isolate intermediate phenotypes
Screens
• Conditions that allow growth of both the desired
mutant and the parent
Examples
lacY
-lacZYA operon allows lactose to be used as a carbon source
lactose
Growth of MacConkey Plates
-Has alternative carbon source: allows lac+ and lac- cells to grow
-Lac+ cells produce acid and lowers the pH. Colonies turn red (See plate)
-Can screen 100-200 colonies per plate
Screens
• How many colonies do you have to screen to find a mutant?
106 colonies
1 mutant
x
1 plate
200 colonies
=
5000 plates
1 mutant
Requires a lot of work and plates to isolate a mutant
Brute Force
• Used by Cairns and DeLucia to isolate the polA mutant
• Requires a lot of work, labor intensive- not a great way to find a mutant
(See handout)
• Cairns and DeLucia
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–
–
–
–
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Isolated mutant E. coli colonies
Grew mutant cells and made a crude extract
Did in vitro DNA synthesis assays on every extract
Isolated 1 mutant colony/thousands
Identified DNA polymerase I encoded by the polA gene, involved in DNA replication
Not real replication enzyme but rather a DNA repair enzyme
How to Isolate a TS lethal
Mutagenize Colony
Plate at 30 on LB
WT and TS grow
Replica Plate at 42 for several hours
Only WT grow
Shift to 30
Only WT grow as TS mutant is dead
Example of how to screen for mutants defective in DNA synthesis
Screens
• Advantages
• Can isolate intermediate phenotypes
• Disadvantages
• Screen lots of colonies
• Labor and time intensive
Enrichments
• Enhance survival of mutants relative to parental cell
Example
-Penicillin Enrichment (Not selection)
-Only kills growing cells by inhibiting cell wall synthesis (Cells not growing survive)
Handout
Suicide Enrichments
• Isolate mutants in a transporter
• Incubate cells with radioactive compound
– WT accumulate the compound and dies
– Mutant does not update compound
– Leave in fridge for a few weeks
– One mInute write: What would happen to the
mutant cells after a few weeks? What would
happen to the WT cells?
Out
Radioactive
compound
In
Suicide Enrichments
• Isolate mutants in a transporter
• Incubate cells with radioactive compound
– WT accumulate the compound and dies
– Mutant does not update compound
– Leave in fridge for a few weeks
– Only mutants alive
– Plate and screen colonies
– John Cronan pioneered this technique
Out
Radioactive
compound
In
Things to Consider during Screen, Selections, or Enrichments
Siblings
Colonies isolated from the same plate may contain the same mutation if they
derive from the same cell
-Use multiple cultures to avoid this problem
Some uses of Mutants
• Define metabolic pathways
• Identify essential functions like DNA or protein
synthesis
• Regulation
• Chemotaxis
• Recombination
• Pathogenesis
How do you isolate mutants of phages?
• Some phages are lytic
only (T4, T1, ᴓx174)
Infect cells, lyse 100-500
phage
Use EM to see phage, as they are so small
Can also use a Plaque Forming Assay
• Some are temperate
(Lambda, P22, P1)
Infect cells, either lytic or
lysogenic
Plaque Forming Assay
Plaques
Take Lysate
Spot of lawn of bacteria on a plate (top agar)
Isolate Plaques
Lawn
-Each plaque results from 1 phage
-Each phage makes a plaque of unique size, shape, and
turbidity
-Some are clear or turbid
-Range in size
Lambda makes turbid plaques since some cells in the middle are lysogens. Can isolate
mutant clear plaques if mutations are in genes responsible for lysogeny
Isolate Mutant Plaques
• Isolate TS, CS Plaques
• Isolate Nonsense
Mutagenize the Phage
Plate at 30 on LB
WT and TS grow
Replica Plate at 42
Look for the Absence of Plaques
Mutagenize the Phage
on Suppressor Strain
Plate at 30 on LB
WT and TS grow
Replica Plate onto
a strain without
suppressors
Look for the Absence of Plaques
HA works well on phage
Amber Mutants
•
Some lethal mutants only grow in some strains of E. coli
– CR63- has information suppressors (decodes UAG)
– B lacks this function
• Amber mutations can be decoded by the tRNA and insert an amino
acid
• Named after the mother of the discover Bernstein: his mothers
name was Amber in German
• Talk about in few lectures
• Handout
Some Strategies for Finding Mutants
• Handout
• Penicillin Enrichment