Isolate MutationsContinued.HW 2 Due Next TueTueLecture: Suppression
How to Isolate Mutants
SelectionScreensEnrichment
Selection
Only allows the growth of the mutant
Resistance to Streptomycin-Plate bacteria on plates with and without streptomycin-On plates without = lawn-On plates with = only resistant colonies
Example
The mechanism of resistance is mutations in therpsLgene which is a ribosomal proteinMutants are rare because only changes in a few amino acids yield a resistant phenotype without affecting its ribosomal activity
Selection
AdvantagesCan examine large number of bacteria for rare mutantsVery powerful“A selection is worth a thousand screens”DisadvantagesUsually hard to develop experiments with direct selectionsCan not isolate intermediate phenotypes
Screens
Conditions that allow growth of both the desired mutant and the parent
-lacZYAoperon allows lactose to be used as a carbon source
Examples
Growth of MacConkey Plates-Has alternative carbon source: allows lac+ and lac- cells to grow-Lac+ cells produce acid and lowers thepH.Colonies turn red (See plate)-Can screen 100-200 colonies per plate
lacY
lactose
Screens
How many colonies do you have to screen to find a mutant?
106colonies1 plate5000 plates1 mutant 200 colonies 1 mutant
x
=
Requires a lot of work and plates to isolate a mutant
Brute Force
Used byCairns andDeLuciato isolate thepolAmutantRequires a lot of work, labor intensive- not a great way to find a mutant (See handout)Cairns andDeLuciaIsolated mutant E. coli coloniesGrew mutant cells and made a crude extractDid in vitro DNA synthesis assays on every extractIsolated 1 mutant colony/thousandsIdentified DNA polymerase I encoded by thepolAgene, involved in DNA replicationNot 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
AdvantagesCan isolate intermediate phenotypesDisadvantagesScreen lots of coloniesLabor 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
Isolate mutants in a transporterIncubate cells with radioactive compoundWT accumulate the compound and diesMutant does not update compoundLeave in fridge for a fewweeksOnemInutewrite: What would happen to the mutant cells after a few weeks? What would happen to theWT cells?
Suicide Enrichments
In
Out
Radioactive compound
Isolate mutants in a transporterIncubate cells with radioactive compoundWT accumulate the compound and diesMutant does not update compoundLeave in fridge for a few weeksOnly mutants alivePlate and screen coloniesJohnCronanpioneered this technique
Suicide Enrichments
In
Out
Radioactive compound
Things to Consider during Screen, Selections, or Enrichments
SiblingsColonies 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 pathwaysIdentify essential functions like DNA or protein synthesisRegulationChemotaxisRecombinationPathogenesis
How do you isolate mutants of phages?
Some phages are lytic only (T4, T1, ᴓx174)
Some are temperate (Lambda, P22, P1)
Infect cells, lyse 100-500 phage
Infect cells, either lytic or lysogenic
Use EM to see phage, as they are so smallCan also use a Plaque Forming Assay
Plaque Forming Assay
Take Lysate
Spot of lawn of bacteria on a plate (top agar)
Isolate Plaques
-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
Lawn
Plaques
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
Mutagenize the Phage
Plate at 30 on LB
WT and TS grow
Replica Plate at 42
Look for the Absence of Plaques
Isolate Nonsense
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. coliCR63- has information suppressors (decodes UAG)B lacks this functionAmber mutations can be decoded by the tRNA and insert an amino acidNamed after the mother of the discover Bernstein: his mothers name was Amber in GermanTalk about in few lecturesHandout
Some Strategies for Finding Mutants
HandoutPenicillin Enrichment
0
Embed
Upload