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Isolate Mutations Continued. HW 2 Due Next Tue Tue Lecture: Suppression

This article explores various methods for isolating mutations, including selection screens, enrichment techniques, and plaque forming assays. It also discusses the advantages and disadvantages of each method and provides strategies for finding mutants.

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Isolate Mutations Continued. HW 2 Due Next Tue Tue Lecture: Suppression

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  1. Isolate Mutations Continued.HW 2 Due Next TueTue Lecture: Suppression

  2. How to Isolate Mutants • Selection • Screens • Enrichment

  3. 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

  4. 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

  5. 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

  6. Screens • How many colonies do you have to screen to find a mutant? 106 colonies1 plate 5000 plates 1 mutant 200 colonies 1 mutant = x Requires a lot of work and plates to isolate a mutant

  7. 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 • 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

  8. 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

  9. Screens • Advantages • Can isolate intermediate phenotypes • Disadvantages • Screen lots of colonies • Labor and time intensive

  10. 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

  11. 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? In Out Radioactive compound

  12. 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 In Out Radioactive compound

  13. 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

  14. Some uses of Mutants • Define metabolic pathways • Identify essential functions like DNA or protein synthesis • Regulation • Chemotaxis • Recombination • Pathogenesis

  15. 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 small Can also use a Plaque Forming Assay

  16. Plaque Forming Assay Plaques 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 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

  17. Isolate Mutant Plaques • Isolate TS, CS Plaques • Isolate Nonsense Mutagenize the Phage Mutagenize the Phage on Suppressor Strain Plate at 30 on LB Plate at 30 on LB WT and TS grow WT and TS grow Replica Plate at 42 Replica Plate onto a strain without suppressors Look for the Absence of Plaques Look for the Absence of Plaques HA works well on phage

  18. 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

  19. Some Strategies for Finding Mutants • Handout • Penicillin Enrichment

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