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

2. How to Isolate Mutants 1.Selection 2.Screens 3.Enrichment

3. 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 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 -lacZYA operon 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 the pH. Colonies turn red (See plate) -Can screen 100-200 colonies per plate lacY lactose

6. Screens How many colonies do you have to screen to find a mutant? 10 6 colonies 1 plate5000 plates 1 mutant200 colonies1 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. 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? Suicide Enrichments In Out Radioactive compound

12. 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 Suicide Enrichments 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 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

17. 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

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