1. Identifying essential genes in phage

2. Screens for essential genes To identify essential genes, you can screen for what types of mutations?

3. Screens for essential genes To identify essential genes, you can screen for what types of mutations? conditional lethal mutations

4. 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?

5. 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

6. 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

7. 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?

8. 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

9. Screens for essential genes STEPS of temperature sensitivity screen: 1.Mutagenize phage particles phage

10. Screens for essential genes STEPS of temperature sensitivity screen: 2. Add mutagenized phage to E. coli at a low MOI E.coli phage

11. Screens for essential genes STEPS of temperature sensitivity screen: 3.Plate and incubate overnight at permissive temperature Mutagen- ized phage E.coli Mutagenized phage

12. Screens for essential genes STEPS of temperature sensitivity screen: 3.Plate and incubate overnight at permissive temperature Why first incubate at permissive temperature? Mutagen- ized phage E.coli Mutagenized phage

13. 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 Mutagen- ized phage E.coli Mutagenized phage

14. 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 Mutagen- ized phage E.coli Wild type phage E.coli Mutagenized phageControl: Wild type phage

15. 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 Mutagen- ized phage E.coli Wild type phage E.coli Mutagenized phageControl: Wild type phage What is the reason for wild type control?

16. 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 Mutagen- ized phage E.coli Wild type phage E.coli Mutagenized phageControl: Wild type phage What is the reason for wild type control? to ensure that the temperature change is not lethal to all phage

17. 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 Permissive temp Non-Permissive temp mutagenized

18. 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 Permissive temp Non-Permissive temp Permissive tempNon-Permissive temp mutagenized Wild type

19. 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 Permissive temp Non-Permissive temp Permissive tempNon-Permissive temp mutagenized Wild type Temperature- sensitive mutant

20. Screens for essential genes STEPS of nonsense-suppressor screen: 1.Mutagenize a known concentration of phage particles phage

21. 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

22. 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

23. 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

24. 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

25. 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 Suppressor E.coli Wild type E.coli Nonsense mutant

26. Complementation test Complementation tests allow you to determine whether your isolated mutations affect the same gene.

27. 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) am1 - am2 - Wild type E.Coli strain

28. 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? am1 - am2 - Wild type E.Coli strain

29. 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 am1 - am2 - Wild type E.Coli strain

30. 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

31. 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

32. 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

33. 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 am1 - + suppressor E.coli am2 - + suppressor E.coli wt phage + suppressor E.coli

34. 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 am1 - + suppressor E.coli am2 - + suppressor E.coli wt phage + suppressor E.coli

35. 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 am1 - + suppressor E.coli am2 - + suppressor E.coli wt phage + suppressor E.coli

36. 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 am1 - + suppressor E.coli am2 - + suppressor E.coli wt phage + suppressor E.coli

37. 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.

38. Mapping STEPS 1. Infect suppressor strain with your two different mutants at high MOI am1 - am2 - Suppressor E.Coli strain

39. Mapping STEPS 2. Allow infected cells to produce phage (1 hour) Suppressor E.Coli strain 1 hour

40. 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 Wild type E.coli 100 phage Suppressor E.coli phage buffer ~1000 phage

41. Mapping STEPS: 4. Count the # of plaques formed on the suppressor strain and on the wild type strain. Wild type E.coli Suppressor E.coli

42. Mapping STEPS: 5. Calculate the frequency of recombination: Frequency of recombination = total recombinants/total phage Wild type E.coli Suppressor E.coli

43. Mapping What is the genotype of the plaques formed on the wild type E.coli strain? Wild type E.coli Suppressor E.coli

44. Mapping What is the genotype of the plaques formed on the wild type E.coli strain? Wild type E.coli Suppressor E.coli am1+, am2+

45. Mapping Recalling that we are dealing with essential genes, do the total recombinants = # of plaques formed on the wild type E.coli strain? Wild type E.coli Suppressor E.coli

46. Mapping Recalling that we are dealing with essential genes, do the total recombinants = # of plaques formed on the wild type E.coli strain? No Wild type E.coli Suppressor E.coli

47. 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? Wild type E.coli Suppressor E.coli

48. 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 Wild type E.coli Suppressor E.coli

49. Mapping Why multiply by 2? Wild type E.coli Suppressor E.coli

50. Mapping Why multiply by 2? To account for the recombinants that cannot grow on wild type E.coli: am1-, am2- Wild type E.coli Suppressor E.coli

51. Mapping The farther the genes are from each other, the higher the frequency of recombination is between them. Wild type E.coli Suppressor E.coli