2.3 Mutant Methodology

1. Process where a physical or chemical or biological agents are used to induce genetic changes in an organism, creating mutants

mutagenesis

2. These are organisms with altered phenotypes compared to the wild type

mutants

3. Ways on isolating mutants

screening, enrichment, isolation

4. This process Utilizes growth conditions where both mutant and wild type are able to grow and would be distinguished phenotypically

screening

5. What are the disadvantages of screening

labor intensive, inefficient because mutants usually occur at a very low frequency compared to the wild type

6. For a 109 assay, it would take how many years to screen if you are doing nothing but assays 24 hours a day 7 days a week

31.7 years

7. the identification of mutants by visually or experimentally of each colony for their differences in phenotype

Brute force screen

8. This plates contains dyes that changes its color in response to metabolic activity

indicator plates

9. What medium is Used to recognize mutants defective in sugar fermentation

MacConkey Agar (MCA)

10. What are the results in MacConkey Agar (MCA)

lac+ utilizes lactose and produces acidic products = red to pink colonies lac- utilizes peptone and produces basic products = white or colorless colonies

11. What medium is used for screening Screening for lac+ and lac- colonies

MacConkey Agar

12. This type of plating is used To screen for auxotrophic mutants

replica plating

13. This plating is used to detect colonies that fail to grow on a given medium

replica plating

14. Plating Technique used to differentiate mutants and wild type strains based on their ability to grow in the absence of certain amino acids

replica plating

15. Mutants obtained following mutagenesis (using physical or chemical agents) are grown in a plate containing complete medium called

master plate

16. Process of replica plating

(1) mutants are grown in a master plate (2) Master plate is gently pressed onto a sterile velvet pad to pick up bacterial cells from each colony (3) The velvet is gently pressed onto replica plates containing complete medium in one plate and another medium lacking amino acid in another plate * Cells are transferred in a pattern identical from the master plate to multiple test plates with different nutrient compositions (5) incubate (6) compare results

17. Conclusion for replica plating

a colony that grows only on the supplemented medium has a mutation in a gene that encodes the synthesis of an essential nutrient

18. Advantage of replica plating

we can determine mutants that were missed during the selective plating process as selective plating can miss mutants tahtbonly show up as a loss of growth

19. This process of isolation Utilizes growth conditions that favor the growth of a mutant over the parental strain

enrichment

20. What is the goal of enrichment process

to increase the proportion of mutants in a mixed population before attempting isolation as mutagenized cultures contains only a small fraction of mutants

21. How does enrichment work usually

Often works by adding a compound that kills a proportion (but not all) of the growing cells in a population. Mutants here are unaffected, so they can be further analyzed

22. True or false: the enrichment process does not directly identify mutants

true

23. This process only tries to reduce the dominance of the wild type cells, thereby facilitating downstream screening on confirmation techniques

enrichment process

24. It is an intermediate step between mutagenesis and the precise confirmation or characterization of mutant phenotypes

enrichment

25. What are the different types of enrichment

(1) antibiotic enrichment (2) limited enrichment (3) filtration enrichment for auxotrophic fungi (4) enrichment by heat inactivation for Bacillus auxotrophs (5) incorporation of metabolic precursors ( radioactive suicide )

26. This type of enrichment uses Antibiotic to add to bacteria actively growing in minimal medium

antibiotic enrichment

27. Effect of addition of antibiotics on antibiotic enrichment procedure

Prototrophs are killed by the addition of antibiotics and the non-growing auxotrophs survive as these are in their dormant state and are protected from antibiotic lethal effects

28. Process of antibiotic enrichment

(1) addition of antibiotics (2) antibiotic is removed by washing (3) surviving cells are plated on a complete medium and replica plated to isolate auxotrophic mutants

29. Process of penicillin enrichment

(1) outgrowth ( growth in minimal medium + His ) where both His+ and His- grows, (2) Enrichemnt ( growth in minimal medium his + penicillin ) His + grows and die , his- cannot grow ratio of mutant increase (3) repeat (4) dilute (5) outgrowth minimal medium + his (6) replica plating onto MM w/out histidine and w/histidine

30. This type of enrichment Involves the use of minimal medium with amount of amino acid smaller than the amount required by the wild type ( there is enough aa for auxotrophs to grow but insufficient for the faster growing wild type)

limited enrichment

31. This type of enrichment takes advantage of the differences in nutrient requirements between wild type and mutant strain

limited enrichment

32. Results of limited enrichment

Results in the formation of smaller mutant colonies due to them being slower growing

33. True or false: for limited enrichment Nutritional deficiency should be known beforehand

true

34. Why is it that the wild type in limited enrichment bigger

the wt cells consumes fhe limited supply of amino acid and growth is restricted . The limitation of nutrients does not kill the wt cells but simply limits their growth

35. This enrichment process is for filamentous auxotrophic fungi

filtration enrichment

36. This enrichment process relies on the difference in growth behavior between the wild type and auxotrophic mutants in liquid culture

filtration enrichment

37. Process of filtration enrichment

(1) Fungal culture grown in minimal medium that allows the growth of prototrophs (2) Prototrophs are filtered out and non-growing auxotrophic mutant spores remain in the filtrate (3) plated in a complete medium (4) replica plated

38. Why will auxotrophs not grow in the minimal medium for filtration for phototrophs

because the minimal medium lacks the nutrient requirements, thus they will remain as ungerminated spores or small germlings which would pass through the filter

39. Enrichment by heat inactivation is usually for what group of microorganisms

Bacillus auxotrophs

40. This method of enrichment takes advantage of the difference in thermostability of spores and vegetative cells ( the actively growing cells are more heat sensitive )

Enrichment by heat inactivation

41. True or false: Heat inactivation kills most germinated prototrophs and allows the concentration of mutant spores

true

42. Process of enrichment by heat inactivation

(1) bacillus wt and auxotrophs in Mm (2) Bacillus wt grow while mutants are not growing (3) heat at 80 degree celsius for 10 min (4) wt killed while mutants are unaffected

43. Also known as incorporation of radioactive metabolic precursors

radioactive suicide

44. What enrichment process allows Wild type and mutants to be grown in minimal medium to which a radioactive metabolic precursor (3H, 35S, or 32P) is added, which will be incorporated to the cellular components

radioactive suicide

45. Concept behind radioactive suicide

Prototrophs are killed and the auxotrophs that did not grow in minimal medium remain unaffected by the radioactive metabolic precursor. Auxotrophs cannot incorporate radioactive precursors

46. This is the most efficient strategy for isolating mutants

selection

47. An experimental condition in which only the mutant cells grow. Wild type either die or survive but fail to grow

selection

48. How to contrast between screening, enrichment with selection

for screening and enrichment, both WT are initially present and must be distinguished or separated after

49. What are examples of selection for resistance to a metabolic inhibitor

(1) production of enzyme that inactivates the antibiotic via hydrolysis or modification of inhibitor molecule (2) intracellular molecule to which the inhibitor binds may be altered such that the inhibitor no longer binds, or it can bind but is unable to inhibit (3) active pumping out (4) inhibitor cannot enter the cell

50. Function of beta lactamase

causes the hydrolytic deactivation of the beta-lactam ring in penicillin and cephalosporins. Inactivation of penicilloic acid inhibits binding to PBPs (penicilling binding proteins), thereby protecting the process of cell wall synthesis

51. Mutations in the ribosomal protein S12 that prevent binding of

streptomycin, streptomycin interacts directly with the small ribosomal subunit, mutations in the rpsL gene encoding the S12 polypeptide generate resistance against streptomycin.

52. Tetracycline resistance can be due

either preventing antibiotic entry or active efflux of the antibiotic.

53. Example of the use of molecules that cannot be used by the wt organisms

E. coli cannot grow on short chain fatty acids (with less than 10 carbon atoms) as the sole carbon source. plating E. coli cells on medium with short chain fatty acids results in the growth of a few fadR mutant colonies and those ilvG mutants

54. What are fadR mutant colonies

expresses the beta oxidation pathway constitutively, allowing them to utilize fatty acids as sole carbon source, even in the absence of an inducer

55. ILVG mutants

valine resistant, wt are inhibited by blocking isoleucine synthesis through feedback inhibition, resistant of the mutant is acquired by bypassing the biosynthetic patwhay