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Exam Prep 2.0 > Organisms Prac > Flashcards

Organisms Prac Flashcards

1
Q

transfer and freeze time shift assays to get

A

ancestral and evolved strains on which you can conduct fitness comparisons at 1:1 abundance

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2
Q

Measuring real-time evolution to make

A

predictions

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3
Q

Q1: is mutation selective or spontaneous?

A
  • measure using phage resistance, where infection = death
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4
Q

Predictions for selective mutations

A

1) similar terminal no.
2) similar across replicates

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5
Q

Predictions for spontaneous mutations

A

1) different no. (potentially none)
2) occur at different times (early/late)
3) replicate differences

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6
Q

How can you elucidate observed data?

A

compare w models

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7
Q

Pseudomonas fluorescens

A
  • abundant soil and water bacterium related to P. aeruginosa
  • pyroverdine pigments
  • deltamutS SBW25 isolate from sugar beet
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8
Q

P. aeruginosa

A

important human pathogen

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9
Q

Questions to address

A

1) at what rate to bacteria spontaneously acquire mutations?
2) what are the costs of a new trait and can these be minimised?
3) does environmental heterogeneity drive adaptive radiation?

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10
Q

DeltamutS

A
  • higher rate of mutation (bad at DNA mismatch repair)
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11
Q

Protocol:

A
  • rifampimicin
  • rpoB SNP alterations prevent binding (mutation rate of AR); also hinder RNAP
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12
Q

rifampimicin

A
  • target RpoB beta-subunit of RNAP
  • disrupts protein synthesis
  • bacterio-static/cidal
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13
Q

plates need to be

A

dry

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14
Q

pre-cultures

A
  • grown w/o rifampimicin
  • no prior selection for mutation
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15
Q

investigation into mutation rate

A

1) single clone culture in King’s B agar
2) single clone culture in King’s B agar + rifampimicin

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16
Q

+rifampimicin

A
  • only resistant bacteria grow
    ; estimate spontaneous mutation rates
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17
Q

Determining no. of AR bacteria

A
  • exclude susceptible cells via selection plating on KB+rifampimicin
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18
Q

Determine no. of initial bacteria

A

1) dilute for a countable density by several factors separate for each strain

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19
Q

countable

A

spot plating

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20
Q

spot plating

A

3x3 of 3X10microlites dried close to flame and cultured with lid slightly ajar

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21
Q

how to dilute

A
  • vortex with a known diluent and serialise w tip ejections
  • 10^-5/10^-7
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22
Q

Does ecological opportunity and competition promote adaptive radiation?

A

1) inoculate bacteria from single KB wt colony
2) grow in static/shaken tube
3) plate out culture and inoculate for 4 days
4) estimate colony diversity

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23
Q

adaptive radiation

A
  • new resources/challenges/niche opportunities in an heterogeneous environment
  • selects for diversity
  • depends on niche positioning and preference
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24
Q

static tube

A
  • microcosm of liquid medium that creates an oxygen gradient
  • can be plated onto solid media
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25
Q

static tube morphs

A

1) smooth ancestor
2) wrinkly spreader at air:liquid (can collapse)
3) frizzy spreader at bottom

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26
Q

Describe the morphs

A
  • distinct w relative fitness
  • frequency dependent: most morphs can invade when rare
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27
Q

frequency dependent morph selection

A
  • when rare, ws smothers other phenotypes
  • when abundant, surface biofilm collapses
28
Q

diversity observations:

A

1) P/A of biofilm and where

29
Q

Further diversity measurements

A

1) vortex (breaks biofilm)
2) grow diluted samples on agar plates (40microlitres at 10-5/-6)
3) count and categorise morphotype ratios (100 from each plate)
4) calculate diversity

30
Q

calculating diversity

A
  • Gini-Simpson index
  • 1 - lamba
  • lambda - sigma pi^2
  • pi = proportion of ith morphotype
31
Q

Measuring costs of AR

A

1) limit access of AB
2) enzyme-AB-inactivation
3) modify/protect receptor (rpoB)

32
Q

naive prediction

A

reversion

33
Q

actual prediction

A

compensatory adaptation more likely

34
Q

Protocol for AR

A

1) grow susceptible wt for 50g without antibiotic, to create susceptible evolved
2) do the same with the mutant strain

35
Q

How to grow for 50 g?

A
  • 24 hours of King’s B
  • 100 fold dilution
  • fresh media
  • x7
36
Q

To measure relative fitness, compare:

A

wild type susceptible and mutant resistant

37
Q

To measure cost, compare:

A

wild type susceptible and mutant resistance

38
Q

To measure compensation, compare:

A

evolved susceptible and evolved resistant

39
Q

Relative fitness comparisons

A

1) against a lacZ marked ancestor
2) 30microlitres of each plus 6ml King’s B
3) oxygenic growth for 48hours, at 28 degrees C, and 200rpm

40
Q

lacZ visual marker

A
  • unmarked = white
  • lactose -beta-galactosidase-> glucose + galactose
  • X-gal analogue creates blue dye
41
Q

Relative fitness (W) comparisons observations

A
  • calculate density with spot plating: average number of colonies per spot
  • check in R; if -ve, wrong!
42
Q

Wstrain =

A

mustrain/mulacZ

43
Q

mu =

A

ln(final/starting density)

44
Q

Determining conc. of inoculant

A

(average no. colonies in spot/volume of spot) x dilution factor

45
Q

Determining no of cells inoculated

A

conc. of inoculant x volume of inoculant

46
Q
A
47
Q

Determining initial density

A

(no. of cells added) / (total media + inoculant vol)

48
Q

Determining final cell conc

A

initial conc x e^(growth rate x time)

49
Q

growth rate =

A

1/time

50
Q

Determining final cell densities

A

(no. of colonies counted/volume spread) x unit of conversion x dilution factor

51
Q

Statistical analyses

A
  • test significance
  • combinatorial and interactive factorial ANOVA for R2 goodness of fit
52
Q

How to calculate mutation frequency?

A

ratio of before and after in CFU/ml

53
Q

Strain dynamics

A
  • in both strains, any cells that developed spontaneous resistance early could have continued to grow and divide, producing large numbers of resistant cells
  • w/o AB, resistant mutants competing against wt; lower fitness means out-competed
  • ΔmutM culture accumulates mutants faster, reducing competition
54
Q

What happens when you have resistant mutants and wt in the same plate?

A
  • if antibiotics: mutants survive
  • if no antibiotics: mutants outcompeted due to energy expenditure
55
Q

AR mutations

A
  • affinity
  • inhibition
  • what happens in the absence of A? Is it costly?
56
Q

When asked “why” something happens

A

think about evolution. For example, high mutation rate is handy in hostile or varying environments, but bad in stable ones where it is energetically costly

57
Q

Relative frequency

A

think about relative fitness and frequency dependence, for example social cheating from EC public goods

58
Q

What happens if relative fitness is higher?

A

every day culture is replaced, frequency will increase

59
Q

When thinking about dynamics

A

think about what might have happened - might something have been driven to extinction?

60
Q

What can frequency dependent selection cause?

A

stable persistence

61
Q

To get high marks

A

think about what might happen. What if antibiotic concentration was increased? what if it was decreased?

62
Q

how to get rid of expense due to high selection pressure?

A
  • if its chromosomal, mutate
  • if its a plasmid, cure it
  • evolve to express only in A presence; selected for alternation
63
Q

What might increase mutation rate?

A
  • presence of multiple stressors where the pathogen must mutate to survive
64
Q

think about mutation rate as a tradeoff

A

it is good when you need it to survive, but bad when you don’t

65
Q

fluctuating environment select for

A

high intrinsic mutation rates; more genetic variation per generation; more variability for natural selection

66
Q

Why can you use antibiotic treatment to test for mutation rate?

A
  • you can determine no. of resistant cells in pre-culture, because one colony on the antibiotic corresponds to one CFU single cell in the pre-culture
  • this is a proxy for mutation frequency
  • assumes all isolates grow at same rate, antibiotic sensitivity, and spontaneous acquisition in a null environment
67
Q

Has it evolved resistance?

A

think about the time frame - resistance takes a long time, especially if its a large cellular process being modified. If its point chromosomes, probably not. If its TE shifts, maybe. If it is HGT (transformation, transduction, conjugation), maybe.

Exam Prep 2.0 (33 decks)

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