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Bio 142 Lab > Final Exam > Flashcards

Final Exam Flashcards

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1
Q

What were the three projects?

A

C. elegans
S. Candidas
Bean Beetles

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2
Q

What was the research question for the C. elegans project?

A

Do c. elegans evolve avoidance or resistance in the presence of a pathogen?

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3
Q

What type of research approach was the c. elegans project?

A

Experimental design

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4
Q

What are the biological replicates in the study design for the c. elegans project?

A

Each student’s passage

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5
Q

What are the technical replicates in the study design for the c. elegans project?

A

Three avoidance plates and three survival plates

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6
Q

What makes c. elegans a good model organism?

A

genetically divers, easy to induce mutations, grows quickly

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7
Q

In the avoidance assay (c. elegans), what were the dependent and independent variables?

A

Independent: time point, evolved or control worms
Dependent: c elegans (# or %) on both sides of the plate at different time points

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8
Q

In the survival assay (c elegans), what were the independent and dependent variables?

A

Independent: evolved or control worms
Dependent: c elegans (# or %) on the e coli side after 48 hours

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9
Q

What are the three steps for washing and passaging worms?

A

Label, wash, plate

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10
Q

What happens during the labelling step in washing/passaging worms?

A

Label date, name, lab section, lab room, and passage number on the agar side

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11
Q

What happens during the washing step in washing/passaging worms?

A
  • Wash c elegans off the plate using 1mL M9 buffer and collect the worms in eppendorf tubes
  • Centrifuge the tube and remove the supernatant
  • Add 800 microliters of M9 buffer to the tube and repeat the centrifuging and supernatant removal steps for two more times
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12
Q

What happens during the plating step in washing/passaging worms?

A
  • Resuspend in 200 microliters
  • Plate 20 microliters worms in the middle of the plate
  • Leave agar side down in the incubator for one week
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13
Q

What is the procedure for plating the bacteria in your avoidance assays for your abstract?

A

-5µL of s marcescens and e coli were plated on two sides of the plate using sterile techniques
-Plates were left agar side down until dry. Then plates
were left for incubation under room temperature with agar side up for one week

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14
Q

C elegans project: The protocol says the optimal worm concentration for our survival assay is around 100 worms/10µL.
You took out 5µL from your 500mL of worm solution, and found around 25 worms on your glass slide
under the microscope. How would you adjust the concentration? Which equation would you use to determine this?

A

Centrifuge the tube, take out 250mL supernatant, flick to mix well then pipette
5µL onto a glass slide and recheck the worm concentration
-c1v1=c2v2

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15
Q

If you’re going to run statistic analysis on your survival assay results, what type of test would you run?

A

Two-sample T-test

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16
Q

If you’re going to run statistic analysis on your avoidance assay results, what type of test would you
run?

A

Binomial test

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17
Q

At the 40 minutes timepoint, you observed 40 worms on e coli and 8 worms on s marcescens across
all the plates, fill in the blanks using numbers provided for the binomial test (successes=?, # trials=?, prob=?)

A

We would consider all the total worms on e coli side for all plates at one time
point as “successes”, because we’re testing if the worms have evolved to avoid the pathogens. Each
worm is “one trial”. The hypothetical result is the result we get when our null hypothesis holds true,
which means, we would see equal amounts of worms on either side of the plates. Thus the
hypothetical probability is 0.5

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18
Q

Upon running two-sample t-test, you got a p-value of 0.424. In the specific context of survival assay,
how would you address the statistic significance of the assay in your scientific poster?

A

A p-value of 0.424 is not below 0.05, the p-value of statistical significance according to scientific convention. We failed to reject our null hypothesis and therefore, at 48 hours time point, the number of worms survived in the evolution group is not statistically different than the number in the control group

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19
Q

What was the research question for the bean beetle project?

A

Does diet affect the internal microbiome of an organism?

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20
Q

What were the biological and technical replicates for the bean beetle project?

A

Biological: each individual beetle
Technical: Multiple plates with the same plate type and same beetle suspension (concentration)

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21
Q

What are two types of replicates within a study design?

A

Biological and technical

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22
Q

What makes bean beetles a good model organism?

A

Easy to maintain, easy to manipulate, controlled diet, short life cycle, and small size

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23
Q

What is the purpose for sterilizing beetles before crushing them?

A

To isolate only their interior microbes

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24
Q

Why 16s rRNA gene is often used to identify species?

A
  • When sequencing the genes, we are looking for a region of genome that is present in all microbes but have variable areas that are different in different species so we can distinguish species.
  • 16s rRNA gene is conserved across all bacterial species with conserved and variable regions, so we’ll use primers against conserved regions to amplify around 1500 base pairs of this gene
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25
Q

What are the materials needed for PCR rxns?

A

DNA polymerase, Template DNA, Primers, Nucleotide triphosphate, Buffer

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26
Q

What are the three steps for PCR reactions?

A
  1. Denaturation
  2. Annealing
  3. Extension
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27
Q

What happens in the denaturation step in a PCR rxn?

A

Heat up the rxn (94) to cause DNA strands to separate

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28
Q

What happens in the annealing step in a PCR rxn?

A

Cool the rxn (40-60) to allow the primers to base-pair (anneal) to the template

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29
Q

What happens in the extension step in a PCR rxn?

A

Taq DNA polymerase copies and extends the template DNA

-70-74

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30
Q

In gel electrophoresis, describe the purpose of the DNA ladder

A

DNA molecules of known sizes so that we can compare the sizes of DNA

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31
Q

In gel electrophoresis, describe the purpose of the SybrGreen

A

It fluoresces under UV light when bound to double-stranded DNA

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32
Q

In gel electrophoresis, describe the purpose of the Agar

A

It creates a “grid” for DNA to travel through

33
Q

In gel electrophoresis, describe the purpose of the Loading dye

A

Makes the sample heavier and helps us visualize the movement

34
Q

Why do you see bubbles when you run your gel?

A

Cathode: H2O+2e- —> H2(g) + OH
Anode: H2O —> 2H+ + 1/2 O2 (g) + 2e

35
Q

What are the two measures of biodiversity?

A

Species richness and species diversity

36
Q

What is species richness?

A

The number of species present in a given community

37
Q

What is species diversity?

A

A weighted measure that incorporates a species’ relative abundance

38
Q

What is Inverse Simpson’s Index a measure of?

A

-Inverse Simpson’s Index is a measure of species diversity

39
Q

How would you calculate the inverse Simpson’s index?

A

=1/sum(# of ind in a species/# of total ind in a species)^2

40
Q

What are the two ways of identifying species?

A

Morphology of bacterial colonies and 16s DNA Sequencing

41
Q

What materials are needed for Sanger sequencing?

A

Template, dNTPs, polymerase, buffer,1 primer, modified ddNTPs —> ddNTPs are usually Flourescently labeled: green ddATP, red ddTTP, black ddGTP, blue ddCTP

42
Q

What are the functions of ddNTPs and why are they fluorescently labeled?

A
  • ddNTPs do not have a 3’ hydroxyl group, hence no further chain elongation can occur once this is on the chain
  • Each of the four ddNTPs chain terminators is labelled with fluorescent dyes, each of which emit light at different wavelengths
  • –Products are run out on a gel for separation by size and the detector reads the fluorescence of each band on the gel, telling us what base is the last base in each product size
43
Q

What components differ between Sanger sequencing and PCR reactions?

A

Sanger uses only 1 primer and has ddNTPs

44
Q

How do you determine a “messy” chromatogram sequence?

A
  1. There are multiple peaks for same base position
  2. Peaks are not much higher than background levels
  3. There are no breaks between peaks, in which can’t tell when one peak ends and next begins
45
Q

Suppose you obtain a Sanger sequencing read that’s comprised entirely of overlapping peaks.
What does this suggest about the sample you sent for sequencing?

A

It was probably a mix of two species

46
Q

What are the two main experimental applications of NGS?

A
  • whole genome sequencing of single species

- 16S sequencing of microbial communities

47
Q

What is an NGS “cluster”?

A

It is the collection of identical DNA sequences in a flow-cell. NGS flow-cells contain 100s of thousands of clusters, which can all be sequenced at once using a laser

48
Q

How can NGS be used to calculate species diversity?

A

The proportion of reads for each individual species corresponds to the proportion of that species present in the population - calculate Inverse Simpson’s using proportions of species reads

49
Q

What are some advantages of NGS compared to the traditional sequencing methods like Sanger
sequencing?

A
  • It allows scientists to sequence many pieces of DNA simultaneously
  • –because can get thousands of sequence ‘reads’ back in single reactions and can sequence a genome by breaking genome into pieces and sequencing the pieces
  • It gives us a more comprehensive result
  • –because keep in mind that Sanger sequencing only sequences the species that can be grown on the cultures; however, we needn’t culture the microbiome here for NGS
50
Q

What is the research question of the c albicans project?

A

How does ploidy affect mutation rates of c albicans?

51
Q

What are the replicates of the c albicans project?

A

Biological: different cultures of yeast
Technical: each group’s project from the same stock of yeast

52
Q

What makes candida albicans a good model organism?

A

-Present in ~50% of human gut microbiomes
-Has a relatively high mutation rate, because ploidy naturally varies under different stress
conditions, so it’s easily genetically manipulated

53
Q

How would you calculate mutation rates of candida albicans?

A

Mutation Frequency = revertants / total yeast viable cells

54
Q

After one week’s incubation, we found in total five revertant cells on across five His- plates. Also, we
counted 75 colonies on the 10^5-diluted plate. Calculate the mutation rate in this yeast stock

A

We’ve plated 100µL of 10^5 diluted solution on the plate, so if we found 75 colonies on this plate, we can get an estimate count for the original undiluted 100µL solution: 7510^5 colonies. We started with 2.5mL undiluted solution and 100µL25=2.5mL, so the estimate in starting undiluted culture is 257510^5 cells. So mutation rate = 5/(257510^5) = 2.5*10^5

55
Q

What were the growth medias for the bean beetle project? What are they?

A
  • Phenylethyl Alochol Agar (PEA): used to selectively cultivate Gram + microbes
  • Eosin Methylene Blue (EMB): used to selectively cultivate Gram- microbes
  • Blood Agar (BA): contains mammalian red blood cells
56
Q

Why were three different growth medias used in the bean beetle project?

A

We use different types to favor different nutritional requirements of bacteria

57
Q

What were the growth medias for the candida project? What are they?

A
  • YPD Plates: Yeast Extract Peptone Dextrose Plates, a culture for yeast growth
  • His Plates: Yeast plates without Histidine, so only mutants can grow on them
58
Q

What are the basic components of a figure?

A

Title, x and y axis, units, data, error bars (and caption, if needed)

59
Q

What does it mean to normalize data?

A

Convert units (usually convert to percentage)

60
Q

What is data combination?

A

The act of combining the replicates’ data (avg the replicates)

61
Q

What chart do you use for qualitative data?

A

Bar plot

62
Q

What chart do you use for quantitative data?

A

Scatter plot

63
Q

The higher the inverse simpson’s index, the ____ biodiverse

A

more

64
Q

In which direction does the DNA travel in the gel?

A

Cathode (-) to anode (+)

65
Q

What do low mutation rates lead to?

A

Little variability in population and little ability to adapt to changing conditions

66
Q

What do high mutation rates lead to?

A

High rates of detrimental mutations in individuals and population

67
Q

What causes evolutionary death?

A

No adaptability

-low mutation rate

68
Q

What causes immediate death?

A

Genetic breakdown

-high mutation rate

69
Q

Why use auxotrophs?

A

Auxotrophs are unable to synthesize a compound that they need to survive

70
Q

Why are the ends of a chromatogram often messy?

A

It is difficult to resolve (clearly separate) very short or long fragments in the gel

71
Q

What does the coverage measure in a blast output?

A

How much of your query sequence aligns with the match

72
Q

What does the E-value measure in a blast output?

A
  • “Expected”

- # sequences aligning this well you would expect by chance (given complexity of search sequence & size of database)

73
Q

What does the identify value measure in a blast output?

A

What percentage of aligned bases match

74
Q

What are biological replicates?

A

Measurements of biologically distinct samples that capture random biological variation

75
Q

What are technical replicates?

A

Repeated measurements of the same sample

—represent independent measures of the random noise associated with protocols or equipment

76
Q

Why use biological replicates?

A

Helps control for noise in biological response

77
Q

Why use technical replicates?

A

Helps control for noise in measurement

78
Q

How is the outside of a beetle sterilized?

A

The outside of the beetle is sterilized by dunking it in bleach, then water, then ethanol, and finally in water

79
Q

What is the major difference that separates gram positive and gram negative bacteria?

A

Gram positive bacteria have a thick peptidoglycan layer outside of the plasma membrane, whereas gram negative bacteria have a thinner peptidoglycan layer sandwiched in between two plasma membranes

Bio 142 Lab (1 decks)

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