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Lab Test 2 Flashcards

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1
Q

Describe the differences between the processes of cellular respiration & fermentation;
-use of oxygen is required
-reactions
- amount of ATP

A

Cellular respiration
-requires oxygen
- produces 36-38 ATP
C6H12O6 + 6O2 -> 6CO2 + 6H2O + Energy + heat

Fermentation
- anaerobic /no oxygen)
- produces 2 ATP
C6H12O6 (ethanol) -> 2 C2H5OH + 2CO2 + Energy
= for yeast cells
C6H12O6 (lactic acid) -> 2 C3H6O3 + Energy
= for muscle cells

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2
Q

Describe the process of cellular respiration, knowing
-chemical equation
-what conditions it occurs in
- location in the cell
-ultimate goal.

A

C6H12O6 + 6O2 -> 6CO2 + 6H2O + Energy + heat
36-38 ATP
aerobic conditions

begin in cytoplasm (glycolysis)
then matrix of mitochondria (Krebs cycle)
then inner membrane of mitochondria (ETC)

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3
Q

Briefly, know the steps of cellular respiration and know what is produced at each step.

A

glycolysis (1 glucose)
net gain = 2 ATP, 2 NADH, 2 pyruvate

pyruvate oxidation
net gain = 2 acetyl con, 2 NADH, 2 CO2

krebs cycle
net gain = 2 ATP, 6 NADH, 2FADH2, 4 CO2

ETC
net gain= 32/34 ATP, water

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4
Q

Describe the process of fermentation, knowing the chemical equation, what conditions it
occurs in, location in the cell, and give the ultimate goal.

A

alcohol fermentation
C6H12O6 (ethanol) -> 2 C2H5OH + 2CO2 + Energy
= for yeast cells

lactic acid fermentation
C6H12O6 (lactic acid) -> 2 C3H6O3 + Energy
= for muscle cells

occurs in cytoplasm
anaerobic conditions
goal = regenerate NAD+ from NADH (to produce ATP by glycolysis only)

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5
Q

Do you know the three main pathways that metabolize pyruvate? Which one oxidizes
pyruvate? Which ones reduce pyruvate?

A
  1. pyruvate oxidation
    -> pyruvate is oxidized
  2. alcohol fermentation
    -> pyruvate is reduced into ethanol and CO2
  3. lactic acid fermentation
    -> pyruvate is reduced to lactic acid
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6
Q

Do you know the difference between alcohol and lactic acid fermentation in terms of the
steps and products formed?

A

alcohol fermentation
1. glycolysis produces pyruvate & NADH
2. pyruvate -> acetaldehyde + CO2
3. acetaldehyde -> ethanol + NAD+
products = ethanol, CO2, NAD+, 2 ATP

lactic acid fermentation
1. glycolysis produces pyruvate & NADH
2. pyruvate -> lactic acid + NAD+
products = lactic acid, NAD+, 2 ATP

difference -> no CO2 in lactic acid fermentation

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7
Q

Can you explain the relevance of the R.Q. ratio (respiratory quotient) in terms of types of substrates
being used (carbohydrates or fats) in cellular respiration?

A

RQ quotient = CO2 produced / O2 uptake

higher RQ = more CO2 produced for less O2 taken
= “more efficient” = substrate needs less oxygen

low RQ (ie fats) = need more oxygen for oxidation

-> RQ indicates the energy source efficiency

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8
Q

For the pea seedlings demonstration of cellular respiration, do you understand the setup with the
soda lime?

A

soda lime absorbs the CO2 produced
-> we can measure only on the O2 uptake
-> good measuring of rate of aerobic uptake

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9
Q

For the pea seedlings demonstration what would a positive gas pressure indicate?

A

soda lime absorbs CO2
no soda lime = CO2 is produced
# CO2 produced = # O2 consumed
= pressure of gas is constant (straight line)

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10
Q

For the pea seedlings demonstration what was the purpose of covering the
seedlings with foil?

A

dark
= prevents photosynthesis by blocking light
= we measure only respiration and not oxygen production from photosynthesis

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11
Q

if you switch from aerobic to anaerobic conditions, what happens to the rate of glucose consumption?

A

it increases, bc you produce less ATP and don’t do all the long steps of cellular respiration -> only glycolysis
= faster
= need more glucose to equilibrate the amount of ATP

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12
Q

explain the Pea seedlings experiment

A

Pea seedling experiment

goal : measure oxygen consumption in cellular respiration in germinating pea seedlings

as the seeds use oxygen for respiration, the gas pressure in the test tube decreases bc oxygen is being consumed
= help understand how much O2 is used

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13
Q

Explain the fermentation in yeast experiment

A

goal: to observe + measure the rate of fermentation in yeast with different sugars

yeast is mixed with a sugar and as it ferments the sugar, it proves ethanol + ATP + CO2
the gas pressure sensor measures CO2 produced

= help compare the efficiency of yeast with different sugars

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14
Q

what is sucrose, maltose, lactose broken down into?

A

sucrose = glucose + fructose
maltose = glucose + glucose
lactose = glucose + galactose

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15
Q

 Can you explain why the fermentation rates for glucose and sucrose are relatively higher than
maltose and lactose?

A

yeast metabolizes glucose immediately
= fast rate

yeast metabolizes sucrose immediately
-> bc most yeast have enzyme sucrase

yeast metabolizes maltose slowly
-> maltose enter the cell by non-specific transport mechanisms
-> once inside cell: they stimulate the synthesis of maltase + maltose permease
-> then more maltase can make more maltose enter the cell
-> then maltose -> 2 glucose -> ethanol

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16
Q

why did you measure a second maltose fermentation rate after a 15 minute incubation?

A

to give the time for maltose to enter through non-specific transport mechanisms and stimulate the formation of maltase and maltose permease

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17
Q

why is maltose fermentation rate slow?

A

yeast metabolizes maltose slowly
-> maltose enter the cell by non-specific transport mechanisms
-> once inside cell: they stimulate the synthesis of maltase + maltose permease
-> then more maltase can make more maltose enter the cell
-> then maltose -> 2 glucose -> ethanol

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18
Q

where is pyruvic acid oxidized to CO2 in aerobic oxidation?

A

mitochondria in the matrix

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19
Q

why is lactose fermentation rate slow?

A

lactose is mostly found in milk products
yeast naturally produces enzymes to help hydrolyze sugars in its environment (not lactose)
since lactose isn’t normally found in yeast, we don’t think that yeast will have the enzyme for it.

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20
Q

If fat were the main respiratory substrate would the O2 uptake and CO2 output be equal (seedling experiment)?

A

no, much more C-H bonds to break and convert into electron carriers for the ETC in fats
= consumes much more oxygen than glucose

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21
Q

Do you know these technical/analytical skills:
o Given a data set, be able to draw a graph depicting O2 uptake or CO2 emission over time. o Given a graph of fermentation rates or a graph of cellular respiration rates, be able to
calculate the rate of O2 uptake or CO2 emission (depending on the scenario).

A
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22
Q

function of cuticle

A

top - very first waxy layer
prevents water loss
protect the plant for pathogens

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23
Q

function of upper epidermis

A

outermost layer
protects leaf
prevent water loss
often covered by a cuticle

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24
Q

label and give the function of cuticle

A

waxy layer that protects the leaf and prevents water loss

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25
Q

function of lower epidermis

A

protects the leaf
contains stomata

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26
Q

label and give the function of upper epidermis

A

right under the cuticle
protect & prevent water loss + give structure

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27
Q

function of palisade mesophyll

A

cylindrical cells just below the upper epidermis
contains cells rich in chloroplasts
main site of photosynthesis

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28
Q

label and give the function of lower epidermis

A

layer on lower surface of the leaf

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29
Q

function of spongy mesophyll

A

below the palisade mesophyll layer
has loosely arranged cells of different shapes with air spaces

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30
Q

label and give the function of stomata

A

system made up of pores + guard cells
where gas exchange occurs
located on lower surface of the leaf

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31
Q

label and give the function of mesophyll

A

functional tissue between the upper and lower epidermis (does not include the vascular tissues)
+ many chloroplast within the mesophyll

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32
Q

function of guard cells

A

specialized cells around the stomata
regulate opening/closing of stomata
= control gas exchange + water loss

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33
Q

function of stomata

A

pores on the leaf surface
allow gas exchange (CO2 in / O2 out)
allow release of water vapour

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34
Q

function of vascular tissue

A

the plant’s transport system
moves water, nutrients, food through the plant
contains the xylem + phloem

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35
Q

function of xylem

A

tissue
upper portion
stained red

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36
Q

function of phloem

A

tissue
lower portion
stained green

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37
Q

hydrophyte’s adaptations

A

Hydrophyte’s adaptations
1. reduced cuticle
= easier gas + water exchange
2. large pockets where air can be trapped
= help leaf float
= better gas exchange of water
3. stomata present only in upper epidermis
= leaf floats on water surface = good gas exchange in upper epideermis

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38
Q

why do hydrophytes have large flat leaves

A

increase surface area (capture more sunlight)
better stability/floating to access sunlight and gas exchange with air

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39
Q

xerophyte adaptations

A
  1. thicker cuticles
    = limit water loss
  2. multiple layers of epidermis
    = barrier to reduce water loss trough transpiration
  3. stomata at the lower epidermis
    = capture evaporating moisture + keep humid environment
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40
Q

why do xerophytes have extensive root systems?

A

dry environment
= need to go deeper in soil to reach water
= retain as much water as possible

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41
Q

would you expect xerophytes to have large and flat leaves?

A

no
dry environment= want lower surface area to volume ratio to prevent water loss

42
Q

what are the 3 leaves and for which specific habitats ?

A

hydrophytes
= adapted from life in water

xerophytes
= adapted for life in very dry habitat

mesophytes
= adapted for life in moderate habitat

43
Q

 Are you able to identify a mesophyte, xerophyte or hydrophyte leaf in a microscopic cross section by characteristics and identify their components

A
44
Q

o When are guard cells turgid?

A

turgid
blue light absorbed
H+ is pumped into the EXM
K+ is pumped into the intracellular fluid
= increases osmolarity inside the cell
= lack of water inside cell
= water comes inside the cell by osmosis
= stoma opens (turgid)

45
Q

when are guard cells flaccid?

A

too much water inside the cells
= osmosis makes water leave the cell
= as it leave, stoma closes (flaccid)

46
Q

o Why do guard cell have chloroplasts?

A

chloroplasts make ATP

-> provide the energy for the opening/closing of the stomata

47
Q

o How do the proton pumps in guard cells affect osmosis and pore-opening and closing?

A

bc guard cell play a role in photosynthesis which help them regulate opening/closing of stomata

daylight
guard cells = photosynthesis (produce ATP)
ATP = used to pump ions into guard cells = opens stomata

night
no photosynthesis = no ATP
= nothing being pumped
= stomata closes

48
Q

Do you understand the difference between absorption & reflection of light, & that the light that is absorbed “powers” photosynthesis

A

chlorophyll b absorbs blue/red light and reflects green light

blue light absorbed is what powers photosynthesis

49
Q

 Do you understand how paper chromatography separates the pigments (what other molecules can chromatography separate?

A

the molecules are separated based on their solubility to a solvent

50
Q

 What is the stationary phase and mobile phase of chromatography? Why is selecting the appropriate eluent so important to chromatography? What eluent was used in this experiment?

A

stationary phase = adsorbent
mobile phase = eluent solution

a pigment will either stay attached to the adsorbent (coat on top of the paper) or go in the solution and move along the paper

adsorbent is often polar = polar molecule will stay in stationary phase

the eluent must be less polar than the adsorbent
& less polar than the molecules being separated

51
Q

most non polar molecules will be in mobile phase or stationary phase

A

mobile phase, with the less polar eluent

52
Q

the most polar polar molecules will be in the stationary/mobile phase?

A

the stationary phase, close to the polar adsorbent

53
Q

explain the experiment in this lab (chromatography)

A

we can observe different migration of the pigments during chromatography

  1. we extracted pigments from spinach
    Rf , from lowest to highest:
    chlorophyll b, chlorophyll a, xantophyll, xanthophyll 2nd, carotene

conclusion:
non-polar pigment will follow the less solvent and migrate a lot
polar pigment will stay near the adsorbent and won’t migrate

54
Q

Why doesn’t chlorophyll a fluoresce in an intact plant?

A

fluorescence = when a molecule absorbs light and releases than energy as light of a longer wavelength

chlorophyll a absorbs light, but uses it for photosynthesis
= no release of light = no fluorescence

55
Q

Given a completed chromatography paper, be able to calculate Rf values. What does a larger Rf value indicate for a pigment? A smaller Rf value?

A

Rf = distance travelled by compound / distance travelled by the solvent

large Rf = compound travelled far = non polar
small Rf = compound stayed low = polar

56
Q

what structural difference between chlorophyll a and b explains the difference in migration?

A

chlorophyll A and B = more polar structure than carotene
(carotene = more straight chain C-H bonds
chlorophylls = more bulky)

chlorophyll B = more polar than chlorophyll A
(bc B has CHO head and A has only CH3)

57
Q

what is the absorbance / reflection of carotenoids (+ wavelength peaks)

A

carotenoid has an absorption peak at:
425
= absorbs blue & green light
= reflects yellow, orange, green

58
Q

what is the absorbance / reflection of chlorophyll A (+ wavelength peaks)

A

chlorophyll A has an absorption peak at:
425nm & 675nm
= absorbs blue & red light
= reflects green

59
Q

what is the absorbance / reflection of chlorophyll B (+ wavelength peaks)

A

chlorophyll B has an absorption peak at:
475nm & 650nm
= absorbs blue & red light
= reflects green

60
Q

why do the absorption spectrum for chlorophylls and carotenoids not overlap completely ?

A

each absorb different wavelength of light
= more diverse range of light for photosynthesis
= maximizes available light

61
Q

What is a transgenesis technique?

A

transgenesis technique
= add a foreign gene to an organism
->technique:
1. the gene is isolated
2. the gene is cloned (amplified)
3. the gene is modified
4. the gene is inserted into a new organism

= alter the phenotype in the organism
= organism can pass on the new phenotype to its offspring

62
Q

What was the transgenic organism you created in this lab?

A

use of CaCl2 transformation
= to make E.Coli artificially competent

add the gene GFP to E.Coli using the pGLO system
GFP = green fluorescent protein

= make E.Coli fluorescent basically

63
Q

why are bacterias good candidates for transgenesis?

A

reproduce asexually = divide rapidly
easily cultured + easily manipulated
unicellular

64
Q

what was the goal of lab 7?

A

add the gene of a bioluminescent jellyfish species to a bacterial species
=use bacterial transformation & pGLO system
= observe green fluorescence under UV light

65
Q

horizontal gene transfer vs vertical gene transfer?

A

horizontal
->bacteria transfer genes (DNA) to organisms that are within a population/species (not parent/offspring)
= genes go “sideways”

vertical
-> genes are passed from parent to offspring during reproduction (DNA inherited)
= genes do “down” a generation

66
Q
  • Be able to define what bacterial plasmids are and what their purpose is in bacteria.
A

bacteria contain small circular DNA called plasmids

the genes on a plasmid can be transferred by horizontal gene transfer to other bacterias
-> lead to adaptation

67
Q

what is an insert in lab 7? what does it make?

A

a DNA fragment that is added to a specific spot in the plasmid
= make a “recombinant plasmid” called pGLO

68
Q

what is the relationship between the GFP, beta lactamase (bla) and ara
C genes on the pGLO plasmid?

A

bla gene ensure that only the bacteria with the plasmid (selection marker) grow

araC regulates expression of GFP

together; work to make transformed bacteria resistant to ampicillin and grow under UV light when arabinose is present

69
Q

function of GFP gene

A

transcription Unit of gene

encodes the amino acid sequence of the GFP protein

70
Q

function of araC protein gene

A

Transcription Unit of Gene

encodes the amino acid sequence of the AraC protein

71
Q

function of bla (beta-lactamase gene)

A

Transcription Unit of Gene

encodes the amino acid sequence of the beta-lactamase enzyme

72
Q

what does the araC protein do?

A

it is the gene expression regulatory protein

-> ParaBAD promoter is present, then;
-> araC protein can either:
-> activate (in presence of arabinose) or ;
->repress (in absence of sugar) the expression of the gene

73
Q
  • What phenotype does the bla gene gave to transformed bacteria? Is this gene constitutively
    expressed or regulated in the pGLO plasmid?
A

selectable marker gene, which encodes the bata-lactamase gene

beta-lactase transforms beta-lactam antibiotics
-> transformed cells now have ampicillin

74
Q
  • How does the ara C gene regulate the GFP gene? Briefly describe this arabinose operon.
A

absence of arabinose
= araC binds to DNA control elements
= araC represses transcription from ParaBAD
= GFP is not expressed

presence of arabinose
= arabinose binds to araC protein -> change shape
= araC is now activated
= araC activates transcription from ParaBAD
= GFP is expressed

75
Q

are ParaBAD and araC naturally found in bacterial genome or artificially made?

A

naturally found in bacterial genome as part of the arabinose operon

76
Q

what are operons? example arabinose operon

A

gene regulation systems found in bacteria
= regulate genes
= allow cells to adapt to changing environment and avoid wasteful production of unneeded proteins

ie: arabinose operon is deactivated when there is no sugar as food source = unnecessary work

77
Q

where is the GFP protein taken from? what is its characteristic ?

A

from a bioluminescent jellyfish

fluoresces bright green under UV light

78
Q

What is the difference between a bacterial lawn and a bacterial colony?

A

colony
= visible group of bacteria from a single cell
= many small circles of different morphology

lawn
= uniform layer of bacteria covering the entire agar plate

79
Q
  • Why was the –pGLO in LB plate a positive control for this experiment?
A

this is a control experiment
= we expect normal conditions
= we expect to see grow of bacteria (colonies)

if no growth, something is inhibiting the natural growth of bacteria

80
Q
  • Why was the –pGLO in LB + ampicillin plate a negative control experiment?
A

no plasmid DNA in the bacterial cell
only plasmid DNA have the amp resistance gene (bla)
= without, it the bacterial cell will not grow
= the amp will inhibit the growth of the bacteria

= we expect no growth to check if the amp works correctly
= confirms that the other growth results will be due to correct transformation, and not contamination/ unexpected amp resistance

81
Q
  • Can you explain from the results of the four bacterial plates why the pGLO plasmid was successfully
    transformed in this experiment?
A

-pGLO LB/ amp = no growth
= confirms the only the pGLO is resistant to amp
= confirms the growth is due to transformation and not contamination

-pGLO LB = growth
= confirms bacteria works normally and no contamination

+pGLO LB/amp = growth
= confirms good transformation
transformed bacteria will have the bla gene and resist to amp = survive amp and grow

+pGLO LB/amp/ara = growth + glow
= confirms araC gene is activated -> glow
= confirms the expression of the GFP gene

82
Q
  • What does transformation efficiency measure? How would you determine this efficiency measure
    from the four plates in this experiment?
A

goal of experiment is to do express a new phenotype
-> you want to know what your starting point is
=

83
Q

why did you take 6 colonies of bacteria and transfer it into a tube? why not just take a swab of a bacteria (strike the loop through the agar)? what happens if you are not gentle with the colonies?

A

you want high transformation efficiency
= must take individual colonies, one at the time, to ensure they can replicate well
= you will choose the biggest ones = most efficient
(if you break are too harsh with the colonies, the cells will break open and die)

84
Q

What was the purpose of the transformation solution, CaCl2?

A

it neutralizes the repulsive negative charges in:
- phosphate backbone of the DNA
- phospholipids of the cell membrane

= allow DNA to enter the cells during heat shock

85
Q
  • What was the purpose of the heat shock procedure (ice-heat-ice)?
A

it makes the bacterial cell membrane more permeable

  1. ice
    = slows down cell activity
    = DNA can come in contact with bacteria
  2. heat shock
    = bacteria cell membrane is more fluid
    = plasmid DNA can enter the cell
  3. ice
    = stabilize membrane & prevent plasmid from leaving the cell
86
Q
  • Why was conducting the experiment with aseptic technique (as much as possible) so important?
A

prevent contamination of bacterial plasmid
if contamination:
= react with the genes plasmid = unwanted

+ ensure that only wanted bacteria is present when reading the gel at the end

= better int

87
Q

define palindromes

A

sequence of base pairs that are identical when read forward and backward

5’ - GAATTC - 3’
3’ - CTTAAG- 5’

when read from 5’ to 3’ = same thing

88
Q

Explain the use of restriction endonucleases by bacteria to destroy viral DNA and how they protect their own DNA by methylation?

A

bacteria use restriction enzymes to cut foreign dangerous DNA
= defense mechanism

methylation
= bacteria protect their DNA by adding methyl groups to the bad DNA

89
Q
  • Can you describe what a recognition site is and restriction fragments?
A

restriction enzymes recognize specify restriction sites

the enzyme come and cut the sugar-phosphate backbone at 2 defined location within this restriction site (1 cut in each strand)
= create restriction fragments

90
Q
  • Can you explain what are “sticky ends” and “blunt ends” when a restriction site is cut by its enzyme?
A

restriction enzymes create fragments
= either sticky or blunt

sticky
restriction enzyme cuts each strand at different points within the restriction site
= form 2 uneven DNA ends
5’- G AATTC -3’
3’- CTTA A -5’
(overhand is 5’, where the shortest end is)

blunt
restriction enzyme cuts both strands at the same point within the restriction site
= form 2 straight and even DNA ends
GG CC
CC GG

91
Q
  • When preparing the pGLO plasmid for digestion with various restriction enzymes, why was a buffer
    added?
A

to create optimal environment fo the enzyme to work efficiently
= maintains same pH
= makes sure the enzyme do not denature

92
Q
  • Why were the restriction enzyme(s) + pGLO plasmid samples placed in a 37oC bath and then heat-
    shocked at 65oC?
A

37 degrees = optimal pH for enzyme
65 degrees = outside the range for an enzyme = denature the protein

93
Q

what did gel electrophoresis procedure separate?

A

gel electrophoresis separates DNA fragments produced by restriction enzyme

94
Q

how does the charge affect the displacement of the DNA in gel electrophoresis ?

A

charge
= determines the direction of molecule
(DNA = negatively charged -> move down the gel, towards the positive electrode)

95
Q

how does the size/shape affect the displacement of the DNA in gel electrophoresis ?

A

size
= smaller DNA move faster through the gel
= large DNA fragments stay near the wells (slow)

shape
= coiled DNA move faster (more compact)
= flat DNA move slower

96
Q

What is the unit of size for DNA?

A

base pairs

97
Q

what were the 2 purposes of adding 6X loading dye to each digested sample before loading them onto a gel? how do you know when to stop

A

to see the DNA
use 2 dyes :
1st: loading dye to allow us to track the DNA movement and stop the experiment before DNA escape from the gel
2nd: fluorescent dye to allow us to see differentt sized band (under UV light) afterwards

98
Q

What was the purpose of loading a lane in the gel with uncut plasmid? why were there two bands in this lane?

A

it is a control experiment
plasmid are artificially made; make sure that they were correctly made circular

uncut plasmid
-> exists in 2 forms : relaxed plasmid and coiled plasmid
-> coiled will travel further = bottom band
-> relaxed will travel less far = top band

99
Q

why would DNA bands move towards the positively charge electrode in a gel when there’s current running through the gel?

A

because the DNA is negative = attracted to positive electrode

100
Q

why are smaller DNA bands migrating further down the gel ?

A

small DNA = less resistance as they move through the gel

101
Q

what happens if you don’t pay attention to the gel and leave it for hours? where to DNA bands go?

A

it will leave the gel and go in the buffer solution

102
Q

why is the gel covered by a buffer solution in electrophoresis?

A

maintain stable pH good for stable enzyme and DNA

to allow ions to carry electric current through the gel
= allow DNA to move across the gel

keeps the gel from drying out (stop the migration)

Biology 2 (6 decks)

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