BIOL-4101 Exam 1

What is magnification?

ratio of (dimension of virtual image) to (dimension of object)

What is resolution?

the ability of a lens or a lens system to distinguish two objects as separate

T/F : Working distance and magnification are inversely proportional.

True

What is working distance?

distance from the lens of the objective to the coverslip when the specimen is in focus

What is the field of view?

diameter of circle of light in virtual image when mapped onto object

T/F : Magnification and field of view are inversely proportional.

True

What is contrast?

difference in light intensity between the image and the adjacent background relative to the overall background intensity

Why care about contrast?

Because good imaging means good contrast!

What is a spectrophotometer?

measures the amount of light absorbed by a sample

What is the light absorbed by a sample measured in?

absorbance (A)

What is the specific wavelength that many cell types, like yeast cells, absorb at?

600 nm

What reagent was used during lab one?

Lactophenol Cotton Blue

What is Lactophenol Cotton Blue?

Type of dye

Phenol - kills live organisms

Lactic Acid - fixative

Cotton Blue - binds to chitin and reflects blue light

What is the use of Lactophenol Cotton Blue?

Preferentially binds to the chitin in the fungal cell walls, ensuring visualization under the microscope

What happens to the area of the field of view when you increase the objective magnification from 4X to 40X?

decreases by a factor of 10

Why did you subtract the control (blank) A600 value from those of all wells that had samples in Lab 1?

because there might be a background level of absorbance that has nothing to do with cells

Why should you agitate a yeast solution before you draw from it?

it will ensure a uniform and repeatable concentration of cells

What is the purpose of sterile technique?

prevent contamination

What are the three types of yeast growth media?

YPD

SDC

SD-HIS

What is YPD yeast growth media?

rich, undefined media

What are the components of YPD media?

-Yeast Extract

-Peptone

-Dextrose

What is SDC yeast growth media?

minimal, defined media

What are the components of SDC media?

-Synthetic Defined Complete

-yeast nitrogen base with amino acids

-dextrose

What is SD-HIS yeast growth media?

drop out media

What are the components of SD-HIS media?

-Synthetic Defined, minus histidine

-yeast nitrogen base without amino acids

-all amino acids EXCEPT histidine

-dextrose

What are auxotrophs?

organisms that need to obtain a particular nutrient from their environment to grow

What was the auxotroph we looked at in Lab 2?

Yeast strain BY4741

What is the genotype of yeast strain BY4741?

-genetically modified to lack histidine, methionine, uracil, and leucine

-carries deletions of that gene

What is the phenotype of yeast strain BY4741?

HIS-, meaning it is an auxotroph for histidine and requires histidine in the growth media

What are prototrophs?

organisms that can synthesize all amino acids

What is the prototroph we used in Lab 2?

Baker's Yeast

What is the phenotype of Baker's Yeast?

HIS+, meaning it does not require histidine in the growth media

What were the three different ways we analyzed cell concentration in log vs. stationary cells during Lab 2?

1. spot plates

2. spectrophotometer

3. hemocytometer

What are the four phases of yeast growth?

1. Lag phase

2. Log (exponential) phase

3. Stationary phase

4. Death (decline) phase

What is the purpose of spot dilutions?

used to calculate the number of cells in cultures and to observe the growth of strains on different media

What does each row on a spot plate represent?

dilution series from a different yeast culture

What is a hemocytometer?

modified glass slide, into which a grid (counting area) has been etched

What is the purpose of using a hemocytometer?

estimate the concentration of cells in a solution

-counting cells

Of the three yeast strains you streaked on plates (S. cerevisiae BY4741, S. cerevisiae BY4741 HIS3+, and Baker’s yeast), which strains do you expect to grow on SD-HIS media? Explain your answer.

Baker's yeast is a prototroph for histidine, meaning it can make its own histidine and will grow on SD -HIS

Why is ethanol fermentation important?

to refuel glycolysis

-oxygen is not needed for glycolysis BUT a problem is generated

-regenerates NAD+, because glycolysis converts NAD+ to NADH which limits the cellular pool of NAD+ molecules

What are the bad products of fermentation?

carbon dioxide and ethanol

Does ethanol inhibit cell budding or increase cell death?

Yes

What is Trypan Blue?

infiltrates ruptured membranes of dead cells and stains the cytoplasm

What is a hypothesis?

must make testable predictions

What is a null hypothesis?

no effect

p > 0.05

-there is NO statistically significant difference between the treatment groups

What is an alternative hypothesis?

has an effect

p < 0.05

-there IS a statistically significant difference between the treatment groups

In Lab 3, does the control group or the experimental group receive the treatment?

experimental group

What is a positive control?

receives treatment

-This is just for the sake of knowing; we did not have to do a positive control for Lab 3.

What does the p-value represent?

p is the probability of getting a particular result if the null hypothesis is true

The trypan blue causes what?

-viable cells to be left unstained

-apoptotic cells to be stained

Once you calculate the mean and standard deviation, you proceed to the Shapiro-Wilk test. You obtain two results with a normally distributed data set (meaning you obtain two results with p > 0.05). What test will you do next?

T-test

Once you have calculated the mean and standard deviation, you proceed to the Shapiro-Wilk test. You obtain one result that is normally distributed and another that is not (meaning you got one result as p < 0.05 and the other as p > 0.05). What test will you do next?

Wilcox test

What is our gene of interest (GOI)?

YLR327C

Where is the Green Fluorescent Protein (GFP) from?

Jellyfish Aequorea victoria

What is GFP in terms of our project?

Reporter gene - makes a cell with a specific gene glow green

-encodes green fluorescent protein

What is HIS3?

Histidine biosynthesis gene that will allow selection of transformants by growing in a medium lacking histidine

-auxotrophic marker

What are the three steps of a Polymerase Chain Reaction (PCR)?

1. denaturation at 98°

2. annealing at 59°

3. elongation at 72°

cycles: 1 -> 35 -> 1

What all is required for a PCR?

-template DNA

-primers

-Master Mix: DNA Polymerase, dNTPs, MgCl

What is a protein family?

proteins shared a common evolutionary origin, as reflected in their related functions, sequences or structure

What is a protein domain?

distinct functional, structural or sequence units that may exist in a variety of biological contexts

What are homologs?

evolutionary similar genes

What are paralogs?

-type of homolog

-duplication event

-new/related function

What are orthologs?

-type of homolog

-speciation event

-same function

What is the name of the plasmid used in Lab 4?

pFA6a - GFP - HIS3

Steps of making GFP fusions?

-There is an insertion happening

-forward primer -> GFP -> HIS3 -> reverse primer

-The forward and reverse primers will make those two areas the GOI after PCR

-removal of stop codon

What is the purpose of the removal of the stop codon in a GFP fusion?

to ensure GOI and GFP are translated into one large protein

What are dNTPs, and what is their purpose in PCR?

-deoxynucleotide triphosphates

-building blocks of DNA

-provide the four individual nucleotides (dATP, dCTP, dGTP, and dTTP) needed to construct new DNA strands during PCR

What does the 35 cycles of PCR represent?

-making billions of strands

-amplification

T/F : If you have a HIS+ gene, it will be unable to grow on an SD-HIS culture.

False

Which cultures do we use in Lab 2 - Yeast Culturing Techniques?

log and stationary phase yeast

What instrument is used to measure optical density (OD)?

spectrophotometer

T/F : Yeast can respire with or without the presence of oxygen.

True

Under which conditions, does yeast undergo fermentation and produce ethanol?

oxygen absent or low

T/F : Yeast cells are eukaryotic and replicate by budding, fission, or spores.

True

T/F : Under stressful conditions, such as nutrient depletion, diploid yeast cells can reproduce sexually via a process called sporulation.

True

What are the effects of an optical density drop?

1. decrease in cell division

2. cells could be dying

What is a negative control?

negative control does not receive treatment

What is the function of the final readout in Lab 3?

percentage of live and budding cells

When would you reject the null hypothesis?

p < 0.05

-means alternative is favored to be true

When would you fail to reject the null hypothesis?

p > 0.05

-means null is favored to be true

What is the purpose of using trypan blue in Lab 3?

to show dead cells

What specific test is used after finding mean and standard deviation on RStudio?

Shapiro-Wilk test

Why is Lab 4's YLR327C clinically or environmentally important?

-We know it binds to ribosomes but do not know function

-could be a stress response gene, because it is up-regulated in stressful situations.

What does each part of this gene mean?

YLR327C

Y - yeast gene

L - letter in alphabet -> chromosome number

R - right arm

327 - genes away form centromere

C - Crick strand

What does ORF stand for?

open reading frame

What is the template DNA in Lab 4?

plasmid pFA6a

What is the amplification sequence in Lab 4?

GFP and HIS3

How did we confirm that PCR worked in Lab 4?

agarose gel electrophoresis

How did we confirm that gel electrophoresis worked in Lab 4?

band of fluorscence

T/F : HIS+ strains can make their own histidine.

True

T/F : HIS- strains cannot make their own histidine.

True

What is an ori?

Origin of replication for making more plasmid in bacteria.

How are primers designed?

-Primers bind to the start and end of the GFP-HIS3 sequence on pFA6a.

-But extra sequences ("tails") are added to the 5′ ends of the primers:

a. The forward primer tail matches DNA upstream of your GOI's stop codon.

b. The reverse primer tail matches DNA downstream of the stop codon.

-These tails make the final PCR product compatible with your GOI in the yeast genome.

-During PCR, the tails become part of the amplified fragment.