BIOL 4101- Exam 2

what was the goal of the transformation technique?

to introduce GFP-HIS3 DNA construct into Saccharomyces cerevisiae strain BY4741 to create a GFP fusion with the gene of interest

What was in the transformation mix for lab 5?

-PEG 3350

-Lithium Acetate (LiAc)

-single stranded carrier

what does LiAc do in the transformation technique?

permeabilizes the yeast cell wall to allow DNA entry

what does PEG do in the transformation technique?

facilitates DNA uptake by bringing DNA close to the yeast membrane

what does the single stranded carrier DNA do in the transformation technique?

protects and enhances transformation efficiency

describe the transformation technique process

1.two tubes of competent BY4741 were centrifuged, then the supernatant was removed.

2. transformation mix (containing PEG and LiAc) was added to each tube

3. the PCR was added to the GOI tube, then the tubes were incubated at 30 degrees Celsius for an hour.

4. once tubes were centrifuged and suspended in sterile water, they were plated on SD-HIS plates

How did we select for transformants in the transformation technique?

• After transformation, cells were plated on synthetic dropout media lacking histidine (SD -HIS).

• The HIS3 gene in the GFP fusion construct acts as a selectable marker.

• Only yeast that successfully incorporated the GFP-HIS3 DNA can synthesize histidine and therefore grow on SD -HIS plates.

• Non-transformants (no DNA added) cannot make histidine and will not grow.

how did we interpret results in the transformation technique?

• Colonies on SD -HIS plates → successful transformants (they took up and expressed the GFP-HIS3 construct).

• No colonies → transformation failed or plasmid not integrated.

• In later labs, fluorescence microscopy or growth under stress conditions will test how GFP expression (and thus GOI expression) changes in different environments.

what was the negative control in the transformation technique?

transformation performed without DNA (PCR)

• confirms that growth on SD-HIS is due to successful DNA uptake, not contamination or spontaneous mutation restoring HIS3 functino

what was the positive control in the transformation technqiue?

the PCR added tube (it has a strain known to carry a His3+ plasmid)

what is transformation?

the uptake of DNA by a cell

why was the stop codon removed in the GFP gene fusion?

to ensure GOI and GFP are translated into one large protein

what was the purpose of the spot dilution assay?

it tests how the GFP-fusion yeast strain and WT yeast respond to various stress conditions. it helps you observe cell survival and growth and infer gene function under stress

describe how to make the dilution series in the spot dilution assay

1. add 100 microliters of the yeast culture to column 1 of each row

2. add 90 micro liters of sterile water to columns 2-5

3. serial dilution:

• transfer 10 microliters from column 1 to column 2

• the transfer 10 microliters from column 2 to column 3

• continue through column 5

• each step gives a 10 fold dilution

in the spot dilution assay how would you interpret strong growth across all dilutions?

strain is resistant/tolerant to the stress

in the spot dilution assay, how would you interpret growth only in undiluted spots?

strain is sensitive to that stress condiiton

in the spot dilution assay, how would you interpret the gene fusion growing better than the WT?

the GOI may promote stress resistance

in the spot dilution assay how would you interpret the gene fusion growing worse than the WT?

the GOI may be involved in stress sensitivity

What is fluorescence?

occurs when a photon excites an electron to a higher energy level. the electron loses energy through rotation and vibration before returning to its ground state, emitting a photon of lower energy (longer wavelength) in the process

what fluorophores did we use?

DAPI and GFP

what does DAPI label?

the nucleus

what were we trying to observe from fluorescence microscopy?

1. successful GFP fusion

2. protein localization

3. protein abundance, using fluorescence intensity as a proxy for abundance

describe how fluorescent microscopy works

1. a mercury lamp emits large quantities of photons across the UV and visible spectrum

2. the excitation filter will block most of the light that is not among the excitation wavelengths

3. the excitation wavelengths then strike the dichroic mirror and are reflected along a path that sends them out of the objective lens

4. the objective lens will then focus the excitation wavelengths on the prepared sample

5. emitted light will the be captured by the objective lens and pass through the dichroic mirror

what are excitation wavelengths?

those wavelengths that can be most efficiently absorbed temporarily by the flurophore