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bronzemink469Lv1

28 Sep 2019

**Experiment 2: Analysis of phosphate acquisition mutants of E. coli.

Present the table from the handout on which you recorded your results. (No marks, will be used as a guide for marking Q2) â¨

Strain

Growth on Pi plates(yes/no)

Growth on G3P plates (yes/no)

AP-ase assay colour

1

No

Yes

Yellow

2

No

Yes

None

3

Yes

Yes

Yellow

4

Yes

Yes

None

Use your results to determine the genotype of each strain (bacteria are haploid, so you would use the terms â(loss of function) mutantâ or âwild typeâ) and complete the table below. (1 mark) â¨

Genotype

Strain

Alkaline phosphatase

Pi transporter

1

2

3

4

Explain how you reached your conclusions for the genotype of each strain. (1 mark) â¨

Why is growth on G3P is an important control for this experiment? (2 marks) â¨

Suppose the DNA sequences for the alkaline phosphatase and the Pi transporter genes from â¨strain 1 were determined. If both genes are found to contain a single base change compared to the wild type sequences, what could you conclude about the effect of each mutation on the encoded protein? (2 marks)** â¨

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28 Sep 2019

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Related questions

yeast population dynamics

Procedure

1. Work in pairs on this lab, so 12 tubes per pair of students. And share a tube rack with one other pair of students

2. Turn on your spectrophotometer. It needs at least 15 minutes to warm up to give you good readings.

3. Add 5 mL of yeast extract solution (YECM) to each of 12 tubes. (The yeast extract provides vitamins and amino acids for yeast growth and will be the same for all cultures). The tubes should be labeled with your initials, treatment, and tube number. Tape or Parafilm down the lids of 3 tubes, and label them âCONTROLâ.

Do not touch the insides of the tubes or lids! Try to keep these as sterile as possible!!

4. Add 50 mL live yeast culture to each of the remaining 9 tubes.

5. Add the varying volumes of sugar and/or ethanol using Table 1 below.

6. Use Parafilm to close the tops of each tube, making sure the Parafilm is tight and no air can get in, and label each tube with the following:

Amount of sugar added (mL) Amount of ethanol added (mL)

Name of your group Tube number

Table 1: setup yeast tubes (remember, 1 mL = 1000 mL)
Tube number	Yeast culture medium? (5 mL)	Live yeast culture? (50 mL)	Sugar added (mL)	Ethanol added (mL)
1 â control	YES	NO	0	0
2 â control	YES	NO	0	0
3 - control	YES	NO	0	0
4	YES	YES	0	0
5	YES	YES	0.25	0
6	YES	YES	0.5	0
7	YES	YES	0	0.25
8	YES	YES	0.25	0.25
9	YES	YES	0.5	0.25
10	YES	YES	0	0.5
11	YES	YES	0.25	0.5
12	YES	YES	0.5	0.5

Procedure for measuring absorbance (in absorbance units, or AU)

7. Calibrate the spectrophotometer:

Turn on the spectrophotometer and let it warm up for 15 minutes. You will get erroneous results if you donât let it warm up first.

Be sure the spectrophotometer is set to read at the wavelength of 550 nm

With no tube in the spectrophotometer and the lid closed, use the left-hand knob to adjust the reading to 0% Transmittance/push zero button to calibrate

Insert a CONTROL tube (making sure it is clear, without bacterial contamination which would make it cloudy), and use the right-hand knob to readjust the spectrophotometer to 100% Transmittance.

When reading the absorbance, be sure to line up the needle on the spec with its reflection.

8. Immediately before reading any tube, vortex the tube so that the spinning column reaches the bottom of the tube for several seconds. This is critical! The yeast cells are heavy and will tend to sink to the bottom of the tube, so you must vortex the tubes to resuspend them: otherwise, your spectrophotometer readings will be erroneously low. If the vortex is not enough to suspend the pellet of yeast cells at the base of the tube, take a piece of Parafilm and cover the top of the tube, then cover this with your thumb and shake the tube vigorously. The pellet should dislodge and the yeast cells should be easily resuspended after doing this. Use a Kimwipe to wipe down the outside of each tube, to remove fingerprints and other smudges that could affect the absorbance reading. (COULD BE A POTENTIAL ERROR)

9. Record the absorbance (in absorbance units, AU) for the tube on your data sheet.

10. Repeat steps 5 and 6 for every tube.

11. Leave the spectrophotometer turned on for the next user.

Figures you should include are:

Average absorbance vs. time for the no ethanol (0 mL) treatment

Average absorbance vs. time for the 0.25 mL ethanol treatment

Average absorbance vs. time for the 0.50 mL ethanol treatment

Sugar added vs. average carrying capacity (K). Use different symbols to denote each of the three alcohol concentrations

A. After a limit, the increasing concentration of sugar decreases the carrying capacity and growth rate. This is because at higher sugar concentrations, the medium becomes hypertonic and the yeast cells loss water towards the medium.With increasing concentration of the ethanol, the carrying capacity and the growth rate decreases. Why does this happen?

B. Is there any interaction between the effects of adding sugar and alcohol on yeast?

C. why do some cultures not reach K?

D. What are the potential sources of error and assumptions made in this experiment?

E. What do these results mean in a more general (non-yeast) context?

greenporcupine374

Table 1: Assay Results
Filter Paper Section	Colony	Color Change?	Oxidase Positive or Negative?
Exp. 1 skin	1	yellow to purple Purple	Positive
Exp. 2 shoes	1	Orange to yellow	negative
Exp. 3 phone	1	Yellow to purple	positive
Control	1	No color change	n/a

Insert pictures of your plates after incubation, and the results of the âDrySlideâ for each of the plates below.

Post-Lab Questions:

Which bacterial plates contained oxidase positive bacteria? Which plates contained oxidase negative bacteria? Did both of the colonies from each plate show the same color change? Why or why not? How would you interpret this?

Which bacterial samples have cytochrome c oxidase as the terminal enzyme of their ETC? Which bacterial samples do not have cytochrome c oxidase as the terminal enzyme of their ETC?

3. Is the control test in this experiment positive or negative control? What is the purpose of a negative control?

4. What is the purpose of a positive control?

5. How are mitochondria in eukaryotic cells similar to bacteria that use aerobic respiration?

Question 1

1 pts

Cystic fibrosis is caused by nonsense and missense mutations in the CFTR gene, which encodes for a chloride channel. You are studying cystic fibrosis patients to determine what mutation they possess in the CFTR gene. The difference between the mutant and wild type CFTR genes can be uncovered by examining the CFTR:

	DNA
	mRNA
	protein
	tRNA

Question 2

1 pts

You decide to identify the CFTR mutation by analyzing the genomic DNA of your patients compared to healthy individuals. You specifically are looking to see whether a specific 3' gene truncation has occurred in the patients. You will determine this using hybridization techniques with samples from healthy and CF patients. Which of the following will allow you to accomplish this?

	Using an RNA probe complementary to the region not removed by the truncation.
	Using an RNA probe complementary to the region removed by the truncation.
	Using an DNA probe complementary to the region not removed by the truncation.
	Using an DNA probe complementary to the region removed by the truncation.

Question 3

1 pts

You would like to ensure that this experiment (to determine whether patients have a specific CFTR gene truncation using hybridization) is properly controlled. Which of the following samples must you test?

	The genomic DNA of a healthy individual who does not have cystic fibrosis.
	The genome of a CFTR patient known to have the specific truncation you are trying to identify.
	The genome of a CFTR patient with a missense mutation but full length gene.
	The genome of a healthy individual married to a CFTR patient with the specific truncation you are trying to identify.
	The genome of a patient with muscular dystrophy, which can be due to a trucation in the dystrophin gene.

Question 4

1 pts

To conduct the hybridization experiment, you are trying to decide between using a DNA or RNA probe. Which would be ideal to use and why?

	As both are composed of nucleic acids, using either would result in identical results.
	An RNA probe because RNA has uracil bases.
	An RNA probe because it could also be used in a translation experiment.
	A DNA probe because it is more stable than RNA.
	A DNA probe because RNA cannot bind to DNA.

Question 5

1 pts

Which of the following will lower the Tm of a given DNA strand?

	Increasing the percentage of GC base pairs.
	Raising the pH of the solution from neutral to basic.
	Decreasing the buffer concentration from 50mM NaCl to 5mM NaCl.
	None of the above.

Question 6

1 pts

One step of the Hershey/Chase experiment involved blending the virus/cell mixture before centrifugation and probing the pellet for radioactivity. Why was the blending step necessary?

	To collect the bacteria at the bottom of the tube.
	To break open the bacteria to release the genome.
	To separate the bacteria from the bacteriophages.
	To be able to detect the radioactivity.

Question 7

1 pts

Imagine Hershey/Chase had used an RNA virus (genome composed of RNA) instead of a DNA virus in their experiment. Would radioactivity still have been found in the pellet?

	No, because only DNA can be labeled with radioactivity.
	No, because the RNA genome would not enter the bacteria upon infection.
	No, because while DNA and RNA nucleotides are similar, they are not identical.
	Yes, because DNA and RNA nucleotides are similar.
	Yes, because genome in any form (DNA, RNA, protein) would be labeled similarly.

Question 8

1 pts

Griffith and Avery's transformation experiments allowed us to identify that DNA is our genetic information. Which of the following scenarios would result in bacterial cells that are capable of killing mice upon injection?

	Heat killed non-virulent bacteria is added to a live virulent bacteria strain.
	Heat killed virulent bacteria is added to a heat killed non-virulent bacteria strain.
	A heat killed virulent bacteria that is treated with a nuclease, is then added to a non-virulent bacteria strain.
	Heat killed mouse cells are added to a non-virulent bacteria.

Question 9

1 pts

The human genome consists mostly of non-coding DNA. Which of the following are benefits of this?

	Random DNA mutations generally won't affect RNA and protein function.
	It is faster to duplicate the genome when these are present.
	The existence of introns can lead to multiple variations of proteins encoded by a single gene.
	It is unlikely transposons would exist in the genome if there was too much protein coding DNA.

Question 10

1 pts

Andrew Murray's sister, Andrea, is adding to her brother's work on chromosomes. She is using cells that are unable to synthesize adenine (âade) and histidine (âhis). The plasmid she is currently working with consists of an origin of replication and the Ade gene.

Following her transformation of the plasmid into her yeast, what media will the cells be plated on to select for cells that have picked up the plasmid?