Microsoft word - ssq1rev.doc

Student-written practice questions: Midterm 1
1. You are running an experiment in which you want to study the metabolism of E. coli. You know that you have a pure culture because you’ve done a nice series of isolation streaks, and you’re excited to inoculate a new broth culture so you can perform your assays the next day. You perform a series of standard assays including OF medium and motility, although you notice unusually reduced growth in your broth tube. When you come in the next day you see that, contrary to every other motility test performed on E. coli, your bacteria just haven’t moved. Nor have they grown as much as you expected. Both your oxygenated and deoxygenated OF tubes show growth but the tube exposed to oxygen shows no more growth than that with an oil cap. Similarly, a broth culture of strictly aerobic bacteria prepared at the same time as your E. coli broth shows significantly less growth than normal. You look at your labmate’s bench and notice three empty bottles labeled “resazurin,” “dinitrophenol,” and “GTP.” Although you know what resazurin and GTP are, you look up dinitrophenol and find that it is a hydrophobic molecule that can reversibly bind to H+. After thinking about your results, you realize that your untrustworthy labmate must have sabotaged your experiment by adding one of the three substances to your tubes. a) What are resazurin and GTP?
b) Which substance do you think your labmate added to your tubes? Explain why this
addition would cause the growth and motility phenotypes you observed.
2. Killer Bacteria! The National Institute of Health (NIH) needs your help. The director of the
NIH, who unfortunately has never taken Microbiology, recently learned that hyperthermophiles
can survive in environments inhospitable to humans. Now he is afraid that hyperthermophiles
have the potential to take over the Earth, and has called for the research scientists of the NIH to
begin developing antibiotics to use against these “robust invaders.” The director argues that if
these microorganisms have taken over such hostile environments such as the hot springs of
Yellowstone National Park and deep-sea vents, it seems that nothing should be able to stop them
from colonizing the Earth. Fortunately, you manage to catch the director right before he calls for
a press conference, and it’s your job to convince him that his fears are completely unfounded.
a) Describe three characteristics of hyperthermophiles that would make them incapable of
colonizing the Earth.
b) Briefly make a case why the NIH should provide funding to study these microorganisms
further, rather than undertaking an ill-planned campaign of mass-extermination.
c) If there was a legitimate cause for developing antibiotics that target hyperthermophiles
specifically, how could such a drug work?
3. You and your lab partner are world-renowned marine biologists and avid hydrothermal vent
enthusiasts. On one of your recent dives, you discovered an organism that inhabits extremely hot
hydrothermal vents. You name this organism Rachiamisus and are currently in the process of
writing up your scientific paper that will announce your findings to the scientific community.
Thus, you are attempting to further characterize attributes of this new species.
After running a thioglycollate test, you find that your bacterium grows primarily below the pink
line. You also find that upon running an oxidation-fermentation test using glucose as your sugar
source, you observed no growth of your bacteria, and no color change in either test tube. During
your testing, you also performed a gram stain, and based on the results of this test, you decided
to examine whether your bacteria was susceptible to treatment of penicillin, and find that it is.
Based on the environment in which it was found, and the above listed test results, what
bacterial characteristics will you describe in your ground-breaking Rachiamisus
publication in Nature?

4. You and your lab partner are investigating the function of RNA in protein production in
bacteria and eukaryotes. You find that a bacterium and eukaryotic cell that produce the same
protein, Protein X. You manage to isolate the gene that encodes for protein X from the gDNA of
each cell. You also isolate the mRNAs for this gene from each organism. Your lab partner
suddenly faints and needs to go to the hospital. He forgets to label gDNA and mRNA tubes
before he leaves.
a) What do gDNA and mRNA stand for?
b) Describe how you would differentiate between the two gDNAs and the two mRNAs.

5. You have managed to isolate a pure culture of a mysterious bacterium that you are
investigating. You decide to grow a broth culture of this bacterium for an experiment, but forget
to label the tube before putting it in the shaker to grow overnight. One of your lab mates is
investigating two different kinds of bacteria, one of which is the same bacterium that you are
studying. He is also growing broth cultures, but he mistakenly inoculates the same tube with
both bacteria. While he realizes his mistake almost immediately after, he is in a rush to get out
of lab and so decides to put the mixed culture broth in the shaker overnight and deal with it the
next morning. Unfortunately, he also forgot to label the tube.
The next morning, both you and your labmate arrive to remove your broth cultures and realize
that neither of you remembers which one is yours. However, you do know that your bacterium is
sensitive to ampicillin, while your lab mate is certain that the second type of bacterium he is
studying does not demonstrate this property.
Using this information, explain one possible way that you could determine which tube
contains your pure broth culture and which contains his mixed culture as well as the
results that you would expect.

6. On your first day at your new lab, the lab supervisor tasks you with culturing a new species of
bacteria, Thermus alkaliphilus, recently isolated from volcanic hot springs. As the name implies,
the bacteria grows only at high temperatures and high pH. You place a sample in a high pH
glucose broth with all the necessary proteins needed for typical bacterial growth and incubate at
70oC. After several days, you notice something is amiss; there is almost no growth.
Remembering the properties of alkaliphiles, you realize that your broth is likely lacking enough
of one simple ion.
a) What is this ion and why is it needed for alkaliphilic growth?
After obtaining a stable culture, you collect genomic DNA and order primers designed to
amplify a stretch of DNA that codes for a protein common to most bacteria in the Thermus
genus. To perform PCR you add your template, the primers, dNTPs, Taq polymerase, Mg2+, and
the necessary buffer solution. Then your lab partner adds a very small amount of
dideoxynucleotides to the reaction tube. You notice him doing this and try to stop him because it
isn’t on the list of things to put into your reaction but it’s too late. Your lab partner insists that it
won’t do any harm so you put your tube into the thermocycler and run the reaction. The next
day you return to lab and decide to run a gel on your PCR product.
b) What would you expect to see on this gel and why? Describe any differences from the
results you would have expected had you done the experiment without your lab partner
“helping.”
You repeat the PCR experiment, following the protocol carefully this time, and then decide to
sequence your PCR product. Unfortunately, both you and a fellow lab member forgot to label
your samples. Luckily, his DNA sample comes from a mesophile found in normal pH conditions.
You decide to go ahead with sequencing, labeling one sample A and the other sample B. After
sequencing, you search a data base and find multiple stretches of sequence A that are associated
with beta sheet formation in protein and several stretches in B associated with alpha helix
formation.
c) Using this data alone, which sample is likely from your Thermus bacterium? Explain
your answer.
d) If you wanted to find further evidence for your assertion, what types of amino acids
would you expect to be most coded for in your sequence? Why?
7. You’ve been selected to partake in a new reality T.V. series “The Lab,” where this season 8 microbiologists have been chosen to live together and compete in various scientific contests with the hope of winning a year in a fully funded lab, with the added bonus that you may make a new scientific discovery witnessed by all the world. Your first contest involves altering a strain of bacteria that when plated produces colonies with a unique phenotype. Everyone was provided with the well known and sequenced bacteria B. bear. So far, your colonies are not looking so hot, but you notice that your lab mate Jody is really chugging along and has created B. bear bacteria that form spotted red colonies. You realize that this is brilliant and you need to figure what he has done to create his spotted bacteria with the hopes that it will inspire you to come up with something equally as amazing. Therefore you sneak into lab while everyone is attending the evening lecture and find Jody’s lab materials. You find several things: 1. A plate with the red spotted colonies of B. bear 2. A large stack of unlabeled plates, you can tell they are not just standard full media plates 3. A sheet with the names and chemical structures of 3 molecules: Aba, Daba, and Doo 4. Four glass slides labeled "Bb genome chip" 5. A “cDNA synthesis and labeling kit” To make sure that the spotted colonies were produced from a pure sample of B. bear, you do several isolation streaks on Jody’s special plates and indeed all colonies are spotted. When you look at live cells from a spotted colony under the scope, you see some white and some red cells. “How interesting,” you say to yourself, “even though all cells contain the gene Jody manipulated, they have different phenotypes!” a) Briefly explain why you know that both white and red cells within a colony contain
Jody’s special gene.
b) What is the most plausible explanation for how both white and red cells can have the
same gene but display different phenotypes?
Next, you need to figure out the mechanism behind this phenotype it in order to stop Jody from sweeping the contest and taking the fully funded lab out of your reach. You assume that Aba, Daba, and Doo have some significance to the specific phenotype so you isolate a colony and grow it in a broth culture, incubated at 37°C overnight. You come back early the next morning and plate the broth culture on seven different plates that you have created as shown in the table below. After incubating the plates at 37°C, you return at night and see the colonies have grown sufficiently and you observe the results shown in the table. Plate # Aba Daba Doo
Colony phenotype
spotted red&white
For your next experiment, you scoop cells from plates 2, 4, 5, 6 and 7 and use the cDNA kit to label cDNAs from cells on plate 6 red and cDNAs from cells on the other plates green. What does cDNA stand for?
What type of molecule do you need to isolate from the cells to synthesize cDNA?
Describe two reagents that must be found in the cDNA kit .
You divide the plate 6 cDNAs into four tubes and add the same amount of one of the other cDNAs to each tube. Next you incubate each cDNA mixture on its own glass slide, wash the slides, then put them through the chip scanner in your lab. Your results are shown on the following table. all yellow spots
all yellow spots with one red/yellow spot
all yellow spots with one red spot
all yellow spots with one red/yellow spot
Interestingly, the red and red/yellow spots are all located in the same place on each slide. d) What do these results tell you about gene expression by Jody’s strain on each of the
plates you tested (#2, 4-7)?
e) Using the results of your two experiments and bearing in mind that the structures of Aba
and Daba look very similar but not identical, describe what you can infer about the gene
Jody created in his strain, including why it produces spotted colonies on his special plates.

Source: http://bowdoin.us/faculty/a/amcbride/courses/bio218/dissemination/SSQ1.pdf