You next decide to use transposon mutagenesis to identify by gN4R51


									Name ______________________________________________________
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                                           (Please Print)
PID Number __________-__________

                                       HOUR EXAM II

                                        BIOLOGY 108

                                          FALL, 2004

                             In the spirit of the honor code, I pledge that I have
      neither given nor received help on this exam.










10 _______

Use the following information for the questions on the exam.

amino acids            vitamins               sugars                 antibiotics
threonine (thr)        biotin (bio)           glucose (glu)          streptomycin (sm)
leucine (leu)          thiamine (thi)         lactose (lac)          rifampicin (rif)
histidine (his)                               maltose (mal)          ampicillin (amp)
tryptophan (trp)       nucleic acid bases     arabinose (ara)        tetracycline (tet)
arginine (arg)         adenine (ade)                                 neomycin (neo)
                       cytosine (cyt)

1. (9 points) Above is a list of ingredients that you have available to prepare bacterial growth
media for an experiment. You can make complex medium or minimal medium (no carbon
source); be sure to specify which you are using.

You perform a conjugation between two E. coli strains with the following genotypes:
      F-: rifR, leu--, trp--, bio--, lac+, mal--, cyt--
      Hfr: rifS, leu+, trp+, bio+, lac+, mal+, cyt+
What medium would you use to select transconjugants of each of the following genotypes?




2. (12 pts)You perform an interrupted mating between two E. coli strains with the following
F-: RifR leu—thi—lac--
Hfr: RifRleu+thi+lac+

The conjugates are plated on the following minimal medium at the times indicated:
Medium 1: glucose, leu, rif
Medium 2: lactose, leu, thi, rif
Medium 3: glucose, thi, rif

The number of bacteria which grow on each medium are counted are as follows:
                 0             5           10           15           20            30
Medium 1         0             4           17           30           43            56
Medium 2         0             0            5           17           29            41
Medium 3         0             0            2            7           12            17
Graph these data on the following page. Label the axes and indicate which line represents which
medium and mutant.
Draw a diagram below to show the gene order and relative positions in minutes.

 Transferred last                                                     Transferred first

If you replica plated colonies from the medium 1 15 min plate to medium 3 would most of them
Yes or no (circle one)

If you replica plated colonies from the medium 3 15 min plate to medium 2 would most of them
Yes or no (circle one)

What medium would you use to replica plate onto to test whether the leu+ bacteria are lac+?

3. ( 8 points)You wish to determine the gene order and relative distances of three genes from E.
coli. You prepare DNA from E. coli which is his+ met-- ade+ and use it to transform E. coli
which is his—met+ ade--. You select for cells which are his+ or ade+ and test them for the ability
to make his, ade or met. You obtain the following results:

Selected            Percentage of selected cells which are
                    his+         met+          ade+
his+                100          10            30
ade+                20           70            100

Draw a diagram to show the gene order and relative positions

What medium did you use to test whether the ade+ bacteria are his+?

Why didn=t you plate the original transformation on media to select for met+ cells?
4. (8 points) If you were to attempt to infect appropriate host cells with the naked RNA of each
of the following viruses would you obtain virus growth in the cells? Why or why not?

virus                 viral growth            why or why not?
                      obtained (yes or no)

brome mosaic



5. (5 points) RNA viruses that grow in plant or animal cells have to solve the problem that
eukaryotic cells generally only make one protein using one piece of mRNA. Different viruses
have used different strategies to circumvent this problem. Describe the mechanism(s) by which
TYMO virus is able to make more than one protein.

Which mammalian virus uses a mechanism similar to part of that used by TMYO? ___________
 6. (9 pts)You recently visited the state fair and during your visit you won a gold fish. A few days
later you discover your gold fish looks more green than gold. After further examination you find
your goldfish is covered in green spots. So using your superior microbiology skills you isolate
the bacterium which caused the green spots which you name Greenas spottii. You next decide to
use transposon mutagenesis to identify the major genes involved in the pathogenesis of this

Fully stocked supply room with reagents and restriction enzymes
Competent E. coli
Greenas spottii
Competent Greenas spottii
E. coli strain UNC-27 pir+ tra+ his – (carries plasmid pQL5, rifR which contains Tn5 (neoR), pir-
depedent origin of replication)
E. coli strain UNC-18 (carries plasmid pBLT, AmpR, Multiple cloning site (MCS), pir-
independent origin of replication)
Plasmid p108
Phage P1
Toxic chemical
Clone of a toxin gene from another known bacteria which causes green spots
Minimal medium, complex medium
X-gal, rifampicin, neomycin, ampicillin
Glucose, Lactose
Mice, rabbits, gold fish, trout, monkeys, sheep
Antibody to whole cell Greenas spottii

Fill in the missing blanks in the protocol shown below using the reagent list supplied.

1. Grow ___________________ and ___________________________.

2. Mix the two cultures and allow them to _______________________.

3. Select the resulting bacteria you desire by plating on _______________ medium containing


4. Test the surviving bacteria to determine which ones have mutations in genes required for

       virulence by ______________________________________.

   This procedure selects/ screens (circle one) for bacteria which carry the transposon and

   selects/ screens (circle one) for bacteria which are mutant in genes required for virulence.
7. ( 10 points) As a summer project in a microbiology research laboratory, your professor needs
you to clone the gene responsible for the production of shieldin from the bacterium Maximus
resistianii. Shieldin is a protein that neutralizes toxin X. Toxin X is produced from the bacteria
Getchareala sicka, which causes both mice and humans to develop purple blotches all over the
skin. Sadly, you can’t read your professor’s handwritten protocol and must fill in the missing
You have the following materials:

                              plasmid vector pB4Q
E.coli, M. resistianii, G. sicka
competent E.coli, competent M. resistianii, competent G. sicka
mice, sheep, hamsters
all enzymes, chemicals, and buffers needed
the following antibiotics: ampicillin, tetracycline, and rifamicin
X-gal, minimal medium, complex medium
any other desired medium components, except purified toxin X (way too expensive)

Protocol to clone the gene responsible for neutralizing toxin X.

1. Grow_______________________________and purify________________from it.

2. Digest the________________________DNA with________________________.

3. Digest the vector DNA with____________________________.

4. Mix the two digests together and add______________________(enzyme). Incubate.

5. Transform the mixture into_________________________________________.

6. Grow the resultant bacteria and plate them on _______________________________.

7. The colonies on your plate that contain a cloned insert for shieldin appear

8. Identify those clones that produce functional shieldin by
8. (7 points) Cholera toxin is the major virulence factor of Vibrio cholerae. Describe the
biochemical mechanism of action of cholera toxin in the intestinal lumen.

9. ( 10 points) Diphtheria toxin can be fatal in many animals as well as humans. Some rodents
such as rats and mice and most animals other than mammels lack the receptor for the toxin on the
surface of their cells. What do you think would be the effect if you injected diphtheria toxin
subdermally (under the skin) in

monkeys____________                   Why? __________________________________
rabbits ______________                Why? __________________________________
mice_____________                     Why? __________________________________
wheat plants_______                   Why? __________________________________

of adding toxin to a culture of E. coli______________         Why? __________________

You make an extract of each of the above organisms which is capable of carrying out in vitro
protein synthesis as measured by the incorporation of leucine into protein. What do you think
would be the effect of adding diphtheria toxin to the reaction on the activity of the extract from

monkeys___________________            Why? ____________________________________
rabbits ____________________          Why? _____________________________________
mice ______________________           Why? ___________________________________
wheat plants _________________        Why? _____________________________________
E. coli ____________________          Why? ____________________________________
10. (13 pts)

Suppose that you are working as a researcher for the Center for Disease Control. An emerging
infectious disease has recently been discovered to be caused by a bacterium (called for short NB).
 The infection causes a significant number of deaths from severe diarrhea and dehydration. You
are asked to design a new vaccine to protect against this bacterium. Describe 2 methods by which
you might design an immunization against this new bacterium using the reagents supplied below.
NB bacteria
toxin from NB
mice, rats, hamsters, sheep, monkeys
vaccinia virus
sequence for the gene encoding the toxin responsible for this disease
yeast cells
E. coli, NB cells
bacterial plasmid containing a promoter recognized by human RNA polymerase
bacterial plasmid containing an origin of replication recognized by E. coli RNA polymerase
T cells
all necessary bacterial, viral, and yeast growth media



What type of immunity will each of these immunizations provide? ________________________

How many different antibodies can a single plasma cell produce? one two       a few many
(circle one)

 How many different antibody molecules could combine with the surface of a molecule such as
tetanus toxin? one two a few many (circle one)
11. (9 points) On a Thursday night a group of UNC cheerleaders meet for a practice session.
One of them brings her pet hamster to the practice and she and some of her friends play with the
hamster before practice. Susie refuses to play with the hamster because she doesn’t like to touch
animals. The next day the hamster becomes ill with large red spots. By Saturday some of the
cheerleaders have also developed large red spots. (Susie is the only one who is completely
unaffected). The NC State game is in one week and the cheerleaders must get rid of the bright red
spots. They ask you as a microbiologist to figure what is causing this distressing disease. What
do you do? (You have available the contents of the reagent shelf shown above in question 10 for
your experiments).

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