Name ______________________________________________________ First Last (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. ______________________________ Signature 1________ 2_________ 3_________ 4_________ 5_________ 6_________ 7_________ 8________ 9________ 10 _______ 11______ 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? bio+____________________________________ mal+____________________________________ leu+____________________________________ 2. (12 pts)You perform an interrupted mating between two E. coli strains with the following genotypes: 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: Time 0 5 10 15 20 30 (minutes) 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 grow? Yes or no (circle one) If you replica plated colonies from the medium 3 15 min plate to medium 2 would most of them grow? 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 marker 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) TMV brome mosaic polio influenza 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 bacterium. Reagents: 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 bits. 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 _____________because__________________________________________________________ 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. Reagents: NB bacteria toxin from NB mice, rats, hamsters, sheep, monkeys vaccinia virus formaldehyde methanol 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 1. 2. 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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