Name First Last (Please Print) PID Number - HOUR EXAM II BIOLOGY 422 FALL, 2010 In the spirit of the honor code, I pledge that I have neither given nor received help on this exam. 1 Signature 2 3 4 5 6 7 8 9 10 1
1. Selection (8 points) You performed a conjugation experiment between two strains of bacteria with the following genotypes Hfr: val + tyr asn + mal + lac suc tet S amp R F : val tyr + asn + mal lac + suc + tet R amp S A. (6 points) What would you add to minimal media to select for recombinants with the following genotypes? Check the boxes of the ingredients you will add to the media. Genotype Val Tyr Asn Glu Mal Lac Suc Tet Amp val + suc + amp R B. (2 points) A friend in another lab has offered you some media she thinks might be helpful. What recombinants would you select for with the following media? (2 points) Minimal media with val, tyr, mal, and tet: 2. (7 points) You are interested in the relative positions of the following genes ade, trp, thi. You grow the phage P1 on an E. coli strain which is ade --, trp +, and thi +. You then use the phage to transduce the genes into an E. coli which is ade +, trp --, and thi --. Gene Selected % selected colonies which are ade + trp + thi + trp + 10 100 30 thi + 80 30 100 Make a diagram showing the gene order and relative positions. Why didn t you select for cells which were ade +? 2
3. A. (10 points) TYMO viruses infect plants. Describe the mechanism by which a TYMO virus produces its proteins. Be specific but concise and mention the steps and enzymes involved. B. (2 points) One of the animal viruses we studied is similar to TYMO virus in the way it makes proteins. Which virus? How is it similar? 3
4. Transposon Mutagenesis (12 points) You plan to identify the mechanism used by a newly identified bacterial species (Pseudomonas detoxicas) to breakdown toluene (a toxic benzene based chemical found in many paints, paint thinners, adhesives. It is a byproduct from refining petroleum). You plan to introduce a transposon into P. detoxicas via a plasmid (containing the transposon and transposase) that cannot replicate in P. detoxicas. You plan to construct the transposon plasmid in E. coli, purify the plasmid from E. coli, and then introduce the purified plasmid to P. detoxicas via transformation. A) What origin(s) of replication would you put in the transposon plasmid? E. coli, P. detoxicas, neither or both? Why? (2 points) B) Name another important feature of the plasmid. You cannot use the transposase enzyme or origin(s) of replication. Discuss the purpose of this feature and how it is important for creating or identifying transposon mutants. (4 points) C) Once you preform the mutagenesis correctly (you know there were no technical errors because you tested for and found mutants with leucine auxotrophy at the expected frequency), there is a possibility that you will not find single transposon insertion mutants that fail to degrade toluene. What is a possible reason for this? (2 points) D) You test 9,000 potential mutants and identify 65 mutants that failed to degrade toluene. There are 3,000 genes in the bacterial genome. Roughly how many genes are required for the breakdown of toluene? (1 point) E) Besides the enzymes in the pathway that directly breakdown toluene, what is another gene you might identify as required for toluene breakdown in the screen? How does this influence toluene breakdown? (3 points) 4
NOTE. FOR THE PROTOCOLS FOR QUESTIONS 5 AND 6 YOU MAY ONLY USE REAGENTS LISTED ON THE LAST PAGE OF THE EXAM. 5. 14 points You decide to use one of the transposon mutants you isolated in question 4 above to study the genes required for the degradation of toluene. The transposon you used encoded resistance to tetracycline. It sequence is known and when you look it up you find no sites for the restriction enzymes EcoR1 and BamH1. How do you obtain a clone containing the site of insertion of the transposon? (7 points) Your clone was successful. How do you obtain a clone of the intact gene into which the transposon had inserted? (3 points) You grow E. coli containing a plasmid carrying a gene required for toluene degradation behind the lac promoter. What medium do you use to grow these bacteria to obtain expression of this gene? (2 points) You discover that the expression of this gene in E. coli does not allow E. coli to degrade toluene. What is one possible reason? (2 points) 5
6. (10 points) Imagine that you are working as an infectious disease specialist and clinical microbiologist at a local hospital. In the last few weeks, eleven patients have arrived in the emergency room with a form of pneumonia that you have never seen before. You have tested samples from many of the patients and have discovered no evidence of a known pathogen. You have concluded that the disease is being caused by a new pathogen. In addition, it has come to your attention while taking patient histories that several of your patients have recently purchased pet rabbits from the same pet shop. When you ask family members of these patients, you discovered that the rabbits are also showing signs of pneumonia and you suspect a bacterial pathogen. Design a protocol, using the reagents on your reagent shelf, to identify and isolate the pathogen. Remember that you must satisfy the principles necessary to determine that the organism you isolate is the pathogen causing the disease. Make you protocol as a bulleted list. (8 points) What is one factor that can make satisfying the principles mentioned above difficult? (2 points) 6
7. Bacterial Toxins (11 points) Fill in the following chart with the designated information Toxin Modification (e.g. methylation of enzyme Q) of host cell entities by the toxin Molecular effect on the cell (1-2 sentences each) Cholera Organ(s) affected Botulism XXXXXXXXXXX XXXXXXXXXXX XXXXXXXXXXX Diphtheria XXXXXXXXXXX 7
8. (12 points) Innate Immune Response: The innate immune system has several different levels of defense against pathogens. A. List three surface or barrier defenses that are used to stop microbes from colonizing a host. (3 points) 1. 2. 3. B. What are two ways that bacteria can be killed in the phagosome? (2 points) 1. 2. C. Fill in the blanks. (7 points) Phagocytes can recognize invading microbes using that recognize on the microbes. This recognition leads to the production of, which cause, the key features of which are,, and. 9. (6 points) Eukaryotes can sometimes obtain advantages from association with a prokaryote. Fill in the following table indicating two different types of such advantages for two different eukaryotes and prokaryotes. Prokaryote involved in association Eukaryote involved in association Advantage to eukaryote 8
10. Metabolism (8 points) You have E. coli growing in minimal medium under the following conditions: 1) Added glucose, Aerobic 37⁰C 2) Added glucose, Anaerobic 37⁰C 3) Added lactose, Aerobic 37⁰C 4) Added glucose, Aerobic 20⁰C 5) Added glucose, Aerobic 55⁰C Complete the following questions. CIRCLE ONE (1 point each) A) The growth rate of condition 2 is: (FASTER EQUAL SLOWER) compared to condition 1. B) The growth rate of condition 3 is: (FASTER EQUAL SLOWER) compared to condition 1. C) Growth rate of condition 4: FAST SLOW NO GROWTH D) Growth rate of condition 5: FAST SLOW NO GROWTH Fill in the following chart (2 points per row) Organism and Growth Medium conditions E. coli, Minimal medium with Glucose and NO 3 - Thiobacillus H 2 S 2 O 3, CO 2 and salts Anaerobic 37 o C Anaerobic 37 o C Growth Rate (Fast, Medium, Slow, None) Biochemical Process producing energy Electron Donor Final Electron Acceptor 9
Use the following list of reagents to answer questions for this exam. Amino acids vitamins sugars antibiotics threonine (thr) biotin (bio) glucose (glu) streptomycin (sm) leucine (leu) thiamine (thi) lactose (lac) asparagine (asn) maltose (mal) ampicillin (amp) valine (val) sucrose (suc) tryptophan (trp) nucleic acid bases tetracycline (tet) tyrosine (tyr) adenine (ade) neomycin (neo) cytosine (cyt) kanamycin (kan) minimal medium without carbon source complex medium chemicals X-gal IPTG toluene Supplementary Reagent shelf for use in questions 5 and 6 dogs, cats, mice, rabbits, monkeys, parrots, goldfish, maize plants non-specific media for growing bacteria (plates) P. detoxicas competent P. detoxicas P. detoxicas DNA a library of P. detoxicas DNA in a plasmid encoding kan R in E. coli E. coli competent E. coli E. coli arg - tra + rif R carrying punc5 E. coli arg - tra + rif R carrying pam22 DNA of plasmid punc5 mob + amp R which possess a Mini-Tn5 transposon that encodes kan R and has one EcoR1 site and an origin of replication which is only recognized by E. coli DNA of plasmid pam22 that has one HindIII site, one BamH1 site, and two EcoR1 and BglII sites and an origin of replication which is only recognized by E. coli and encodes amp R all the biochemicals and media on the list above all restrictionn enzymes DNA polymerase, DNA ligase, ATP, topoisomerase all enzyme buffers pcr primers and a pcr machine gel electrophoresis apparatus kit to extract DNA kit to label DNA pink kit to extract proteins kit to label proteins blue 10