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P 1.) You Have Three Compatible Plasmids and You Want to Know If Name_______________________________ Bacterial Genetics, BIO 4443/6443 Studentp ID#__________________________ Fall Semester 2003 Exam III 1.) You have three compatible plasmids and you want to know if they are conjugative, mobilizable, or nonmobilizable. Plasmid A has an ampR gene (ampicillin resistance) Plasmid B has a kanR gene (kanamycin resistance) Plasmid C has a tetR gene (tetracycline resistance) You use a recipient E.coli that is resistant to rifampicin and perform the following experiments. i.) A culture of cells that contains plasmid A is mixed with the recipient, No allowed to incubate for several hours, and then the mixture is plated on Colonies a plate containing ampicillin and rifampicin. ii.) A culture of cells that contains plasmid B is mixed with the recipient, No allowed to incubate for several hours, and then the mixture is plated on Colonies a plate containing kanamycin and rifampicin. iii.) A culture of cells that contains plasmid C is mixed with the recipient, allowed to incubate for several hours, and then the mixture is plated on a plate containing tetracycline and rifampicin. iv.) A culture of cells that contains all three plasmids, A, B and C, is mixed with the recipient, incubated for several hours, and then the mixture is spread on each of the plates shown below. No Colonies ampicillin and rifampicin kanamycin and rifampicin tetracycline and rifampicin 1.) Is plasmid A self-transmissible, mobilizable, or nonmobilizable? Why (4pts) 2.) Is plasmid B conjugative, mobilizable, or nonmobilizable? Why (4pts) 3.) Is plasmid C conjugative, mobilizable, or nonmobilizable? Why (4pts) 4.) Which of the plasmids are likely to have the genes that encode for pilus formation? (4pts) 5.) Which plasmids are likely to have the genes that encode for a relaxase or nickase? (4pts) 6.) Which antibiotic(s) were used to counterselect against the donor cells? (4pts) Name_____________________________ page 2 7.) Describe (or draw) the events involved in the conjugative transfer of a plasmid. Include the following terms if appropriate (not all the terms are appropriate). Donor, Recipient, Transconjugate, Transformant, oriV, oriT, RNAI, Leading strand replication, Lagging Strand Replication, pilus, Dnase, Relaxase, Primase, Transformasome, Tra pore complex. (10 pts) 8.) Strain A is a his- trp+ Hfr strain of E. coli. Strain B is a his+ trp- recipient. You mix the two strains and grow them under conditions where conjugation normally occurs, before spreading the mixture on minimal plates that contain glucose plus the supplements indicated below. Following an overnight incubation, draw how each plate would be expected to appear. BELOW EACH PLATE, EXPLAIN WHAT GROWS AND WHY. (8 pts) Plate 1: Plate 2: Plate 3: Plate4: Histidine Tryptophan Histidine and No additions Tryptophan Rationale: You repeat the experiment above, this time using a his- trp+ strain of E. coli that carries an F plasmid, Strain A, and the same his+ trp- recipient as a recipient, Strain B. After spreading and incubation, draw how each plate would be expected to appear. BELOW EACH PLATE, EXPLAIN WHAT GROWS AND WHY. (4 pts) Plate 1: Plate 2: Plate 3: Plate4: Histidine Tryptophan Histidine and No additions Tryptophan Rationale: Name_____________________________ page 3 You have isolated a new mutant that confers resistance to the antibiotic naladixic acid (nalR) in an Hfr strain and you would like to determine its position on the chromosome. You know that trpA is located at 27 minutes on the chromosome, hisG is located at 45 minutes on the chromosome, and argR is located at 74 minutes on the chromosome and your nalR strain is wild type for all of these markers. You mate the Hfr strain to an E. coli recipient that is rifR, trpA-, hisG-, argR- for 90 minutes and then spread the cells on a minimal plate that contains i) rifampicin, histidine, and arginine. 100 colonies grow up on these plates. When you replica plate these 100 colonies onto plates that contain ii.) rifampicin and arginine, 24 colonies grow. or iii.) rifampicin and histidine, 3 colonies grow. or iv.) rifampicin, arginine, histidine, and naladixic acid, 19 colonies grow. 9.) What gene(s) is selected for in plate (i), (ii), (iii), and (iv)? (5pts) 10.) Based on your results above, fill in the graph below to determine where the nal gene maps on the E. coli chromsome AND indicate the minute position for nal next to the graph (9pts) 100 s t n a n i The nal gene is b m 10 located at approximately co e r ________ minute on the % E. coli chromosome 1 20 30 40 50 60 70 80 Minutes on the chromosome Name_____________________________ page 4 You’ve identified an operon that controls the expression of the genes that are required to synthesize the amino acid tryptophan. You mutagenize the bacteria and isolate 8 trp- mutants that map to this region. Hoping to understand more, you decide to determine how many different genes are represented in your 8 mutants using a complementation analysis. You isolate F’ factors that each carry one of the trp- mutations. One by one, you transfer each F’ into each mutant to see if the different trp- mutants can complement each other. The ability of each transconjugate to grow in the absence of tryptophan is shown below. Recipient mutant #1 #2 #3 #4 #5 #6 #7 #8 which carries an F’ with mutant #1 - #2 - - #3 + + - #4 + + + - #5 + + + + - #6 - - + + + - #7 + + + + - + - #8 + + + + - + - - 11.) Which mutants have mutations that are in the same trp gene. How many different genes have you isolated mutations in? (10pts) You’ve isolated an additional mutant #9 that appears different from the other mutants in your complementation tests. Its results are shown below. Recipient mutant #1 #2 #3 #4 #5 #6 #7 #8 #9 #10 which carries an F’ with mutant #9 - - - - - - - - - 12.) Describe a type of mutation that could produce the above result for mutant #9 and identify where in the operon the mutation occurs? (5pts) Name_____________________________ page 5 The last mutant you isolate, mutant #10, maps to a different portion of the chromosome from all the other mutations and you suspect that it has landed in a regulatory protein for the operon. You isolate an F’ with mutation #10 on it and perform the complementation analysis as before. In addition, you also try complementing the mutant with the wild type strain and obtain the results shown below. Recipient mutant #1 #2 #3 #4 #5 #6 #7 #8 #9 #10 WT cell which carries an F’ with mutant #10 - - - - - - - - - - + 13.) Assuming that mutation #10 does occur in the regulatory gene for this operon, is the mutation dominant or recessive to the wild type gene? (2pts) 14.) Assuming that this mutation inactivates the regulatory gene, is it more likely that the trp operon is regulated by positively or negatively? Why? (3pts) 15.) You have obtained the following four mutant strains of the naturally competent bacteria B. subtilis. For each strain, describe what effect the mutation would most likely have on the competence of the bacteria and WHY. (8 pts) A strain deleted for comX: A strain with a point mutation in comA that renders the ComA protein constitutively active: A strain deleted for comP: Name_____________________________ page 6 You are trying to knockout the lacZ gene in the naturally competent bacterium Acinetobacter calcoaceticus. You have purified a plasmid that contains the lacZ gene with the gene for kanamycin resistance inserted into the middle of the gene to inactivate it. You mix naturally competent culture of bacteria with a solution of plasmid DNA that has been treated in the following way: i.) The plasmid was linearized and denatured into single stranded. ii.) You linearize the plasmid DNA with a restriction endonuclease before you mix it with the cells iii.) You leave the circular plasmid intact without doing anything 16.) Which procedure(s) will most likely yield transformants? Why or why not? (8pts) 17.) Which of the above substrates would yield transformants if you were to induce competence by using electroporation to transform the DNA? (4pts).
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