Solutions to 7.02 Quiz III

Transcription

1 Solutions to 7.02 Quiz III Class Average = 79 Standard Deviation = 12 Range Grade % A B C 20 > 54 D/F 3

2 Question 1 On day 1 of the genetics lab, the entire 7.02 class did a transposon mutagenesis of E. Coli cells that had approximately 4500 genes. Each of the 36 groups produced 18 mutagenesis plates on MacConkey agar. Most of the colonies on these 648 mutagenesis plates were red but a total of 30 colonies were be white. a) Would you expect the red colonies to be blue on Xgal plates? Explain. (3 points) Approximately 1/6 of the red colonies would be blue on Xgal plates. The cells in these colonies would carry the transposon inserted adjacent to a promoter such that the lacz gene is in the correct reading frame and transcribed and translated. Each of the 36 groups produced 1 LB Xgal Kan mutagenesis plate. Most of the colonies were white, but some will be blue. b) What color would the white ones be on MacConkey agar? Explain. (3 points) Most of the white ones would be red on MacConkey agar, but approximately 3/4500 would be white (ara ) on MacConkey agar. There are three genes, araa, arab, and arac, that can be disrupted to give ara cells. There are approximately 4500 total genes in E. Coli. c) What color would the blue ones be on MacConkey agar? Explain. (3 points) Most of the blue ones would be red on MacConkey agar, but approximately 3/4500 would be white (ara ) on MacConkey agar. There are three genes, araa, arab, and arac, that can be disrupted to give ara cells. There are approximately 4500 total genes in E. Coli. You streak your mutants to single colonies and then patch them to different plates. You see the following. M9 Ara Kan M9 Glu Kan LB Xgal Kan LB Ara Xgal Kan Mutant 1 no light blue dark blue Mutant 2 no no blue blue Mutant 3 no dark blue dark blue Mutant 4 no white white d) Predict where the transposon may have inserted in each mutant. Be as specific as you can in terms of gene names (i.e., araa, arab, arac, ara D, genex, etc.). Clearly note any ambiguities. mutant 1 araa or arab (2 points) mutant 2 genex required to grow on minimal (2 points) mutant 3 arac (2 points) mutant 4 araa, arab or arac (3 points) 2

3 Question 1, continued You prove that the Ara phenotype of mutant 3 from the previous page is due to an insertion of the transposon in an ara gene. Suppose you infect strain A (phenotype = KanS, Mal-, Ara+, ΔLac, Thr-) with a P1 transducing lysate made from the Ara- mutant 3 (phenotype = KanR, Mal+, Ara-, LacZ+ constitutive, Thr+). *Mal- indicates that there is a mutation in a gene required to break down Maltose. Thr- indicates that there is a mutation in a gene required to synthesize threonine. Mistakenly, You plate your transduced cells on LB plates instead of LB kan plates and obtain 10,000 colonies. Type LB Kan M9 Glu M9 Ara M9 Mal M9 Mal Thr M9 Glu Thr LB Xgal # of this type 1 grows grows No No 2 No No No No 3 No No No No 4 No grows grows No grows grows blue 20 No grows white 9960 grows grows white 19 No grows white 1 e) What are the phenotypes of the transductants with respect to Kan, Mal, Ara, LacZ, and Thr? If the phenotypes are ambiguous, use a?. For example, if the ara phenotype cannot be determined from the data, write ara? (8 points total) For each row, kan Ara lacz = 1; thr mal = 1) Type Phenotype: Kan Ara Mal LacZ Thr 1 kanr kans + _ 3 kans kans + + f) Circle the correct statement(s). (6 points total) 2 points for each correct, -1 for each extra Ara is linked to mal Ara is linked to thr Ara is not linked to either mal or thr Ara is linked to both mal and thr Ara is linked to kanr Ara is linked to lacz 3

4 Question 2 (points) 7.02 was so much fun that you come back as an undergraduate TA. As a TA you decided to study histidine synthesis rather than arabinose breakdown during the genetics module. The histidine operon is diagrammed below. hisr encodes a regulatory protein that binds near the P E-A and hisa-hise expression is influenced by the presence or absence of histidine. P R P E-A his R hise hisd hisc hisb hisa a) If hisr encodes an activator, when would you expect it to bind near P E-A : when histidine is present or when histidine is absent? (2 points) absent b) If hisr encodes a repressor, when would you expect it to bind near P E-A : when histidine is present or when histidine is absent? (2 points) present You and another undergrad TA use the same approach, transposon mutagenesis, to isolate bacteria that cannot synthesize histidine. The transposon you will use looks like this: tet lacz *Note: the lacz gene has no promoter and no start codon. tet encodes for resistance to tetracycline (tet) and has its own promoter and start codon. c) You deliver the transposon to a strain called TA100 using a modified λ phage, λ1211. When wild type λ phage infects a host cell, what happens to the host cell? Lysis or lysogeny. (4 points) The host cell can facilitate the production of many new phage particles and eventually lyse. The host cell can stably integrate the λ phage genome into its chromosome. How must the modified λ phage, λ1211 differ from a wild type λ phage? (3 points) 1) λ1211 would need to be modified such that it can t lyse our host cells. There must, however, be a host that can be lysed by λ ) λ1211 would need to be modified such that it can t integrate its DNA into the host chromosome. 3) It also needs to have the transposon DNA as part of the phage genome. d) What must be the phenotype of the TA100 cells (the starting strain before mutagenesis) to allow identification of his mutants and cells carrying the lacz translational fusion? (8 points) Histidine + Lac Sensitive to tetracycline Able to express transposase 4

5 Question 2, continued Your stated goal is to identify cells that have a mutation in one of the genes needed to synthesize histidine. e) What media would you plate TA100 cells on directly following mutagenesis with λ1211? Circle the best choice from the list below. (3 points) Mac Ara Kan LB Ara Kan M9 + glucose Ara Tet M9 + glucose M9 + glucose Xgal Ara Tet LB Tet M9 + glucose Xgal M9 + glucose His ii) Does this media allow you to screen for mutant cells? If so, explain how you would identify the mutant cells. (1 point) No. iii) Does this media allow you to select for mutant cells? If so, explain how you would identify the mutant cells. (2 points) Yes. Mutant cells are tetr. iv) Does this media allow you to screen for his cells? If so, explain how you would identify the his cells. (1 point) No. v) Does this media allow you to select for his cells? If so, explain how you would identify the his cells. (1 point) No. f) You retrieve your mutagenesis plates from the warm room and plan to replica plate. You will need two types of plates. Describe the composition of each plate including media type (M9, LB, or Mac) and any additional sugars/amino acids/drugs you add: Plate #1: Plate#2: M9 Glu His (tet) (2 points) M9 Glu (tet) (2 points) g) Complete the table below to show how replica plating using these plates will distinguish your his mutants from other cells. (4 points) Description of colony on plate. Some examples you used in the lab were:, no, red, white, blue, etc. Phenotype of Cell: Plate # 1 Plate # 2 His, tetr grows No His+, tetr grows grows His+, tetr No No 5

6 Question 2, continued You patch mutants on LB X-gal His Tet and LB X-gal Tet plates. You find no blue cells on either plate as shown below. = blue cells = white cells LB Xgal His Tet LB Xgal Tet M9 His Tet M9 Tet h) Which of the mutants could you use to study the expression pattern of the genes required for histidine synthesis? Explain. (2 points) None are expressing lacz, so none would be useful. You isolate six confirmed his mutants (Z5-Z10) from your original mutagenesis and patch them as you did with the previous mutants: Z5 Z6 Z7 Z5 Z6 Z7 Z8 Z9 Z10 \ Z8 Z9 Z10 LB X-gal Tet LB X-gal Tet His i) What do mutants Z7, Z9 and Z10 suggest about regulation of the his operon? (2 points) Mutants Z7, Z9 and Z10 suggest that the presence of histidine in the media represses transcription of one or more of the his genes. j) Z6 likely has a transposon insertion in which gene? (refer to page 4 if needed) (2 points) hisr. 6

7 Question 3 The following is a diagram of the lac operon in E. coli. Enzymes Z and Y are both required for the break down of the sugar lactose. The wild-type operon is regulated by the repressor protein (Lac I), which is continuously produced. P I Gene for repressor a) Name three different components that when mutant could produce a constitutive phenotype. (6 points) P I, laci, or O lacz P ZY O laci Z Y lacy b) What would be the phenotype of a P zy cell? in the m2 mutant? Why? (3 points) P zy would be uninducible. c) A diagram of the arabinose operon in the presence arabinose is shown below. arab, A, D mrna arac O2 I1 I2 arab araa arad = AraC protein Complete the table below. (18 points) The for BW140 is given. If the answer is ambiguous, put a? in the box. Strain without arabinose β-gal AraB AraC β-gal with arabinose AraB AraC BW140 = Δ(lac)U169 low low high low high high Δ(lac)U169, arac::tn10 (lacz, kan) low low low low low low Δ(lac)U169, arac::tn10 (lacz +, kan) high low low high low low Δ(lac)U169, genex::tn10 (lacz +, kan)? low high? high high Δ(lac)U169, genex::tn10 (lacz, kan) low low high low high high Δ(lac)U169, araa::tn10 (lacz, kan) low low high low high High Δ(lac)U169, araa::tn10 (lacz +, kan) low low high high high high 7