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MIT Department of Biology 7.02 Experimental Biology & Communication, Spring 2005
7.02/10.702 Spring 2005
Microbial Genetics Exam Study Questions
These questions—adapted from old exam questions--are meant to help you prepare for the 7.02/10.702 Genetics exam on March 8th, 2005. The exam will likely contain 4 questions based on both lecture and laboratory material. The exam is CLOSED BOOK and CLOSED NOTES , and w ill be held in the lecture hall, during normal lecture time.
These study questions are not meant to be exhaustive, but should give you an idea of what topics you should study. We strongly urge you to work through these questions before looking at the answers, and bring any questions to your Undergrad TA, Grad TA or one of the Instructors.
Answers to these questions will be available on the 7.02/10.702 web site!
Question 1
You are handed an undiluted culture of pNK/KBS1 E. coli , and are told that it contains 4 x 1011 cells/L. You also know that 1 OD550 of pNK/KBS1 = 1 x 10^8 cfu/mL.
a) You want to take an OD550 of this culture. By what factor do you need to dilute the cells to ensure an accurate spectrophotometer reading of 0.25? SHOW YOUR CALCULATIONS.
b) Complete the following sentence to describe how you would make 1 mL of diluted culture with an OD550 of 0.25:
I would add __________ microliters (μL) of culture to __________ microliters (μL) of dilutant to obtain a final volume of 1 mL.
c) If you made a 1:10,000 dilution of a culture with an OD550 of 0.25, and plated 100 μL of that diluted culture onto an LB plate, how many colonies would grow on the plate? SHOW YOUR CALCULATIONS.
To set up an experiment, you mix 3 ml of undiluted pNK/KBS1 cells (titer of 4 x 10^11 cells/L) with 2 ml of P1 phage with a titer of 10^8 pfu/mL.
d) Determine the MOI of this experiment. SHOW YOUR CALCULATIONS.
e) Circle the experiment that the MOI calculated in part d) is more appropriate for:
making P1 transducing lysates OR P1 transduction
Explain your answer in two to three sentences by stating why that experiment requires that type of MOI (i.e. what do you want to happen/not happen in the experiment, and how does this kind of MOI ensure that?).
Question 3
You are interested in studying the ability of a newly isolated E. coli strain to use the sugar arabinose as a carbon source, and decide to perform transposon mutagenesis to identify Ara- mutants. To do this, you infect the E. coli strain with λ702, a modified lambda phage containing a version of the Tn10 transposon diagrammed below:
kan
head+ tail+ att+ int cI+^ P
As seen in the diagram, the modified Tn10 transposon consists only of a kan gene with its own promoter and start codon flanked by two IS sequences (black bars). The λ702 genome contains wild-type head, tail, integrase ( int ), and cI repressor genes, a functional att site, and the transposon. The phage also carries an amber mutation ( P80 ) in a gene required for phage DNA replication. You use λ702 phage to infect an E. coli strain that does not contain an amber suppressing tRNA, but does contain a functional att site in a gene required for motility (swimming). The E. coli strain contains no plasmids. You grow the bacteria, mix the bacteria and phage at an appropriate MOI, and allow the infection to proceed. You then plate on Mac Ara Kan plates, and incubate the plates overnight at 37˚C.
a) Did any bacterial cells get lysed by λ702 during the infection described above? Justify your choice in two sentences or less.
b) Predict whether all, some, or none of the colonies on the Mac Ara Kan plate will be able to swim. Justify your choice in two sentences or less.
c) Predict whether all, some, or none of the colonies on the Mac Ara Kan plate will be white. Justify your choice in two sentences or less.
d) Predict whether all, some, or none of the colonies on the Mac Ara Kan plate will be able to be lysed by an infection with wild-type λ phage. Justify your choice in two sentences or less.
Question 4
On the next page, you will find pictures of four “mystery plates” onto which six bacterial strains have been patched. Use the strain list provided below and the growth/color phenotypes of each bacterial strain to determine the composition of each “mystery plate.”
Please answer the questions listed in the table below for each of the “mystery plates.” Note that each column heading has a list of possible answers to that question, and that a plate may contain more than one added antibiotic, amino acid, and/or sugar.
Mystery Plate #
Type of media?
(LB, M or Mac)
Contains Xgal?
(yes or no)
Must it contain antibiotic(s)? (yes or no)
If yes, which one(s)? (Kan, Strep, Tet, Cm)
Must it contain any added amino acid(s)? (yes or no)
If yes, which one(s)? (Phe, Thr, Leu)
Must it contain any added sugar(s)? (yes or no)
If yes, which one(s)? (Ara, Xyl, Lac)
1
2 NO
STRAIN LIST
(NOTE: all characteristics which are not noted are wild-type)
strain relevant phenotype
MER13 Lac- KanR CmR Phe-
DAK7 Lac-, LacZ+(inducible) Ara- Xyl- Thr- JCA82 Lac- KanR StrepR Xyl-
KTC43 Lac- LacZ+(constitutive) TetR CmR
MSJ50 Lac- KanR Leu-
LMA2 Lac- KanR Leu- Thr-
Question 5
Please mark whether each of the following statements is true or false. If a statement is false , correct it by crossing out and/or substituting words or phrases.
gray (For example: __ False__ The winter sky over Boston is usually blue).
___________ a) λ DNA circularizes upon entering the bacterial host via 12 bp,
complementary sequences.
___________ b) We used the lysogenic life cycle of P1 to generate our P1 transducing
lysates.
___________ c) In 7.02 lab, you used M9 Glu Leu plates to screen for colonies that could not
metabolize the amino acid leucine.
___________ d) Repressors are proteins that bind to DNA and turn off transcription of a gene or
operon.
___________ e) The term phenotype describes the genetic constitution of an organism.
___________ f) The site on the DNA to which RNA polymerase binds to start
transcription is called the promoter.
___________ g) In a selection, both parental cells and mutant cells grow, and can be
differentiated from each other by a visible characteristic.
___________ h) Integration of λ DNA into the E. coli chromosome during the lysogenic life
cycle occurs via homologous recombination.
___________ i) In conservative transposition, the transposon is “cut” out of the donor site and
“pasted” in to the recipient site.
Question 6
You are tutoring one of your hallmates, and he asks you to help them work through the differences between lambda phage and P1 phage. Much to the grad TA’s dismay, your hallmate thinks that “phage are all the same.” Help your friend understand more about P1 by answering the following questions.
(a) When making your transducing lysates, you used P1 phage and not λ phage. What is unique about P1’s life cycle (as compared to λ’s) that allows it to be useful for transduction experiments?
b) In 7.02 laboratory, you performed P1 transduction into KBS1 and C600 strains. For each experimental parameter, circle the correct conditions from the choices given (ONE per parameter!), and explain your reasoning BRIEFLY.
Experimental parameter
Circle the correct condition
Reason for your choice?
MOI of this experiment
HIGH LOW EITHER
shake phage/cells during 30’ incubation?
YES NO EITHER
temperature during 30’ incubation
4˚C room temp.
37˚C
cofactor required
Mg+2 Ca+2 Na+
Question 7
After successfully completing 7.02, you decide to come back and join the teaching staff as an undergraduate TA. During the Genetics module, one of your student groups needs your help in understanding the results of their transposon mutagenesis and P1 transduction.
They explain to you that they started the transposon mutagenesis by mixing 1 ml of E. coli pNK/KBS1 cells with 500 μl of λ1205. To get the titer of the cells, they diluted an aliquot of the E. coli pNK/KBS1 cells 1:25, and took the OD 550 , which they determined to be 0.021. They also titered the λ1205 stock, and found that it contained 10^9 pfu/ml.
a) Assuming that 1 OD550 = 6.3 x 10^7 CFU/ml, calculate the MOI of your students’ transposon mutagenesis? Show your calculations!
b) Do you think that this MOI is appropriate for transposon mutagenesis? Why or why not?
After selecting and screening for putative Ara- mutants, your students characterize one mutant, Q2W1. They find that the mutant has the following growth and color characteristics
M9 Ara Leu Kan
Mac Ara Kan
LB
Kan
LB Xgal Kan
LB Ara Xgal Kan
Phenotype
NG G,
white
G G,
dark blue
G,
dark blue
NG = no growth; G = growth
c) Based on these data, what is the phenotype of the Q2W1 mutant? What gene(s) are likely to have a transposon insertion? Explain your reasoning (i.e. how did you determine the phenotype/genotype?).
Question 7 (continued)
Finally, your students perform P1 transduction using a lysate made from the Q2W1 mutant strain. They infect KBS1 cells with this lysate, and plate the cells on an LB Kan plate. They then patch 20 transductants from the LB Kan plate, and observe the following:
M9 Ara Leu Kan
Mac Ara Kan
LB
Kan
LB Xgal Kan
LB Ara Xgal Kan grid # 1-10 G G, red G G, dark blue G, dark blue 11-20 NG G, white G G, white G, dark blue
They also notice that both their “lysate alone” and “cells alone” control plates are clear (i.e. no growth on either).
d) Is the P1 transduction data above consistent with your expectations? Why or why not?
e) Propose a model that is consistent with ALL the data collected by your students.
Question 9
Debbie asks you to test several modifications of the transposon mutagenesis experiment you did in the GEN module. She wants you to compare the outcome of each experiment to the outcome of λ1205 infection of pNK/KBS1. Here are diagrams of the four different lambda phage she asks you to try:
λ 1205^ kan^ 'lacZ
att site deletion (^) P80(amber mutation)
λ 1305 'lacZ
att site deletion P80(amber mutation)
λ 1405 kan^ 'lacZ
att site deletion P80(amber mutation)
λ 1505^ kan^ 'lacZ att+ P+
You set up the experiments as he asks, and plate the resulting mixture on the same plates you used in 7.02 to select/screen for Ara- transposon insertion mutants.
For each experiment listed below, predict the outcome in terms of number of colonies expected relative to λ 1205 infection of pNK/KBS1 , and explain your prediction briefly.
Phage used
Strain infected
Predicted # of colonies (i.e. none? less? same? more?) Reason for your prediction
λ 1205 KBS
λ 1305
pNK/
KBS
λ 1405
pNK/
KBS
λ 1505
pNK/
KBS
Question 10
Listed below are seven potential strains (A-G) that could result from the transposon mutagenesis performed in 7.02/10.702 lab (using strain pNK/KBS1 and lambda1205). On the chart below, CLEARLY indicate the growth (G or NG) and/or color phenotypes that you would expect on each plate for each strain.
A. The strain was never infected by lambda1205 (and thus did not receive miniTn10).
B. MiniTn10 inserted into the araC gene in the same orientation and reading frame as the araC gene is transcribed.
C. MiniTn10 inserted into the promoter of the araC gene, blocking araC transcription.
D. MiniTn10 inserted into the araA gene in the same orientation, but different reading frame, as the araA gene is transcribed.
E. MiniTn10 inserted into the araB gene in the same orientation and reading frame as the gene is transcribed.
F. MiniTn10 inserts into the gene encoding succinate dehydrogenase (constitutively active promoter, not essential for growth) in the same orientation and reading frame as the gene is transcribed.
G. MiniTn10 inserts into the thrC gene (required for threonine biosynthesis) in the same orientation and reading frame as the gene is transcribed. (Note: thrC transcription is repressed in the presence of threonine.)
A B C D E F G
M9 Ara Leu Kan
M9 Glu Leu Kan
Mac Ara Kan
Mac Lac Kan
LB Xgal Kan
LB Ara Xgal Kan
Question 12
Before performing a transposon mutagenesis, you need to titer the λ1205 that you will use in your experiment. To do this, you perform the following experiment:
- Make 10-5, 10-6, 10-7, and 10-8^ dilutions of the original λ1205 stock.
- Mix 0.2 ml of the 10-5^ phage dilution and 0.8 ml of bacteria in a test tube. Perform this mixture in duplicate (i.e. 2 tubes for the 10-5^ dilution).
- Repeat the mixing of phage and bacteria for each of the other three dilutions (also performed in duplicate), and incubate all 8 tubes on your bench for 30 minutes.
- Take 400 μl from the first 10-5^ reaction tube and plate the phage/cell mix as in 7.02. Repeat the plating for the other tubes, and grow all 8 plates overnight at 37˚C.
- Count the plaques that appear on the plate the next morning.
10-5 dilution 10-6 dilution 10-7 dilution 10-8 dilution
Number of Plaques on Plate # TNTC 79 7 0
Number of Plaques on Plate # TNTC 83 10 1
a) Use the data above to calculate the titer of the original λ1205 stock. SHOW ALL CALCULATIONS!
b) To set up your mutagenesis, you mix 5 ml of pNK/KBS1 cells and 0.25 ml of λ1205. In order for this mixture to produce the MOI you selected in part a), to what OD 550 must you have grown your pNK/KBS1 cells? SHOW YOUR CALCULATIONS.
Conversion factor: 1 OD 550 = 1 x 10^8 cfu/ml
Question 13
You are interested in understanding how the fictional bacterium R. tannyalis regulates genes involved in the metabolism (breakdown) of the sugar rhamnose. You decide to perform transposon mutagenesis to identify mutants defective in rhamnose metabolism. The transposon you use for your mutagenesis--miniTn10- gfp-amp —is diagrammed below. The delivery vehicle for miniTn10- gfp-amp is λ1207—a modified λ phage that can neither lyse nor lysogenize your starting strain of R. tannyalis.
gfp amp
IR IR
Note: gfp encodes GFP, a protein that glows green under UV light; this gene has no promoter or start codon. The amp gene encodes resistance to the antibiotic ampicillin, and has its own promoter and start codon.
a) Name two proteins that the starting R. tannyalis strain must express for your mutagenesis to be successful. Justify your choices.
b) What type of plates would you use to select/screen for putative rhamnose metabolism mutants? What would your desired mutants look like on these plates?
The following table describes the phenotypes of 5 strains isolated from your mutagenesis:
Strain Growth on LB Amp
Growth on M Rhamnose Amp
Growth on M Glucose Amp
**Color on LB Amp
**Color on LB Rhamnose Amp
1 + + + green^ green
2 + - - white^ white
3 + - + white^ green
4 + + + white^ green
5 + - + white^ white
c) Independent of position in the genome, which strain(s) contain translational gfp fusions? Explain your reasoning.
Question 14
After 7.02, you join a laboratory that is interested in identifying E. coli mutants that are defective in chemotaxis (movement toward a stimulant, such as a sugar). You mutagenize a wild type E. coli strain with the miniTn10 transposon from 7.02, and identify an interesting Che- (chemotaxis) mutant. You stabilize the mutation (which occurs in a gene you call cheA ) using P1 transduction, and confirm that the KanR and Che- phenotypes are linked. Your colleagues at another university have identified another E. coli Che- mutant (in a gene they call cheB ). They tell you that cheB maps very close to the his genes, and can also be cotransduced with the trp genes. Using cotransduction, they have deduced the gene order of (and relative spacing and between) cheB, his , and trp to be:
_____ _cheB____his____________________________trp______
To try to determine whether cheA and cheB are the same gene, you decide to map cheA with respect to his and trp. You perform a P1 transduction experiment using the following strains:
Donor: CheA-, KanR, His+, Trp+
Recipient: CheA+, KanS, His-, Trp-
You obtain the following data:
select for His+ (total= 1000) select for KanR (total = 1000)
KanR Trp+ 108 Trp- His- 390
KanR Trp- 212 Trp+ His- 290
KanS Trp+ 5 Trp+ His+ 3
KanS Trp- 675 Trp- His+ 292
a) Determine the gene order of cheA , his , and trp. Show all calculations used , and explain your logic.
Question 14 (continued)
b) Are cheA and cheB the same gene? Justify your answer briefly.
You identify a third Che- strain. The Che- phenotype in this strain arises from a mutation in a gene you call cheC ; the cheC mutation is 100% linked to a gene which confers tetracycline (tet) resistance. You suspect that cheC may be the same gene as cheA , and perform the following P1 transduction experiment to test your hypothesis:
Donor: Che-, KanR, TetS
Recipient: Che-, TetR, KanS
You select for KanR transductants, and test each colony for its sensitivity or resistance to tetracycline.
c) What phenotype(s) (TetR or TetS) would these transductants have if the cheA and cheC mutations were 100% linked (i.e. they are in the same gene )? Explain your answer briefly. (Hint: a diagram may be useful!)
