a) Explain the difference between RNA degradation and RNA denaturation.

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1 Question 1 You have been asked to grow large batches of E. coli cells from which 7.02/ students can purify β-galactosidase during the Biochemistry module. These "fermentations" are conducted at the 10-liter scale inside bioreactors, which provide better control of the cell environment than the shake flasks you used to grow E. coli in the Genetics module. You decide to study β-galactosidase transcription in E. coli during the fermentation process. To do this, you first need to isolate total RNA from cells at different time points of the fermentation. During your RNA isolation, you take precautions to ensure that the RNA in your samples does not get degraded. In addition, you want to be sure that your RNA samples are properly denatured before running them on an agarose gel. a) Explain the difference between RNA degradation and RNA denaturation. Lane 1 of the EtBr-stained agarose gel below shows the "expected" pattern of bands from a bacterial RNA preparation. (Note: bacterial ribosomes contain 23S and 16S rrna subunits, which migrate differently on a gel than eukaryotic 28S and 18S rrna subunits.) MW b) In lane 2 of the gel, draw what you'd expect to see if the RNA in your sample was partially degraded, and explain your answer in one or two sentences.

2 Question 1 (continued) You successfully isolate RNA from samples harvested at three time points during the E. coli fermentation, and perform a Northern blot using a protocol similar to that used in 7.02/ lab. Alas, when you develop the film at the end, you see NO BANDS, not even in your positive control lane. You decide to look back at your notes to figure out what mistake(s) you might have made. For each of the following four scenarios: 1. IDENTIFY the mistake; 2. Explain WHY the mistake resulted in a lack of bands on your film; 3. Propose how you would FIX the mistake if you were to redo the entire procedure. Note: each scenario is independent of the others, and you should assume that all other steps in the protocol were performed "as in lab." c) Probe labeling reaction containing zcyt1 cdna template, Klenow fragment, 1 mm each of datp, dgtp, dctp, 0.65 mm dttp, 0.35 mm DIG-dUTP, random hexamer primers, and buffer (Tris-HCl, MgCl 2, DTT, BSA). d) Second wash conditions: 0.5x SSC, 95 C. e) Antibody step: 30 minute incubation with rabbit antibody specific for β- galactosidase, conjugated to alkaline phosphatase. f) Probe detection solution: NBT/BCIP in detection buffer.

3 Question 1 (continued) g) Your coworker says that you can obtain data from the Northern blot developed with NBT/BCIP without redoing any steps of the protocol. Explain how this is possible. Your labmate has performed the Northern blot procedure correctly using RNA isolated from cells harvested at 1 hour, 4 hours, and 6 hours into the fermentation. She observed the following results: X 2X 4X bands) point point point EtBr-stained gel Nylon membrane (arrows indicate position of MW Lane 1: MW standards (all sizes in kb) Lane 2: RNA from "1 hour" time Lane 3: RNA from "4 hour" time Lane 4: RNA from "6 hour" time Lane 5: βgal cdna fragment Northern Blot Film (arrows indicate position of MW bands) h) What three conclusions can you draw from this Northern blot result? Explain how you arrived at these conclusions by referring to specific lanes of the gel/membrane/film.

4 Question 2 You have identified an interesting zebrafish mutant strain. When compared to wild type embryos at 24 hours post fertilization, these mutant fish have abnormally small brains. You have cloned the gene whose function is lost in your mutant; you name the gene lilbrain. You decide to study the expression pattern of lilbrain during the development of wild type embryos by Northern blot. To do so, you need to create a lilbrain-specific-probe. Unfortunately, you have run out of the random-hexamer labeling kit used to make your probe in 7.02/ lab. a) Describe how you could use PCR to make a lilbrain-specific probe by completing the following table: Template used Amount of template required Primer length DNA polymerase used Denaturing temperature Annealing temperature Extension temperature b) PCR performed with the dntps used in lab will generate a labeled DNA probe, not an RNA probe. Why might an RNA probe be a better choice than a DNA probe? After successfully preparing your probe, you isolate total RNA from wild type zebrafish embryos at the blastula, gastrula, segmentation, pharyngula, and hatching stages of development. After separating the RNA on a denaturing agarose gel, you perform a Northern blot using your labeled, lilbrain specific probe. Your Northern results are consistent with a role for lilbrain in brain development, and you next want to see where lilbrain is expressed in the developing zebrafish embryo. c) What experiment discussed in lecture is the best way to visualize if the lilbrain gene is expressed specifically in the brain?

5 Question 2 (continued) d) Name one thing that this experiment has in common with a Northern blot, and one difference between this experiment and a Northern blot. You perform the experiment named in part d), and you observe lilbrain mrna not only in the brain, as you expected, but throughout the entire embryo. You are not sure if this is due to non-specific binding of the probe, or if the lilbrain gene is really expressed all over the embryo. You decide to alter the stringency conditions to distinguish between these two possibilities. e) In order to reduce non-specific probe binding, one could do any of the following (circle the correct choice from each pair): i. increase / decrease salt concentration of washes ii. iii. iv. increase / decrease temperature of washes increase / decrease formamide concentration during hybridization make a shorter /longer probe v. use a probe with more / fewer mismatches to the lilbrain mrna f) Provide molecular explanations as to how altering the salt and formamide concentrations affects the ability of a probe to bind to a target mrna. After altering the stringency conditions appropriately, you find that lilbrain mrna is indeed found throughout the embryo (and not just in the brain). As mutations in the lilbrain gene only affect brain morphology, you suspect that translation of the lilbrain mrna is somehow regulated. You hypothesize that another protein, encoded by the brainiac gene, normally blocks translation of lilbrain mrna in the parts of the embryo where the Lilbrain protein is not needed. g) How could you adapt the experiment named in part d) to detect the Brainiac protein instead of the lilbrain mrna? Be brief but specific.

6 Question 2 (continued) h) If your hypothesis is correct, where in the embryo would you expect to find the Brainiac protein expressed? Explain your reasoning briefly. Question 3 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) In an agarose gel, plasmid DNA that is supercoiled migrates more slowly than DNA that is nicked. b) Isopropanol was used to remove excess salt from precipitated miniprep DNA. c) Ligation of two fragments of DNA by T4 DNA ligase requires ATP. d) AG1111 cells contain T7 RNA polymerase and will therefore express GFP. e) Ethidium bromide binds to DNA is a stoichiometric manner, i.e. the shorter the DNA fragment, the more molecules of EtBr it binds. f) Calf Intestinal Phosphatase (CIP) catalyzes the removal of 5 hydroxyls from DNA. g) Taq polymerase is denatured each time the temperature is raised to 96 C in the PCR reaction.

7 h) Type II restriction enzymes (like XbaI, SspI, and EcoRI) cut DNA at palindromic sites. i) Two primers that have the same length (same number of base pairs) will always have the same Tm. j) Taq DNA polymerase requires a primer to begin DNA synthesis. k) The extension time (time that Taq polymerase spends at 72 C) determines the maximal size of the product that can be produced in a PCR reaction. l) EDTA is a reducing agent that sequesters divalent cations (like Mg+2) away from nucleases. Question 4 The fictitious T. unafish species normally has two pectoral fins, one on each side of its body. Development of these fins requires the proper expression of the pec gene. Researchers have identified "finless" T. unafish variants which lack pectoral fins. These "finless" fish can arise in two different ways: 1) transposon mutagenesis of both copies of the pec gene; 2) treating the T. unafish with caffeine during development, which drastically reduces pec transcription These effects can be observed by performing a Northern blot and probing for pec mrna, as shown below: total RNA lane 1: molecular weight markers lane 2: untreated fish lane 3: pec::transposon fish lane 4: caffeine treated fish Note: all lanes contain equal amounts of (Note: the mrna from pec::transposon fish is larger because of the transposon insertion)

8 Despite their lack of pectoral fins, these "finless" fish are capable of mating and producing offspring. You isolate total RNA from the offspring of two "finless" fish, and perform Northern blots with a pec specific probe. a) What do you expect to see on the Northern blot if the offspring are the result of a mating of two "pec::transposon" fish, and why? (Be sure to indicate both the size and intensity of the pec mrna band as compared to that seen in "untreated" fish.) b) What do you expect to see on the Northern blot if the offspring are the result of a mating of two "caffeine treated" fish, and why? (Be sure to indicate both the size and intensity of the pec mrna band as compared to that seen in "untreated" fish.) c) What will be the phenotype(s) of these two sets of offspring (wild type or "finless"), and why? Write your answers in the table below: Parental fish Offspring phenotype Explanation pec::transposon caffeine-treated

9 Question 5 You have identified an enzyme (SweT) required to metabolize the fictional sugar sweetose in E. coli. You wonder if this enzyme has been conserved during evolution, so you compare the protein sequence of SweT from E. coli with the SweT proteins from two other bacterial species, B. subtilis and S. aureus. While the three SweT proteins are remarkably similar, you do notice a few differences. The E. coli SweT protein (E) is 140 amino acids in length. The B. subtilis SweT protein (B) has a deletion of 30 amino acids at the C-terminus, whereas the S. aureus SweT protein (S) has two point mutations in the sequence from amino acids These differences are diagrammed below: N amino acids 1-75 amino acids Met-Tyr-Lys-Trp-Glu amino acids C E. coli SweT (E) N Met-Tyr-Lys-Trp-Glu C B. subtilis SweT (B) N Lys-Tyr-Lys-Lys-Glu C S. aureus SweT (S) a) Draw the structure of the following pentapeptide (aa 76-80) at ph= 7.0: N terminus---met-tyr-lys-trp-glu C terminus b) You run the three SweT proteins (E, B, and S) together on a gel filtration column. Which protein will elute from the column last? Explain your answer.

10 Question 5 (continued) N amino acids 1-75 amino acids Met-Tyr-Lys-Trp-Glu amino acids C E. coli SweT (E) N Met-Tyr-Lys-Trp-Glu C B. subtilis SweT (B) N Lys-Tyr-Lys-Lys-Glu C S. aureus SweT (S) c) You load samples of all three purified SweT proteins on both an SDS-PAGE and a native gel. Draw the relative migration position of each SweT protein on each gel. (Note: On each gel, lane E contains E. coli SweT; B = B. subtilis SweT; S = S. aureus SweT). - E B S E B S - + SDS-PAGE GEL + NATIVE GEL d) You accidentally pool the tubes containing purified E. coli SweT and S. aureus SweT. What kind of column would you use to separate them, and why? e) You also test the ability of all three species to grow on plates with sweetose as the sole carbon source (M9 Swe plates). You find that B. subtilis and E. coli can grow on these plates, but S. aureus cannot. Based on what you ve learned about the SweT proteins, how do you interpret this result?

11 Question 6 You are interested in studying the regulation of genes in the E. coli his operon (histidine biosynthesis). You do transposon mutagenesis using minitn10,kan, lacz like in the GEN module, and identify a his::lacz translational fusion. You think your insert is in one of three genes--hisd, hisb, or hisa and decide to use PCR to pinpoint your transposon s location. Here is a map of the his operon (the his promoter is indicated with an arrow, and each gene s size is noted beneath it): 5' 3' hisd hisa hisb 3' 5' 1.6 kb 0.8 kb 1.1 kb a) You first construct three sets of Forward and Reverse primers (HisD-For, HisD-For, HisD-Rev, HisB-For, HisB-Rev, HisA-For, and HisA-Rev). Indicate on the map above approximately where these primers will hybridize. (Assume that these primers correspond to the Ara primers you used in the 7.02 lab) You run the following PCR reactions, and load a sample of each PCR reaction onto an agarose gel. Note that the LacZ primer is the same one you used in the RDM module, and that you allowed a 2.5 minute extension time for your PCR. You observed the following: Lane DNA used Primers used 1 DNA size standard none 2 Wild type HisD-For, HisD-Rev 3 Wild type HisA-For, HisA-Rev 4 Wild type HisB-For, HisB-Rev 5 his::lacz mutant HisD-For, HisD-Rev 6 his::lacz mutant HisA-For, HisA-Rev 7 his::lacz mutant HisB-For, HisB-Rev 8 his::lacz mutant HisD-For, LacZ 9 his::lacz mutant HisA-For, LacZ 10 his::lacz mutant HisB-For, LacZ b) Based on this data, where do you think the transposon has inserted? How confident are you about this conclusion, and why?

12 Question 6 (continued) You notice that none of the PCR reactions using the LacZ primer gave products. You decide to design a new primer specific for the transposon, and use this in a new set of PCR reactions. Here is a diagram of the 4.9 kb lacz kan r minitn10,lacz,kan transposon: KanR (for part d) c) You decide to design a primer that hybridizes to the inverted repeat sequence of the transposon. Your undergrad TA strongly discourages you from doing this. Why? d) Instead, you design the primer KanR-For, which hybridizes as shown on the diagram above. What primers (of HisD-For, HisD-Rev, HisB-For, HisB-Rev, HisA-For, and HisA-Rev) could you use in PCR with KanR-For to help you determine the location of your his::lacz transcriptional fusion? (Assume that you use a 2.5 minute extension time for PCR). What would the size of the PCR product you obtain tell you about where your transposon inserted?

13 Question 7 You have two E. coli strains that are identical in every way except that one has an intact copy of the lacz gene (LacZ+) and one has a deleted lacz gene (LacZ-). The strains are unlabeled, and you need to figure out which strain is which! In the chart below, describe five different types of experiments/techniques that you could use to help you distinguish the two strains. Tell me in what module you learned about the experiment/technique, and what you d expect to see if your strain is LacZ+, and if it is LacZ-!! (Remember: Many experiments learned in 7.02 can be adapted to study any gene or protein of interest!) Experimental description what module? if the strain is LacZ+, I d observe... if the strain is LacZ-, I d observe...

14 Question 8 You perform a transposon mutagenesis using l1205 and pnk/bw140 E. coli cells. You select for mutagenized colonies by plating the cells on LB Kan plates. You then wish to screen the KanR colonies for the following: 1) insertions in any gene required for maltose utilization (malt, malp, or malq); and 2) insertions in any amino acid biosynthetic gene. a) Describe how you would screen the KanR colonies for insertions in a mal gene. b) Describe how you would screen the KanR colonies for those with insertions in any amino acid biosynthetic gene. c) What is the expected frequency of mal mutants and amino acid biosynthetic mutants (approximately) among the KanR colonies? d) What do you predict is the frequency of Tn10 insertions into essential genes (genes required for growth) among the KanR colonies? Briefly explain your answer.

15 Question 9 You are studying an organism that lives in hydrothermal vents (like Thermus aquaticus!). You are working with a gene that keeps proteins folded properly at extremely high temperatures, and you need to map a plasmid containing this gene prior to subcloning it. After digesting the plasmid with two different restriction enzymes (XbaI and BamHI), you get the following results on an agarose gel. M M lane M: 1 kb ladder lane 1: uncut DNA lane 2: XbaI lane 3: BamHI lane 4: XbaI + BamHI lane M: 1 kb ladder Draw a restriction map consistent with these results. Be sure to indicate the location of all restriction sites, the distances between restriction sites, and the total size of the plasmid.

16 Question 10 After 7.02, you decide to redo the minitn10 transposon mutagenesis from the Genetics module and see what other types of mutants you can isolate. One of the mutants you isolate has a remarkable phenotype it turns blue and dies rapidly when the temperature of the incubator reaches 39 C. To begin to understand this phenotype, you decide to obtain the sequence of the DNA where the Tn10 inserted. a) Name the technique that can be used to get the sequence of the DNA flanking your Tn10 transposon. After performing this technique and sequencing the DNA, you find that your Tn10 has disrupted an open reading frame of unknown function. You clone the open reading frame, express it in the mutant bacteria, and find that the bacteria now survive above 39 C! You BLAST the sequence of the E. coli protein through the database and discover a remarkable similarity to a heat shock protein in maize (corn). Heat shock proteins protect organisms from high temperatures, and you wonder if the maize protein is capable of rescuing your E. coli mutation. (In other words, can the maize protein, if expressed in the mutant E. coli, prevent the cells from dying at high temperatures?) To test this, you decide to clone the maize protein into an expression vector using PCR. The expression vector you choose allows you to clone proteins under the control of a galactose-inducible promoter (pgal); these proteins will be produced when the bacteria containing the plasmid are grown in media containing galactose. b) What is the advantage of using a promoter, like pgal, that can be regulated? c) In order to clone your PCR product into pgal you ll need to add the sequence for a restriction enzyme to each of your primers. To which end of the each of the primers (5 or 3 ) should the restriction enzyme site be added? Why?

17 Question 10 (continued) Finally, you need to choose the restriction enzymes that you will incorporate into your PCR primers and that you will use to cut the vector (pgal) DNA. The following are maps of the multiple cloning site of pgal and the DNA sequence that encodes the maize heat shock protein. pgal promoter 5' 3' E S Nd X Nc pgal multiple cloning site B 3' 5' E= EcoRI S= SspI Nd= NdeI Nc= NcoI X= XbaI B= BamHI 5' ATG B E S B TAA 3' restriction map of the open reading frame encoding the maize (corn) heat shock protein d) Another student in the lab suggests that you clone the maize gene by including an XbaI site in your forward primer and an SspI site in your reverse primer. Do you agree or disagree with this strategy? Provide two reasons for your answer.

18 Question 11 You have two E. coli strains, JAF1 and DAK2. JAF1 is phenotypically AmpR, and DAK2 is phenotypically AmpS. You suspect that the AmpR gene is carried on a plasmid in JAF1, rather than on JAF1's chromosome, and want to design an experiment to test this hypothesis. In designing your experiment, you may assume that you have access to the following materials: 1. all reagents, plates, plasmids, and equipment used in the 7.02 RDM module 2. Overnight cultures of JAF1 and DAK2 3. Competent cells of JAF1 and DAK2 a) Describe a simple experiment you could perform to test the hypothesis that the AmpR gene is carried on a plasmid in JAF1 and not on JAF1's chromosome. (Note: there is no need to describe the details of a particular procedure you might use, simply stating what you would do is sufficient e.g. ligate A + B together). b) What specific experimental result will tell you that your hypothesis is correct? c) Identify one positive and one negative control you would include in your experiment above, and how you will use these controls to help you interpret your "experimental" results. (d) If the AmpR gene was carried on JAF1 s chromosome, name a technique that would allow you to transfer this gene to DAK2?

19 Question 12 The fictional bacteria B. experimentus contains multiple DNA polymerases that synthesize DNA using datp, dctp, dgtp, and dttp as substrates. In investigating what chromatography steps to use to purify these proteins, you find that polymerase A elutes first while polymerase B elutes second on each of the columns listed below (a-c). Describe the properties of polymerase A and polymerase B that are indicated by their behavior on each of these columns: a) The column is an anion exchange column equilibrated with a buffer containing Tris at ph 7.5 and NaCl at 100mM. The column was eluted with the same buffer containing increasing concentrations of NaCl up to 1M. b) The column is a gel filtration column equilibrated and eluted with a buffer containing Tris at ph 7.5 and NaCl at 100mM. c) The column is a dctp-agarose column equilibrated with a buffer containing Tris at ph 7.5 and NaCl at 100mM. The column was eluted with the same buffer containing 100mM dctp. d) Starting with 30 ml of a cleared lysate, you wish to purify polymerase B away from polymerase A using the three columns mentioned above, plus ammonium sulfate precipitation. Which of the following would be a logical order of the steps of protein purification? Circle one, and explain your answer briefly. gel filtration --> ammonium sulfate ppt. --> agarose ammonium sulfate ppt. -->gel filtration --> exchange anion exchange --> dttp dttp agarose --> anion dttp agarose --> gel filtration --> anion exchange --> ammonium sulfate ppt.

20 Question 13 Listed below are seven potential strains (A-G) that could result from the transposon mutagenesis performed in 7.02 lab (using strain BW140/pNK and lambda1205). On the chart below, CLEARLY indicate the growth (G or NG) 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 leub gene (required for leucine biosynthesis) in the same orientation and reading frame as the gene is transcribed. (Note: leub transcription is repressed in the presence of leucine.) M9 Ara Kan A B C D E F G M9 Glu Kan Mac Ara Kan Mac Lac Kan LB Xgal Kan LB Ara Xgal Kan

21 Question 14 You perform transposon mutagenesis with a modified minitn10 transposon containing the AmpR gene (instead of KanR) and screen for cysteine mutants. You make P1 lysates from your mutant to stabilize and map its location relative to the mal (maltose utilization) and arg (arginine biosynthesis) genes. In addition to the cys::minitn10 mutation, your mutant is also Mal - and Arg +. a) What characteristics must the strain that you are using to map have? b) You have decided to select for AmpR colonies. What plates will you use for mapping your mutation? c) You collect the following classes and data: AmpR Mal+ Arg- 420 AmpR Mal- Arg+ 190 AmpR Mal+ Arg+ 10 AmpR Mal- Arg- 380 What is the order of the cys, mal, and arg genes? SHOW YOUR WORK!

22 Question 15 You've come a long way since the beginning of the semester! Demonstrate your understanding of the major goals of each of the previous course modules (and the techniques used to reach these goals) by completing the statements given below. Microbial Genetics (GEN) module: 1. In this module, your goal was to create a mutation in a gene involved the process of. 2. These mutations were made using the minitn10 transposon, which created an (a type of mutation) in the gene of interest. 3. The kan gene in the minitn10 transposon made the cells resistant to kanamycin, which allowed you to. 4. The lacz gene in the minitn10 transposon served as a gene; observing told you that your gene of interest was being under those particular growth conditions. 5. P1 transduction was used to stabilize these mutations by moving them away from the enzyme, which is required for. Question 15 Protein Biochemistry (PBC) module: 1. In this module, you learned that ammonium sulfate separates proteins based on differences in, whereas gel filtration, ion exchange, and APTGagarose columns take advantage of the fact that proteins differ in their,, and, respectively. 2. You used ONPG as a substrate for your activity assay (instead of lactose) because. 3. You used SDS-PAGE to separate denatured proteins based on their, and the Bradford assay was used to determine.

23 Recombinant DNA Methods (RDM) module: 1. In this module, you used PCR to. 2. You transferred gfp from the pugfp vector to the pet vector because the pugfp vector lacked a, which is required for of gfp DNA. 3. To accomplish this, restriction enzymes were used to, and ligase was used to. 4. You learned that "transformation" is a method for, and that the "miniprep" protocol allows you to. 4. BL21 cells containing the pet-gfp plasmid glowed green under UV light because these cells contain the enzyme, which is required for.