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. You were given a strain that one friend claims requires threonine and another friend claims that it does not. You are sure you can tell the difference so you streak the unknown strain on a minimal plate. a) If it didn't grow, could you conclude that it does in fact require threonine? Why or why not? b) If not, how would you modify your experiment to allow a conclusion? After you do the experiment correctly you are still confused because the strain can make small colonies without threonine, but it seems to make bigger colonies when threonine is present. c) You do a growth curve to quantitatively demonstrate a growth difference between the WT and the mutant strain. Predict/draw what you would expect the growth of the WT and mutant strains to look like in minimal medium. Predict/draw what you would expect the growth of the WT and mutant strains to look like in minimal medium + thr. d) Assuming you demonstrated a partial requirement for threonine in this strain, what kinds of mutant proteins could cause this phenotype (list 2)? Explain how the mutation could lead to a partial requirement?

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Tod Thiel
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29 Sep 2019

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Related questions

Please answer questions at the end.

In this lab, you will add different volumes of “cell-free supernatant” (CFS) to fresh cultures of V. harveyi and observe bioluminescence. The CFS is prepared by culturing V. harveyi in liquid medium overnight, then separating the cells from the supernatant by centrifugation.

MATERIALS • overnight culture of V. harveyi in AB medium • fresh AB medium • 1 microcentrifuge tube • 3 glass culture tubes

PROCEDURE

1. This experiment will be performed with your lab group. Label the microcentrifuge tube and 3 glass cultures tubes with your group number. Also, label the 3 glass culture tubes as follows: “Media only,” “Cells+fresh media,” and “Cells+CFS.”

2. Using aseptic technique, pipet different volumes of fresh AB medium into the glass tubes as follows: 1 mL in the “Media only” tube, 800 ul in the “Cells+fresh media” tube, and 500 ul in the “Cells+CFS” tube.

3. Pipet 500 ul of the overnight culture into a microcentrifuge tube and centrifuge at maximum speed (>13,000 x g) for 5 min to pellet the cells.

4. After centrifugation is complete, transfer 300 ul of the cell free supernatant (CFS) from the microfuge tube to the glass tube labeled “Cells+CFS”. Be careful to avoid the cell pellet.

5. Remove the remaining supernatant from the microfuge tube using a micropipette and discard as culture waste.

6. Add 500 ul of fresh AB medium to the cell pellet in the microfuge tube and pipet up and down to resuspend until no clumps are visible.

7. Add 200 ul of the cell suspension to the “Cells+fresh media” tube and 200 ul of the cell suspension to the “Cells+CFS” tube. Note the time.

8. Place the 2 glass tubes containing cells in the 30 degree incubator/shaker.

9. After 1 hour and 2 hours, observe your tubes next to the “Media only” tube in a dark room. You will need to let your eyes adjust for at least 30 seconds (count to 30 slowly in the dark) before you can evaluate whether the cultures are luminescent or not. Also, make sure you try swirling the tubes in the darkroom and observe the tubes for a minute after you have stopped swirling. After the observation at the 1 hour time point, put your tubes back in the incubator shaker until the 2 hour time point.

10. Note your observations. At the end of the lab, place your tubes in the tube waste.

Questions

1. What were the expected results? Explain the rationale of the experiment. If you didn’t obtain the expected results, hypothesize why not and describe how the experiment could be improved.

2. What are differences between luminescence and fluorescence?

3. Why do V. harveyi cultures glow more brightly when swirled vigorously? Be specific.

4. Imagine you have a mutant V. harveyi strain that can’t synthesize autoinducer and another mutant V. harveyi strain that makes a defective luciferase. If you streaked both strains near (but not touching) each other on the same plate, would you expect to see light produced? If so, by which strain, where, and why?

5. You have two identical tubes of wild-type V. harveyi (with the same cell concentration in each tube) in fresh media. To one tube, you add CFS from a light-producing V. fischeri culture, and to the other tube you add an equal volume of fresh media. If both tubes of V. harveyi have the same light production, what can you conclude? Explain.

6. Many biomedical research labs study quorum sensing in V. fischeri or V. harveyi. How is studying light production in marine bacteria relevant to human health? List at least 3 different examples.

7. Why do you think it is important for bacteria to be able to control the expression of some genes based on the number of individuals present?

Please I need help answering those questions i always lose bunch of points i'm not doing it correctly.

Use information from Nature Reviews Microbiology to answer the following question.What is the role of EF-G and EF4 in protein synthesis? Under what circumstances does EF4 participate in translation and what is its role?

from this reading The Highly Conserved LepA Is a Ribosomal Elongation Factor that Back-Translocates the Ribosome Qin Y,Polacek N, Vesper O, Staub E, Einfeldt E, Wilson D.N, and Nierhaus N.H. Cell 127: 721-733 (2006).

Q2. In which species is Lep A found and in which ones is it absent? Is the result expected based on your knowledge of the endosimbiosis theory?

Q3.

3a What is the percentage of identity between LepA and its ortholog genes?

3b. (5pts) Indicate what domains are conserved between Ecoli EFG and LepA, and which ones are not.

3c. What do grey and black boxes in the sequence alignment indicate?

Q4. Analysis of in vivo experiments shown in Figure 3. Only analyze graph on the left side of the figure.

4a. What is the difference between the bacteria whose growth are shown by the blue and the green curves? What can be concluded about the effect of EFG on bacterial growth?

4b. Which protein is expressed by bacteria whose growth is shown by the yellow curve? Does the expression of this protein affect bacterial growth? Compare the yellow and blue curves. How can this result be explained?

4c. Explain why the addition of IPTG results in lower bacterial growth. Compare the yellow and red curves?

4d. Why is the green curve an important control of the experiment? The green curve.

The next questions are based on the reading of the article Elongation factor 4 (EF4/LepA) accelerates protein synthesis at increased Mg2+ concentrations.

Elongation factor 4 (EF4/LepA) accelerates protein synthesis at increased Mg2+ concentrations Pech M, Karim Z, Yamamoto H, Kitakawa H, Qin Y, and Nierhaus K. PNAS 108: 3199-3203(2011)

Studies done in E. coli using a knockout mutant of the lepA gene (ΔlepA , lepA gene has been deleted) did not show any phenotype when grown in rich LB medium. This result was unexpected due to the high level of conservation of EF-4 among species.

Q5. Equal amounts of wild type and mutant cells (lacking the lepA gene) were mixed and co-cultured for 24h. The population distribution was determined every 4 hours. Figure 1 indicates changes in the % of mutant cells over several generations.

Q5a. What is the question that researchers addressed in the experiments shown in Figure 1?

Q5b. Which curve represents the control of the experiment? Do the two strains grow at similar rate? How do you know?

Q5c. Do high salt concentration and/or low temperature have any effect on the growth of mutant cells?

Q5d. What can you conclude about the role of LepA on bacteria growth? When is this protein important?

Q6.

6a. (6pts) Where in the cell is EF4 located? Did you expect that this would be the EF4 location? Why or why not?

What is the question that the researchers want to address in the experiment shown in Figure 2?

6c. Analyze the green and red bars graph shown in Figure 2. What technique was used to detect EF4? Is there any change in EF4 localization?

Q7. Summarize the main points of the model shown in Figure 4

The overall goal of the research project that we carried out in Experimental Biology is insertion of GFP into target lapA/EF-4 gene.

1. What is the overall goal of the research project that we carried out in Experimental Biology, and how does the experiment aimed at purification of your fusion protein fit in with the overall goal of our research? How does the subcellular localization experiment fit into the overall goal?

2. What method did we use to determine whether your fusion protein was a soluble protein or a membrane bound protein? What positive control did we perform? What result was obtained from the positive control? Was this expected? Explain.

3. Know the methods used for protein purification in general as described in class, and in the file; “protein purification.ppt”

4. What method did we use to attempt to purify your LepA/EF 4 – GFP fusion protein? Why did we use this method, what advantages are there compared to other protein purification methods?

5. What is “Protein A” and how did we use it?

6. What antibody did we use in our purification experiment and why did we use it? Why can’t we use the anti-EF 4/LepA antibody to purify your fusion protein?

DAY 18, NOVEMBER 7: INTRODUCTION TO IN VIVO FUNCTIONAL ASSAYS; ASSAY FOR EXPRESSION TOXICITY AT 30°C VS. 37°C

Purpose: Compare the growth of pPEM109, pIN III, and candidate plasmids in DH5alpha-F’lacIQ -/+ IPTG to compare toxicity of functional LepA/EF-4 (expressed from pPEM109) to empty vector control (pIN III), and EF-4-GFP fusion proteins.

Background: This assay is based on the observation that E. coli is extremely sensitive to elevated levels of LepA/EF-4 expression. In prior work it was demonstrated that LepA/EF-4 expression from pPEM109 is lethal to cells growing in the presence of IPTG [Yaskowiak and March, 1995). Furthermore, pPEM109 can only be maintained in strains that contain the mutated Lac repressor gene, lacIQ, which increases repression of the operon. The wild-type promoter of the lacI gene is rather weak, resulting in ~10 molecules of Lac repressor protein per wild-type cell. The lacIQ gene contains a “promoter-up” mutation that increases transcription and Lac repressor expression 10-fold, resulting in ~100 repressor protein molecules per wild-type cell (Müller-Hill et al., 1998). Strains that only have normal Lac repressor have a slightly elevated level of “leaky” expression from the lac promoter. It was shown by comparing two identical strains (one contained lacIQ and one contained lacI) that pPEM109 could only be maintained in the lacIQ containing strain.

If you consider the structure of LepA/EF-4, and its proposed function, why do you think it would be toxic if expressed at high levels levels? What kind of mutations in the lepA gene could give rise to a mutated protein that is not toxic when expressed? What do you predict for your LepA/EF-4 - GFP fusion protein, will it be toxic when expressed or not?

-------------------------------------------------------------------------------------------------------------------

DAY 19, NOVEMBER 9: BIOCHEMICAL ASSAY FOR SUBCELLULAR LOCALIZATION OF LEPA/EF-4-GFP (Lysis and Ultracentrifugation).

Background: LepA/EF-4 was originally found in the cytoplasm, membrane and periplasm when the protein was expressed from a plasmid [March and Inouye (1985) J. Biol. Chem. 260: 7206-7213]. The endogenous protein, expressed from the chromosomal gene in the absence of plasmid expression was located in the membrane [March and Inouye (1985) Proc Natl Acad Sci, USA Vol. 82: 7500-7504]. More recently it has been shown that the cellular location of LepA/EF-4 depends on the Mg2+ stress status of the cell [Pech et al. (2011) Proc Natl Acad Sci, USA Vol. 108: 3199-3203].

Here we will examine the comparative distribution of LepA/EF-4 and LepA/EF-4-GFP in cells by subcellular fractionation of native cellular protein extracts. Do you predict that your EF-4-GFP fusion protein will be localized to the cytoplasm, membrane, or both?

In contrast to our previous denaturing extract preparation, the native preparation maintains the overall shape/folding of proteins so they can localize appropriately prior to analysis. Looking ahead, how will the preparation be different in native vs. denaturing extracts?

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DAY 20, NOVEMBER 14: BIOCHEMICAL ASSAY FOR SUBCELLULAR LOCALIZATION OF LEPA/EF-4-GFP (SDS PAGE and Transfer)

Procedure:

SDS PAGE Analysis

1. Each section should have the following control samples to analyze from DAY 19:

Extract Fraction Treatment

pPEM109 Input +IPTG

pPEM109 Sol +IPTG

pPEM109 Mem +IPTG

In addition, each student should have 3 experimental samples for their candidate:

Extract Fraction Treatment

Candidate Input +IPTG

Candidate Sol +IPTG

Candidate Mem +IPTG

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DAY 21, NOVEMBER 16: BIOCHEMICAL ASSAY FOR SUBCELLULAR LOCALIZATION OF LEPA/EF-4-GFP (Western Blot detection)

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DAY 23, NOVEMBER 28: PURIFICATION OF NATIVE LEPA/EF 4 – GFP FUSION PROTEINS BY AFFINITY CHROMATOGRAPHY.

Purpose: In order to examine whether LepA/EF 4 – GFP fusion proteins retain function our prior experiments examined whether fusion proteins retained expression toxicity, and whether fusion proteins localized to the membrane. In order to examine other aspects of function, such as GTP – binding and ribosome - binding, purified protein is required. In this experiment we examine the feasibility of purifying LepA/EF 4 – GFP fusion proteins using affinity purification.

Background: Scientists are often interested in recovering proteins that have been produced in bacteria for research or therapeutic purposes. The process of protein purification from bacteria is medically relevant. It can be used as a method to mass-produce proteins of interest. For instance, the human insulin gene can be cloned into a plasmid and transformed into E. coli. The protein can then be purified and used to treat patients with diabetes.

The process of purifying a protein means separating this protein from other cellular components (proteins, DNA, ions, etc.). Protein purification is described in all biochemistry books, and in Chapter 5, Section A, of the textbook used in the Emmanuel College Biochemistry class (Fundamentals of Biochemistry, Voet, Voet and Pratt). This book is on reserve in the Emmanuel College Library. Here we will test the feasibility of using affinity chromatography, and magnetic beads to purify LepA/EF 4 fusion proteins in one step.

Preparation of magnetic beads for affinity purification of native LepA/EF 4. This procedure will be done the day before by the Emmanuel College Lab staff.

Background. The primary antibody that we employed to detect LepA/EF 4 – GFP in our Western blotting experiments can also be used to purify native, functional LepA/EF 4 – GFP fusion protein. Commercially available Protein A magnetic beads will be used for this purpose. Protein A is a very stable bacterial protein that has a high affinity for IgG produced from any animal species. Protein A magnetic beads contain tiny magnetized beads with Protein A covalently attached to their surface. Remember that the primary antibody that was employed in our prior experiments was affinity-purified rabbit IgG that was specific for GFP epitopes. The application of GFP primary antibody to Protein A magnetic beads will result in a tight, non-covalent attachment of GFP – specific IgG onto the magnetic beads. These beads would specifically recognize and bind to GFP that was present in a crude cell lysate. The application of a magnetic field could then be employed to collect the beads and the bound LepA/EF 4. By this method the collection and wash steps would be very rapid and very efficient. After washing away unbound proteins, purified LepA/EF 4 – GFP could be eluted from the magnetic beads. HOWEVER, the GFP IgG was non-covalently attached to the Protein A, and therefore some will leak out in the elution step. This would contaminate the purified LepA/EF 4 – GFP fusion protein with IgG. To avoid this problem, GFP IgG will be chemically cross-linked onto the Protein A magnetic beads by the procedure described in Sisson and Castor [Journal of Immunological Methods (1990), Vol. 127, 215-220].

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