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ISB 202 Lecture 6: ISB202-lecture6-TWIRLIE Activity

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MSU
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Integrative Studies Biological
Course
ISB 202
Professor
A.Bierema

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4

ISB 202 Lecture 4: -modelling

6

ISB 202 Lecture 6: ISB202-lecture6-TWIRLIE Activity

7

ISB 202 Lecture 7: -Scientific controversies

Document Summary

Till now we have learned about how scientists think, now we should analyze a small incident in the way which scientists think. There is a file attached to this document , it shows some rectangular shapes, cut the shapes at the bold lines. We have to fold the shape in such a way that , when we drop it from certain height it should twirl around and come down, this is our task. Ans:its simple turn sidea and sideb backwards and then fold side c downwards, now fold side d backwards. make sure that side e and side d are foldes in the opposite. When we fold it , it looks somewhat similar to the picture shown above. now fold all the remaining four into the same shape. Fix a particular height and throw them from that height and measure the time it took to reach the ground. Compare the timings and draw some conclusions accordingly.

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Related Questions

As you may recall in the work of Triplett (1898), the presence of others in a bicycle race was found to enhance individual performance. This is called social facilitation (Williams, Harkins, & Karau, 2007). At times, however, the presence of others can detract from individual performance, which is termed social inhibition (Crisp & Turner, 2007). Similarly, social loafing can occur when an individual’s performance within a group decreases due to his or her reliance on others in the group (Crisp & Turner, 2007).

For this Discussion, review the media program, Week 5: The Virtual Office. Focus on the characters in the media and determine which characters displayed social facilitation, social inhibition, and social loafing.

With these thoughts in mind:

Post by Day 3 a description of the characters in the media program demonstrating social loafing, social inhibition, and social facilitation. Explain how you identified them. Then, select one of the characters and, using the current literature, explain two ways you might mitigate their behavior.

Media Program

(Onscreen Text: The employees at the United Cardboard Company often work individually on projects, but as with most environments, they are occasionally asked to participate in group projects. One such group is about to meet right now to plan a presentation for the Board of Directors. Observe the conversation and reactions of various group members to determine which group members exhibit signs of the following: Social Loafing, Social Inhibition, and Social Facilitation.)

(Interior of an office. Ken stands at the front of the conference room.)

KEN: OK, I guess we might as well all get started. As we all know, the big man has requested the five of us to get together and write and conceptualize a presentation to the board on our new packaging system. Maybe we can start by going around and throwing out some initial ideas.

KATHY: Sounds good to me. I think a PowerPoint presentation would do the job well.

GAVIN: I agree. We could follow it up with a physical demonstration of how the packaging looks and folds-- maybe a video of the larger pieces that won't fit into the boardroom.

KEN: Sounds perfect to me. Amanda, Brian, what do you guys think?

BRIAN: Yeah, what you guys said sounds fine.

(Amanda thinks, “I don’t know what to say!”)

KEN: Amanda?

AMANDA: I don't know. I didn't really get much chance to think about this. We only found out we needed to do this together three days ago.

KEN: OK, no worries. Let's focus on the PowerPoint idea. I've been doing a lot of them lately, so I'm happy to take the lead on the design aspect. What do you guys think we should all incorporate into the slides?

GAVIN: I think we should start with where we got the idea for this new packaging -- the company that designed it and the different sizes that it comes in.

KATHY: We should follow that with a graph comparing the old prices to the new ones to illustrate how much we'll be saving over time. Amanda, didn't you have most of that information collected at some point?

(Amanda thinks, “Oh no! What if I deleted that?)

AMANDA: I don't know, maybe, it's been so long. I didn't think I'd need it again for a group project like this.

(Gavin thinks, “I wonder what’s gotten into her. She’s not usually this tense.”)

KEN: That's OK, Amanda, I'm sure we can easily dig it up again. Brian, do you have any other ideas for the PowerPoint slides?

BRIAN: Nah, I think what you guys mentioned sounds right.

(Kathy thinks, “Wow. Way to contribute Brian.”)

KEN: OK then, Kathy and Gavin why don't you send me all of the info and I'll write it up and make the slides. Amanda, don't worry about the cost saving document, I'm sure I can dig it up from my old emails and put it into that slide too.

AMANDA: Are you sure? I'm happy to look. I mean I'm sure I have it somewhere. (Kathy thinks, “She really needs to get it together. Why is she so nervous?”) At least I think I have it somewhere. You won't tell the Board of Directors if I lost it, will you?

KEN: Of course not, we're in it together. So as I was saying, I'll write up and prepare all the PowerPoint slides. Then we can start our presentation with reading and explaining each slide. Then, what?

KATHY: Well, and then I think we should show them the packaging live, and the quick deconstructing techniques.

KEN: Great idea. You and Amanda can demonstrate the folding, while I narrate what you're doing.

(Amanda thinks, “Is he doing too much of the work? And what if I drop everything while he’s talking?”)

GAVIN: And after you all demonstrate the smaller packaging, we could show a quick video of how the really big ones function down at the warehouse. Brian, would you be able to go film that quickly one of these days?

BRIAN: I don't really have any experience with video cameras or anything like that, so...

(Gavin thinks, “I should have seen that one coming.”)

KEN: That's OK. My wife actually just got me this great little camera for our anniversary. It takes great videos. So I can just run down to the warehouse, get some footage, then put it together on this new editing software I just downloaded. Maybe add a little music. It's going to be really slick!

KATHY: Are you sure, I wouldn't mind doing that too.

KEN: Oh no, it's fine. I love this kind of stuff; just leave it to me. But I suppose that's enough planning. Now it's time to get to work and make the presentation a knock out. Go team!

(Everyone except Ken thinks, “Team?”) ©2012 Laureate Education, Inc.

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?

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

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].

Read the following passage focusing your attention on the scientific procedures and scientific abilities illustrated in it. Think about what types of experiments (observation, testing or application) are described. Identify the hypothesis, predictions, outcomes of experiments and assumptions that appear in the passage. The standard explanation of how humans originally acquired AIDS is that HIV (human immunodeficiency virus) originally was transmitted to humans from monkeys or apes carrying SIV, simian immunodeficiency virus. How could the transmission have occurred? Several similar hypotheses involve an African hunter coming in contact with monkey blood. For example this could have occurred when a hunter butchering a monkey, got some of its blood in a cut, or when a human ate undercooked monkey meat, or when a monkey bit a human. Depending on one's assessment of the genetic diversity of HIV over time, this transmission might have occurred only a few decades ago or, alternatively, hundreds or thousands of years ago. If the latter is correct then AIDS could have been restricted to remote African villages until recent decades when trade and international travel led to the current pandemic. There are however other alternative explanations for the spread of AIDS. One of them is that AIDS arose from contaminated polio vaccines used in Africa from 1957-59. Why is this a plausible explanation? Polio vaccines are cultured on apes’ kidneys, so contamination of a batch of vaccine by an SIV is a possibility. There is a precedent: in the 1950s and early 1960s millions of doses of polio vaccine were contaminated by another simian virus, SV-40. The vaccine alleged to be the origin of AIDS was given to hundreds of thousands of people in central and west Africa from 1957-1959. These locations later had some of the world's highest levels of HIV infection. The timing is also right, since the earliest known cases of AIDS date from the 1960s; the earliest HIV-positive blood sample was collected in Kinshasa in 1959. At the time, there was no way of testing for the presence of SIVs, which were only discovered in the 1980s. Another factor adds plausibility to the theory: the vaccine was given to many infants less than a month old, in an extra high dose. Since the immune systems of infants are not fully developed, this is an excellent way to help a virus jump the species barrier. Some scientists argue that the above explanation is not necessarily correct but that it is worth taking seriously. There are a number of ways to test it. One is to analyze stocks of polio vaccine; if the explanation is correct, then it is probable that some of the vaccines will carry SIV. Another is to scrutinize records of early polio vaccination campaigns in Africa. If it is true that the immunodeficiency virus passed to humans by way of those vaccines, the areas where the campaigns took place would be the same as those where the initial sprouts of the disease occurred and where the larger number of cases occurred during the early stages of the pandemic. Another is to analyze blood samples in Africa from the 1950s and earlier (pre polio vaccination): if the immunodeficiency virus was transmitted via the polio vaccine, HIV should not be found. Adapted from “Political refutation of a scientific theory: the case of polio vaccines and the origin of AIDS”. Brian Martin, published in Health Care Analysis, Vol. 6, 1998, pp. 175-179. Now reread the passage and answer the following questions.

5. For each experiment, write an outcome that would disprove the hypothesis of AIDS passing from monkeys to humans through polio vaccines.

6. What outcomes of the experiments would fail to disprove the hypothesis of AIDS passing from monkeys to humans through polio vaccines?explain.

7.What outcomes would prove the hypothesis? Hint: This is not the same as question 6. Think about what would you need to do to 100% prove a hypothesis.

8. For what reasons do scientists need to conduct testing experiments?