Lab 4 Assignment

By next meeting, you will submit both a physical hard copy and an electronic copy of this assignment.

For the last several weeks, you and the other students in your lab section have been collecting data on tyrosinase isozymes found in the common white button mushroom, Agaricus bisporus. You have the extracted the enzyme, measured latent and induced kinetics, quantified protein to make the kinetics results comparable, and have performed literature research using online databases. Previously, you have written Methods and Results sections related to this work. This week you will write three subsections found in all scientific reports: Title, Objectives, and Discussion. In your assignment, give each section its own heading. Even though you will not be submitting a complete report for this work, write as if these sections were part of a complete report. Carefully, follow the guidelines below and consult the Writing Guidelines for general stylistic guidance for each section.

Title and author information

  • At the top of your first page provide (1) a descriptive title of the work, (2) your name, (3) your professional email address where a reader can contact you with questions, (4) indicate your DePaul University affiliation, and (5) the date of manuscript submission.

  • Your title must have the detail and specificity the title of a peer-reviewed research paper. It should convey the objective of your study and mention both the enzyme and organism studied.

Objectives

The objectives are typically found in the final one or two paragraphs of a scientific paper. For context, read the Introduction section of the Writing Guidelines. First, the author states a problem that lacks data in the literature regarding the system under study. Next the author states how following work will contribute to solving the problem. Finally, the author briefly describes the experimental approach to solving the problem.

The following examples are randomly selected from the journal Biochemistry. Visit the source articles to understand how the objective is woven into the text of an Introduction.

Example 1

Article title: "An Alkaliphilic Chitinase Unveils Environment-Dependent Variation in the Canonical Catalytic Machinery of Family-18 Glycoside Hydrolases"

"What is unknown: "To gain further insight into how the complex catalytic machinery of GH18 chitinases (Figure 1) may have adapted to extreme pHs, and to gain insight into novel chitinases with atypical properties,"

Objective: "we have studied the GH18 chitinase from C. alkaliphilus, CaChiA."

Example 2 

Article title: Ion-DNA Interactions as a Key Determinant of Uracil DNA Glycosylase Activity

What is unknown: "Much of our understanding on the relationship between ion-DNA interactions and the activity of DNA glycosylases comes from..." "We were therefore surprised by reports that the activity of the UNG2 catalytic domain was..."

Objectives: "In the present work, we aimed to reconcile the disparate reports of how monovalent and divalent cations influence DNA binding and uracil excision activity of UNG2 and its catalytic domain."

Example 3 

Article title: "De Novo Design of Parallel and Antiparallel A3B3 Heterohexameric α-Helical Barrels"

What is unknown: "To our knowledge, the design of conditional heteromeric assemblies of >4 helices has yet to be achieved."

Objectives: "Thus, we set out to design a heterohexameric, A3B3-type αHB, for which the individual helices were unfolded in solution and coassembled when mixed."

In this week's assignment, you will:

  1. Describe an unresolved problem in the current literature.
  2. State the how your investigation will help to resolve the problem, i.e. give the purpose of the investigation.
  3. Finally, describe the experiments you will present.
  4. Part of this description must explain why it is necessary to measure specific activity and why protein concentration is required for this measurement.

Tips

  • Invent your own objective, drawing on examples discussed in class.
  • Your objective should integrate all collected data: different tissues, basal activity, and detergent-induced activity.
  • Frame the purpose as an effort to answer an open question or discover something new about tyrosinase in Agaricus bisporus.
  • Assume your results offer novel insights to the field.
  • There is little published data on detergent activation and tyrosinase isozyme distribution in mushrooms. Measuring activity in separate tissues is already a unique contribution.
  • Your objective can be as short as two sentences or as long as two paragraphs.
  • For further examples, read the introductions of previously published journal articles that have studied tyrosinase. From these papers, you will learn how other researchers have explained their objectives.

Discussion

Required data:

  1. Table 1: The heart of the report will be this table which compiles complete activity data for your lab section. At a minimum, Table 1 should have headings for mushroom tissue type, Basal Specific Activity (U/mg of protein), Induced Specific Activity (U/mg of protein with SDS), and Inducibility Factor (the unitless ratio of activities +SDS/-SDS). You don't have to name these headings exactly this way. Be sure to follow the guidelines for presentation of measured values. You will receive up to five points of extra credit if you additionally overlay some of the activity data onto a diagram of the mushroom to better illustrate the trends.

Structure of writing:

  1. Briefly reiterate the goals of your work from the Introduction and disclose if you were unsuccessful in achieving those goals.
  1. Comment on the trends (if any) for activity in different tissues (Table 1) as they relate to your purpose.
    • If the trends are unclear to you, a good strategy is analysis (the separation of information into categories). A common analytical approach is to define categories for the highest and lowest extremes. Depending on your data, you may wish additional intermediate categories.
    • At minimum, you must describe at least one trend from two of the three following measurments:
      • Basal Specific Activity (no SDS),
      • Induced Specific Activity (with 0.1% SDS),
      • Inducibility Factor (+SDS/-SDS ratio).
    • Make sure you discuss differences that are actually statistically different (note the error ranges). 
    • You must indicate to your reader how confident you are in these trends. For example, if a value has high uncertainty, you must acknowledge that the role of that data point in the trend is less certain. 
  1. Interpret the trends as they relate to your stated purpose.
    • Interpret any observed trends—or the absence of trends—in relation to the possible function of tyrosinase. Specify whether your interpretation relies more on absolute activity levels or on the induction ratio. For example, if you found that tyrosinase activity is highest in the skin and ring, you might hypothesize that the enzyme's function is to generate pigments that protect the mushroom from UV light upon superficial injury since exposed parts are most vulnerable.
  1. Conclude.
    • To conclude, briefly summarize what you have learned and note any incomplete information or low confidence data that limits your conclusions.
    • You must mention the impact of isozymes on the confidence of your conclusions.
    • If there is a serious problem with any data, direct the reader to the specific aspect of the measurement that had problems (i.e. rate measurement or protein concentration). Explain how the problem could be potentially remedied in the future. Although you do not have access to all the raw data, you do have access to all of the relative activity and protein concentration data. At a minimum, you should be able to determine what part of a specific activity measurement was the likely source of the problem.
    • Finally, suggest follow-up research that will either fill-in the gaps in your data, improve confidence, or take the research into a new direction that builds on what you have accomplished.

References

  • It is critical that you include cited references.
  • You must cite at least three sources from published (unchanging) peer-reviewed literature. Webpages (including these lab lessons) and databases do not count since they change constantly and are not usually peer-reviewed. Read the Appendix C (Citations) from the Writing Guidelines for more details.
  • You may additionally cite secondary references such as Lehninger or Wilson.
  • You should never cite D2L or these lessons in this course.
  • Follow the citation format in the Writing Guidelines.