Lab 4 Assignment: Full Report

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.

Abstract

The purpose of an abstract is to summarize the key components of an article by providing an overview of the system, the methods used, and the results in less than 250 words. Since the abstract is a summary of what you have written, you should write it last. Consult the Writing Guidelines for the rules about abstract style. The following outline provides a framework for writing an abstract for this report. Try to limit yourself to 1-2 sentences for each bullet point below.

  • Background: In one sentence, introduce the tyrosinase/mushroom system and the broad motivation for the work (Why should the reader care about the work?).
  • Objectives: In one sentence, state the aspects of the system you are specifically interested in studying. At minimum, this sentence should mention tyrosinase activity, SDS activation, and tissue types.
  • Methods and Scope: Briefly describe the methods used and what was measured. For this, keep in mind that our work focused on tyrosinase activity. Determining protein concentration is a standard procedure and should therefore not be included within an abstract.
  • Major Results: Qualitatively describe the key experimental results. You should not list any specific values within these sentences. These sentences should just describe the key takeaways from the work.
  • Conclusions: Briefly connect your results to the broader mushroom system. Keep in mind that this sentence should use speculative language. (e.g. “Altogether, this work suggests that _____ may play a role in ______”).

Introduction

An introduction consists of two main parts: (1) the background, and (2) objectives of the experiment.

The text of the introduction should flow naturally without subsections.

For examples, read the introductions of previously published journal articles that have studied tyrosinase. From these papers, you will learn why other researchers have studied tyrosinase.

For additional guidance on format and style read the Writing Guidelines.

Additional requirements

Your Introduction must also include:

  • A chemical reaction scheme: At minimum, the reaction scheme must illustrate the substrates, intermediate(s), and products for the enzyme activity you are studying; it can also include other related reactions. This scheme cannot be a copy-paste of the images provided in the course materials. You are permitted to draw the scheme by hand on the print copy of your submitted report.
  • Figure 1: A digram of a mushroom as reference for any discussion of mushroom tissues. Explain the abbreviations in the caption. You are free to use this mushroom diagram  (CC BY 4.0) provided you attribute the image source in accordance with the  Creative Commons Attribution 4.0 International license  , which requires attribution of the image source in your references.

Tips for writing the background

When writing the background, you first present the system being studied (in this case, mushroom tyrosinase), then provide some broader context of why this enzyme is interesting/important, and finally present any other necessary theoretical background required to understand the system.

Make sure your facts are supported by references to literature. You may not cite the lab lessons as references. However, you are encouraged to use the references given in the lessons themselves. The PDF files of these references are linked on the D2L Main Page.

This week, you used Swiss-Prot and BRENDA to locate references. Consider using some of these in your introduction.

Important background for tyrosinase (or any enzyme) should include the enzyme function, the reaction(s) it catalyzes, natural substrates and products of the reaction catalyzed, features of the active site involved in catalysis, existence of isozymes, and how specific chemicals/enzymes regulate activity.

Beyond the natural reactions of the enzyme, you should give the reader context for the reagents that you will be using, the substrate you are using and product you are measuring. You should also explain the effect of SDS and why you are using it.

It is not necessary to provide your reader with background on the methods you used.

Tips for writing the 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.

When writing the objectives, an author must:

  1. Describe an unresolved problem in the current literature.
  2. State the how their investigation will help to resolve the problem, i.e. give the purpose of the investigation.
  3. Describe the experiments they will present.

To frame your objectives as an effort to answer an open question or discover something new about tyrosinase in Agaricus bisporus, assume your results offer novel insights. Your objective should integrate all collected data: different tissues, basal activity, and detergent-induced activity.

Measuring activity in separate tissues is already a unique contribution. Additionally, there is little published data on detergent activation and tyrosinase isozyme distribution in mushrooms.

Your objective can be as short as two sentences or as long as two paragraphs.

The following examples of objectives were randomly selected from the journal Biochemistry. Visit the source articles to understand how the objective was woven into the text of the 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."

Experimental

  • Each experimental method should have its own subheading, listed below:
    • Sample Preparation: Refer to Figure 1 (see Results and Discussion)
    • Kinetics Measurements: Be sure to update your method from Assignment 2 to include definitions for U/mg (in adition to U and U/mL)
    • Protein Quantification: Only write the procedure for the protein quantificiation method you used for calculating specific activity. You should not mention the methods from Lab 3 that you did not ultimately use.

Results and Discussion

Required figures:

  • Figure 2: A representative plot of kinetics data. Include at least one plot of your data with and without SDS. The purpose of the figure is to convince the reader of the linear quality of your initial rates data. Plot trendlines through the data. Present the linear equations and R2. You may reuse Figure 1 and its caption from Assignment 2.
  • Figure 3: A representative plot for the standard curve of the assay method that was used for calculating the specific activities (Bradford, Lowry, or BCA). Include a trendline through the data. Plot your own data since it is the only data you have. The purpose of this figure is to convince the reader of the quality of protein standard curves. Present the linear equation and R2 value. You may use Figure 1 and its caption from Assignment 3.
  • 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.
  • Additional Figure(s): You will receive up to five points of extra credit if you additionally generate a figure that presents your Table 1 data in a way that contributes to the reader’s understanding of your discussion. This could include bar graphs or data creatively overlayed onto a diagram of the mushroom.

Structure of writing:

  • Reiterate the goals of your work and disclose if you were unsuccessful in achieving those goals.
  • Follow this opening with the subsections described in bold text below.
  • Determination of Specific Activity
    • For this subsection, discuss your experience with the measurement of specific activity and the reasons for this meaurement.
    • Describe the problem(s) inherent in relative activity (U/mL) comparison that are solved by specific activity.
    • Define specific activity.
    • Explain how specific activity was measured.
    • Comment on the quality of your raw kinetics data (Figure 2).
    • Comment on the quality of your protein standard curve that you used for specific activity calculation (Figure 3).
    • Comment on the quality of the dataset for all tissues as a whole (relative uncertainty). State any problems related to these measurements.
      • Refer to the Writing Guidelines for propagating error from a value derived from division.
    • Reference relevant tables and figures.
  • Specific Activity Trends
    • For this subsection, comment on the trends (if any) for activity in different tissues (Table 1) as they are relevant 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 for discussing a parameter that varies is to define categories for the highest and lowest extremes. Depending on your data, you may wish additional intermediate categories.
    • At minimum, you must discuss trends for Inducibility Factor (+SDS/-SDS ratio) and at least one of:
      • Basal Specific Activity (no SDS),
      • Induced Specific Activity (with 0.1% SDS),
    • 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.
  • Interpretations
    • 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 most inducible 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.
    • Note any incomplete information or low confidence data that limits your conclusions.
      • If there was a serious problem with any data, suggest the source of the problem in the measurement (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.
    • You must mention the impact of the absense of isozymes information on the confidence of your conclusions.
  • Conclusions
    • To conclude, briefly summarize what you have learned with respect to your stated objectives.
    • 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 four 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 textbook references such as Lehninger or Wilson.
  • You should never cite D2L or these lessons in this course.
  • Every citation from a journal article must follow the same format.
  • Follow the citation format in the Writing Guidelines.