Assignment

Lab 4 Assignment: Full Report

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.

Introduction

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

The text of an introduction should flow naturally without subsections.

For examples, read the Writing Guidelines for additional guidance on the format and read the introductions of previously published journal articles that have studied tyrosinase. From these papers, you will learn why other researchers have studied tyrosinase.

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:

Describe an unresolved problem in the current literature.
State the how their investigation will help to resolve the problem, i.e. give the purpose of the investigation.
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."

Materials and Methods

Each method should have its own subheading: Preparation of Extracts, Kinetics Measurements (be sure to update your method do that it defines U/mg as well as U and U/mL), and Protein Quantification.

Consult the Report 1 Rubric and Appendix B of the Writing Guidelines for addtional details. Appendix B lists the minimum level of detail required for common techniques.
Make certain that you have updated:

the kinetics methods to explain the calculation of specific activity in U/mg,
the protein concentration assay method if your section chose a different method than the one you wrote in Assignment 4.

Results and Discussion

Required figures:

Figure 1: A diagram of the mushroom (the file is available on D2L) as reference for discussing the other 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 from Assignment 2.

Figure 3: A plot for the standard curve of the assay method that your class selected as reliable (Bradford, Lowry, or BCA). The purpose of this figure is to convince the reader of the quality of your protein standard curve. Plot a trendline through the data. Present the linear equation and R² value. You may use Figure 1 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 (unitless ratio of activities +SDS/-SDS). You don't have to name these headings exactly this way. Also, you may wish to creatively combine Figure 1 and Table 1 by overlaying the data onto the diagram or multiple versions of the diagram. If you do so, make sure that the figure is clear.

Structure of writing:

Reiterate the goals of your work and disclose if you were unsuccessful in achieving those goals.

Follow this opening with subheadings for each experiment you performed (in bold text below).

In each subsection
- Concisely explain what the relevant figures reveal in terms of what we learn from the experiment
- If the results were not of good quality, state this. If you dropped data, justify your decision(s).
- Interpret the trends and be clear about limits of your ability to interpret.
- A simple way to analyze a trend is to focus on smallest and greatest values, and note whether or not the differences are significant in magnitude by comparing them the uncertainty values.
- If there is a serious problem with any of your data and you have no results, discuss what went wrong and how the problem could be remedied in the future.

Determination of Specific Activity
- For this subsection, discuss your experience with the measurement of specific activity and the reasons for this meaurement.
- Define specific activity and explain why we compare it instead of activity (in U).
- 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 (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 propogating error from a value derived from division.
- Make 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 criteria, you may also need to define a category for values that do not fall into your defined categories.
- At minimum, you must discuss trends for Inducibility Factor (+SDS/-SDS ratio) and at least one of:
- State the limits of your conclusions in relation to the quality, nature, and/or amount data you have.
- Reference relevant tables and figures.

Conclusion

Summarize your major conclusions
Answer the "problem" posited in the introduction
Interpret your trends (or lack of trends) in terms of a possible function of tyrosinase. 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 upon injury since exposed parts are most vulnerable. You can decide if absolute activity values or ratio of induction is more relevant to your interpretation.
To conclude, briefly summarize what you have learned and note any incomplete information or low in confidence data.
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 buids 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.

Discussion

Required data:

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.