Soil Enzyme Activity (Alkaline Phosphatase Assay)

Soil Enzyme Activity (Alkaline Phosphatase Assay) Laboratory 9    Soil Enzyme Activity (Alkaline Phosphatase Assay) Introduction Bacteria and fungi that break down insoluble nutrient sources in the soil produce extracellular enzymes.  These are proteins that are produced inside the cell and exported out into the soil solution.  The enzymes are active outside the cell where they catalyze reactions to break down the structure of the nutrient source to make it more accessible.  The amount of an extracellular enzyme in the soil depends on the metabolic abilities of the soil organisms, the number of organisms present, the presence of substrate and the environment of the soil (pH, temp., ionic strength etc.).  Because enzymes are costly for the cells to make, they are tightly regulated.  Enzymes will only be made when they are needed. One example of a common extracellular enzyme in soil is alkaline phosphatase.  This enzyme is produced by many organisms in the soil.  Its purpose is to remove the phosphate molecule from organic compounds such as phospholipids and nucleic acids.  Once the phosphate is cleaved it becomes soluble and can be taken up by the cell. This is a very important activity because phosphate is often the limiting nutrient for microbial growth in soil. In this lab you will be measuring the amount of active enzyme in soil samples by using a chromogenic substrate assay.  In the presence of alkaline phosphatase, the colorless chemical para-nitrophenol phosphate is converted to para-nitrophenol, which is bright yellow.  The amount of product formed can be measured using a spectrophotometer and the amount of enzyme activity can be calculated.  You will also calculate the dry weight of the soil in order to standardize the results.  The soils that you will be analyzing have been kept moist and incubated for ~2 weeks with the following amendments: 1.6g of yeast extract, 0.2g of inorganic fertilizer, 1.6g of yeast extract and 0.1g of inorganic fertilizer, 1.6g of yeast extract and 0.4g of inorganic fertilizer, or no addition. Materials Equipment -    Balance -    Scissors -    Incubator (37C) -    Benchtop centrifuge -    5 ml pipettes and pumps -    Screw-top tubes (wide-mouth) -    16 X 100 mm test tubes -    Balance -    Spectrophotometer (440nm) -    Drying oven (100C) -    Aluminum weighing dishes Supplies -    Plastic flowerpots -    Dry, sieved soil -    Yeast extract -    Fertilizer (15-30-15) -    Markers -    Filter paper Media and Reagents -    Buffer (pH 10) -    2 mM p-nitrophenol -    0.5 M CaCl2 -    PNPP test solution (para-nitrophenol phosphate in buffer) Procedures Constructing Microcosms 1.    Cut out a circle of filter paper to fit the bottom of the flower pot. 2.    Use a glass beaker to measure out ~300ml of soil. 3.    Weigh out the appropriate amount of fertilizer and mix it into the soil. 4.    Put the filter paper circle into the bottom of the flowerpot 5.    Pour in the soil and fertilizer mix until it is ~1/2 inch from the top. 6.    Water the soil until it is all moist. Treatment    Fertilizer    Yeast Extract Organic    0.0 g    1.6 g Inorganic    0.2 g    0.0 g Combined    0.2 g    1.6 g Unamended    0.0 g    0.0 g Phosphatase Assay 1.    Weigh out two 2-gram portions of your group’s soil sample and pour them into screw-cap tubes labeled “test” and “soil blank”. 2.    Label one other screw-cap tube as “reagent blank” 3.    Pipette 5ml of 0.5 M CaCl2 solution into each of the three tubes and shake well. 4.    Pipette 1ml of PNPP solution into the tubes labeled “test” and “reagent blank”. 5.    Pipette 1ml of phosphate buffer into the “soil blank” tube to serve as a control. 6.    Incubate all three tubes at 37C for 1 hour. 7.    Transfer 4ml of the liquid from each tube into labeled 16 X 100mm test tubes. (Be careful to avoid transferring sediment.) 8.    Centrifuge the test tubes for 5 min. at 2500 rpm. 9.    Transfer 1ml of the supernatant into clean cuvettes (re-centrifuge if liquid is at all cloudy). 10.    Set the wavelength on the spectrophotometer to 440nm. 11.    Set the absorbance to zero with the “soil blank” tube. 12.    Read and record the absorbance for the “test” and “reagent blank” tubes. 13.    Set the absorbance to zero with a cuvette containing 1 ml of CaCl2. 14.    Read and record the absorbance of the prepared standards. 15.    Plot the absorbance vs. concentration to make a standard curve. Water Content Analysis 1.    Weigh an aluminum dish and record the weight. 2.    Weigh out ~10g of your soil sample in the aluminum dish.  Record the exact weight. 3.    Put the samples in a 100C oven overnight and let them cool in a desiccator. 4.    Weigh the dried sample and record the weight. Lab #9                                            10 points Name:                                    Date: Alkaline Phosphatase Assay Spectrophotometer Readings Absorbance    Net Absorbance (test – reagent  blank)    Concentration of p-Nitrophenol    Enzyme Activity Organic fertilizer (test) control (reagent blank)        XXXX    XXXX    XXXX Inorganic fertilizer control (reagent blank)        XXXX    XXXX    XXXX Combined control (reagent blank)        XXXX    XXXX    XXXX Unamended control (reagent blank)        XXXX    XXXX    XXXX Standard Curve Concentration    2.0 mM    1.0 mM    0.5 mM    0.25 mM    0.125 mM    0.063 mM absorbance Water Content Analysis Sample    Organic    Inorganic    Combined     unamended Dish weight Wet weight with dish Wet weight of soil Dry weight with dish Dry weight of soil Water content Calculations Water Content of Soil and Dry Weight Water content = (wet weight of soil – dry weight of soil) / dry weight of soil Dry weight of sample = (wet weight of sample / water content + 1) Enzyme Activity One unit of enzyme activity (U) is defined as the amount of enzyme that is able to convert 1 mole of substrate to product in one minute.  For soil assays, activity is reported as U per gram of dry soil 1.    Calculate the amount of p-nitrophenol that was produced using the standard curve (remember that the total volume of liquid was 6ml even though you only measured the concentration in 1ml). _______ µmoles in 6 mls 2.    Divide the amount of product by the number of minutes that the samples were incubated to find the value of U _______ µmoles / minute 3.    Calculate the dry weight of the soil sample that was used in the incubation. _______ grams 4.    Calculate the activity per gram of dry soil. _______ U / gram of dry soil Lab Reports You will be required to write two full lab reports (Labs #5 and #9) during the semester.  Each lab report must be at least 8 pages in length without a title page. Times New Roman, 12 point font, and double spaced with 1” margins. How to Write a Lab Report The lab report should include all of the following sections: Title:  The title should be short, descriptive, and refer specifically to the lab. Abstract:  The Abstract is a summary of the lab and emphasizes the results and conclusions. It should be very succinct (approximately 200 words in length) and briefly present the objective, experimental design, summary of results, and significance of the lab. Introduction:  This section provides the purpose of the experiments performed in the lab and an appropriate literature background. The hypothesis and objectives of the lab should be included in the Introduction. (~1 page) Material and Methods:  This section should provide sufficient information to permit repetition of the lab by others, but avoid unnecessary detail. All methods used in the study must be documented, however, do not copy word-for-word any of the written sources you used. Instead, you should summarize in your own words what you did. (~2 pages) Results:  This section should contain a concise account of the data collected. Once data has been analyzed, a summation should be presented. Tables, figures, graphs, pictures, etc. should be used as appropriate and include figure legends. (~1 page + figures) Discussion:  The Discussion is a critical section of the paper and cannot be well-written until the rest of the paper is in good shape. It should address whether your hypothesis was supported or rejected, how the data compare to work done by others, the biological principles which explain what happened in your study, and what experiments should be done next. (~2 pages) Literature Cited:  Citations are arranged alphabetically. All references cited in the text must appear in the literature cited section, and all items in this section must be cited in the text. The formatting of the references should follow the ASM style (see below). ASM Style Guide References In the reference list, references are numbered in the order in which they are cited in the article (citation-sequence reference system). In the text, references are cited parenthetically by number in sequential order. The following types of references must be listed in the References section: Journal articles (both print and online) Books (both print and online) Provide the names of all the authors and/or editors for each reference; names should not be abbreviated with “et al.”.  Abbreviate journal names according to the PubMed Journals Database (National Library of Medicine, National Institutes of Health), the primary source for ASM style. Follow the styles shown in the examples below for print references. Caserta E, Haemig HAH, Manias DA, Tomsic J, Grundy FJ, Henkin TM, Dunny GM. 2012. In vivo and in vitro analyses of regulation of the pheromone-responsive prgQ promoter by the PrgX pheromone receptor protein. J. Bacteriol. 194:3386-3394. Stratagene. 2006. Yeast DNA isolation system: instruction manual. Stratagene, La Jolla, CA. {Use the company name as the author if none is provided for a company publication.} Forman MS, Valsamakis A. 2011. Specimen collection, transport, and processing: virology, p 1276-1288. In Versalovic J, Carroll KC, Jorgensen JH, Funke G, Landry ML, Warnock DW (ed), Manual of clinical microbiology, 10th ed, vol 2. ASM Press, Washington, DC. Online-only references must provide essentially the same information that print references do. For online journal articles, posting or revision dates may replace the year of publication; a DOI (preferred) or URL is required for articles with nontraditional page numbers or electronic article identifiers. Some examples follow. Bina XR, Taylor DL, Vikram A, Ante VM, Bina JE. 2013. Vibrio cholerae ToxR downregulates virulence factor production in response to cyclo(Phe-Pro). mBio 4 (5):e00366-13. doi:10.1128/mBio.00366-13. Winnick S, Lucas DO, Hartman AL, Toll D. 2005. How do you improve compliance? Pediatrics 115:e718-e724. doi:10.1542/peds.2004-1133. Note: a posting or accession date is required for any online reference that is periodically updated or changed. Wollstonecraft’s “from A Vindication of the Rights of Women” Kant’s “An Answer to the Question: What is Enlightenment?” PLACE THIS ORDER OR A SIMILAR ORDER WITH US TODAY AND GET AN AMAZING DISCOUNT :)-