Enzymes are proteins that act as catalysts in the production of chemicals needed by cells.  Catalysts increase the rate at which chemical reactions go to equilibrium while not being consumed in the reactions themselves.  In one sense, cells can be thought of as bags of biochemical pathways in which numerous chemical reactions are all running continuously with each step in each pathway being catalyzed by enzymes.  Because of the arrangement of enzymes and a continuous supply of energy to cells, chemical reactions in cells do not reach equilibrium as long as the cell is living.  Cells in a state of equilibrium are called “dead” by highly trained scientists.

Enzymes act as tools for doing the work of cells, but there is nothing to stop enzymes from being used as tools to do work for us.  In this very simple exercise, we will look at the difference enzymes can make in a simple everyday product, laundry detergent.  Many difficult to remove fabric stains are composed primarily of proteins. Protein stains are not necessarily easy to get out using surfactants like Sodium Dodecyl Sulphate (SDS).  Protelytic enzymes in detergents break up proteins and thus make those found in stains easier to remove from fabrics.

Procedure:

Day 1 -  Setup

1.      Separate into groups of no less than three and no more than four.  Do the rest of this exercise as a group.

2.      Take a hard-boiled egg and cut it in quarters.

3.      Discard the yolk

4.      Put 100 ml of water into each of 4 beakers.

5.      Add 2 ml enzyme containing detergent to one beaker, 2 ml enzyme lacking detergent to another beaker and leave the third beaker without detergent.

6.      The how you use the fourth beaker is up to you.  You may want to try a different concentration of detergent, or a mixture of both detergents.  Be as creative as you can be within the very narrow restraints of this exercise.

7.      Place 1/4 of the egg white into each beaker.

8.      Clearly mark each beaker, then let them stand at room temperature for 2 days.

Day 2 - Data collection

1.      Compare the egg whites in each beaker.  Record this data.

2.      Cleanup the beakers, then start work on your lab report.

Questions you will want to address in your lab report:

What is the hypothesis you are testing in this experiment?

Which beakers acted as controls in this experiment?

Did the enzymes present in the enzyme containing detergent make a difference to the egg white?

Do the results of this experiment prove that enzyme containing detergents are better at cleaning fabrics than regular detergents?

Would you want to use protelytic enzyme containing detergent on silk fabric?

What other uses might there be for protelytic enzymes?

Why do cells make protelytic enzymes?

Materials:

·        Detergent containing enzymes

·        Detergent that lacks enzymes

·        Water

·        100 ml Beakers (4 per group)

·        Boiled Eggs (1 per group)

·        Kitchen knives

During lab periods set aside for microscopy, you will have the opportunity to use three different types of microscopes - dissecting, compound and the Andrews University Scanning Electron Microscope (SEM).  On October 2, you will be expected to hand in a report with drawings done using both types of light microscopes and a photograph from the SEM.  In the written part of your report, which must not exceed two type written pages, you must include an analysis of the relative advantages and disadvantages of each type of microscope and an explanation of why drawings are sometimes better than photographs.  Remember that you will be given two weeks to compile this report, so the quality of work handed in must reflect two weeks of diligence.

A minimal report (C grade) will include five (5) drawings using a compound microscope: three (3) of microorganisms, one (1) of a prepared tissue slide, and one of your choice.  Also, two (2) drawings using a dissecting microscope must be in the report, one (1) of an insect and one of a subject of your choice.  Only one (1) SEM photograph must be in the report.

            Deoxyribonucleic acid (DNA) contains the genetic code.  It is also a good example of a macromolecule because it is made up of four relatively similar monomers called nucleotides.  The four nucleotides are adenine (A), cytosine (C), guanine (G), and thymine (T).  When joined together, these nucleotides can make some very large molecules.  In humans, each cell contains DNA made from approximately three billion (3 x 10⁹) nucleotides joined together.  When strung together in a linear fashion as they are in DNA, three billion nucleotides stretch almost 1 m, so each human cell has almost 1 m of DNA folded up in the nucleus.

            This laboratory takes advantage of several physical and chemical properties of DNA including its solubility in various solvents and physical length.  Because human beings have not been found to willingly volunteer their tissues for this type of experiment, onions will be used as the source of DNA.  As all organisms have a genetic code, onions have DNA in the nucleus of their cells just as humans have DNA in theirs.  The only difference is in what the DNA codes for, onions on one hand and humans on the other.

            The first step in extraction of any chemical from cells is the disruption of cells so that the chemical is released.  To do this with onion DNA, two techniques are used in this laboratory.  First, physical disruption in a blender, and second, chemical disruption using sodium dodecyl sulfate (SDS) which is a surfactant (detergent) that dissolves the oily cell membrane.  Other chemicals in the homogenization buffer we will use maintain the correct pH, osmolarity, and salt concentration.

            Following cell disruption, as many possible of the unwanted chemicals released by disruption must be removed, this is the second step.  In this laboratory, the ability of chloroform to denature protein so that it is no longer soluble is taken advantage of, consequently protein can be easily removed.  The chloroform has no effect on the solubility of DNA.

            The final step involves the actual removal of DNA from the solution.  This is achieved by taking advantage of two properties of DNA; 1) it is insoluble in alcohol, and 2) it is a large long molecule that will stick to glass.  DNA that precipitates at the interface between the aqueous solution containing DNA and an alcohol phase that has been carefully poured over the top can thus be spooled (wound onto) a glass rod.

Method

1          Cell disruption:

a          Place 50 g of diced (less than 3 mm³) onion into a 250 ml beaker and add 100 ml of homogenizing medium.

b          Incubate in a 60 oC water bath for exactly 15 min.

c          Rapidly cool the solution to 20 oC in an ice bath.

d          Place the cooled solution into a blender and homogenize.

e          Pour the solution back into the beaker (which should be cleaned first) and let it stand on ice for 15 to 20 min.

f           Filter the homogenate through four thicknesses of cheese cloth and save the filtered solution which contains the DNA.

2                    Deproteinization

WARNING: Chloroform should not be inhaled under any circumstances. Do all deproteinization steps in a fume hood that is working.

a          Pour exactly 50 ml of the filtered homogenate into a clean 250 ml flask then add 2 ml of chloroform very gently by pouring it down the side of the flask keeping the water and chloroform phases separate.

b          Very gently swirl the solution being careful not to totally mix the water and chloroform.  You should see a white precipitate appear at the interface of the two solutions.  This is denatured protein.

c          Carefully transfer the upper homogenate layer to a new 250 ml flask leaving behind the chloroform and denatured protein.

d          Repeat steps a through c another four times to ensure that all protein has been removed.

e          Transfer the homogenate to a clean 250 ml beaker being very careful to leave all the chloroform behind.

3          Precipitation of DNA

a          Cool the homogenate to 10 oC on ice.

b          Very slowly add 50 ml of ice cold ethanol by pouring it down the side of the tilted beaker.  It is essential that the ethanol and homogenate form separate layers.

c          Spool out the white stringy DNA that appears at the interface by gently swirling a glass rod around at the ethanol/homogenate interface.  Always turn the rod in the same direction.

d          Place your DNA into the blue microcentrifuge tubes containing 100 microliters of TE buffer provided.

Exercise 2: Qualitative Analysis

            By using the following procedure it is possible to test for the presence of DNA.  A positive reaction with the dipheylamine reagent (Dische) will provide one more item of evidence that the substance you removed from the onion cells actually is DNA.

1          Start a boiling water bath using 100 ml of water in a 250 ml beaker.   Be sure to exercise caution when dealing with boiling water.  If you are burned, run cool water over the burnt area and inform your instructor immediately.

2          Number 3 test tubes 1 - 3.  Into tube 1, place 1 ml of your onion DNA solution.  Into tube 2, place 1 ml of the standard DNA solution (this is a positive control).  Make a negative control with the third tube by placing 1 ml of distilled water into it.

WARNING: The Diphenylamine reagent is very acidic.  Handle it with care.  If you get any on your skin, rinse it at once with large amounts of water and inform your lab instructor.  Remove any clothing that gets diphenylamine reagent on it.

3          Add 1 ml of the diphenylamine reagent to each test tube.  then place all three into the boiling water bath for 10 min.  The test tubes containing DNA should produce a yellowish product with maximum absorbance at 595 nm.  This is best viewed against a white sheet of paper.

Materials

Equipment

Flasks, 250 ml

Ice bath

Pasture disposable long pipets

Test tubes, 10 ml

Thermometers

Water bath, 65 ^o C

Ice bath

Chemicals

Diphenylamine reagent: To make 100 ml

Mix 1 g of fresh diphenylamine with 100 ml of glacial acetic acid and 2.5 ml of concentrated H₂SO₄.  Stable for 6 months at 2 ^o C.

DNA Standard: 5 mg/ml of any DNA (Salmon sperm DNA is readily available).

Chloroform

Ethanol, 95 %

Homogenization buffer

To make 2 liters:

Sodium dodecyl sulfate (SDS)              100 g

NaCl                                                                17.54 g

Sodium citrate                                                    8.82 g

Ethyylenediamine tetraacetic acid (EDTA)           0.584 g

Add distilled water to                                        2 L

Supplies

Cheese cloth

Onions

            Deoxyribonucleic acid (DNA) contains the genetic instructions for all the proteins in our body. These instructions are spelled our in a language called the genetic code.  DNA a good example of a macromolecule because it is a very long polymer made up of four relatively similar monomers called nucleotides.  The four nucleotides are adenine (A), cytosine (C), guanine (G), and thymine (T).  One set of human DNA made from approximately three billion (3 x 10⁹) nucleotides joined together.  When strung together in a linear fashion, as they are in DNA, three billion nucleotides stretch almost 1 m, so each human cell has almost 2 m of DNA folded up in the nucleus. Why 2 m? Because our cells have two sets of DNA, one from our mother and one from our father.

            This laboratory takes advantage of several physical and chemical properties of DNA including its solubility in various solvents and its physical length.  Because most human beings wont willingly volunteer their tissues for this type of experiment, so as a substitute onions will be used as the source of DNA.  As all organisms have genetic information, onions have DNA in the nucleus of their cells just as humans have DNA in theirs.  The only difference is in what the DNA codes for, onions on one hand and humans on the other.

            The first step in extraction of any chemical from cells is the disruption of cells so that the chemical is released.  To do this with onion DNA, two techniques will be used in this laboratory.  First, physical disruption in a blender, and second, chemical disruption using sodium dodecyl sulfate (SDS) which is a surfactant (detergent) that dissolves the oily cell membrane.  Other chemicals in the homogenization buffer we will use maintain the correct pH, osmolarity, and salt concentration.

            The final step involves removal of the DNA from solution.  This is achieved by taking advantage of two properties of DNA; 1) it is insoluble in alcohol, and 2) it is a large long negatively charged molecule that will stick to positive charges on the surface of glass.  DNA that precipitates at the interface between the aqueous solution containing DNA and an alcohol phase that has been carefully poured over the top can thus be spooled (wound onto) a glass rod.

Method

1          Cell disruption:

a          Place 80 ml of water, 10 ml of dish washing detergent (SDS solution) and 10 g of non-iodized salt into a 250 ml beaker.

b          Add 50 g of diced (less than 3 mm³) onion Incubate in a 60 ^oC water bath for exactly 15 min.

c          Rapidly cool the solution to 20 ^oC in an ice bath.

d          Place the cooled solution into a blender and homogenize.

e          Pour the solution back into the beaker (which should be cleaned first) and let it stand on ice for 15 to 20 min.

f           Filter the homogenate through four layers of cheese cloth and save the filtered solution which contains the DNA.

2          Deproteinization

a          Add 1 g of meat tenderizer to the strained homogenate then let stand for 5 minutes at room temperature.  This step should digest most of the protein present in the solution.

3          Precipitation of DNA

a          Cool the homogenate to 10 ^oC on ice.

b          Very slowly add 50 ml of ice cold isopropanol (rubbing alcohol) by pouring it down the side of the tilted beaker.  It is essential that the isopropanol and homogenate form separate layers with the homogenate on the bottom.

c          Spool out the white stringy DNA that appears at the interface by gently swirling a glass rod around at the isopropanol/homogenate interface.  Always turn the rod in the same direction. The DNA will look like a blob of mucus on the glass rod.

Questions you will want to address in your lab report:

What is the hypothesis you are testing in this experiment?

Is DNA a long or short molecule? How do you know this from what you did in your experiment?

Inhalers that deliver DNAse enzymes (enzymes that digest DNA) to the lungs have been used successfully to treat cystic fibrosis. Find out as much as you can about cystic fibrosis and suggest a reason why DNAses might help people with cystic fibrosis?

Why do cells make DNAse enzymes?

Materials

Equipment

Beakers, 250 ml

Ice bath

Pasture disposable long pipettes

Thermometers

Water bath, 65 ^o C

Blenders

Chemicals

Kitchen dishwashing detergent

Isopropanol (Rubbing alcohol)

Meat tenderizer

Water

Supplies

Cheese cloth

Onions

There are three parts to most research projects: 1) Planning, 2) Execution and 3) Reporting.  In the practice project you will be doing for this class, you will participate in all three parts although the emphasis will be on execution and reporting.   Today we will be going over planning a research project, you will then set up your project.  Next project period, you will start collection of data, then the final practice project period will be dedicated to final data collection, data analysis and starting work on data presentation in the form of a poster and written report.  Practice projects will be done in groups of two, so the first thing for you to do is get together with someone you would like to work with for the next few weeks.  Remember that your evaluation in this part of Research Biology will be based on judging of your project by Berrien County Intermediate School District staff.

Before the Expo Project, you will need to do all the planning yourself, for the practice project, most of the planning has been done for you.  One of the most important parts of the planning process is writing a proposal.  Proposals outline why the research should be done, what will be achieved, and how it will be done.  In doing this, a proposal has the following parts:

Introduction - A clear statement of the question being asked, why it is important and what the hypothesis being tested is.  In doing this, it is essentially the same as the introduction of a normal scientific paper.

Proposed Methods – A detailed outline of the procedure that will be used in answering the question posed in the introduction.

Budget – No research is free, thus it is important to know how much it will cost and how it will be paid for before undertaking the research.  Running out of funds halfway through a project would mean that all the funds that have been spent and effort expended were wasted.  One of the main purposes of most proposals is to obtain funding, so this is a very important part of proposals.

Following is the proposal for the practice project you will be doing:

Relative Growth Rates In The First Week Following Germination

In The Presence Or Absence Of Fertilizer

Introduction

Use of commercial fertilizers in farming enterprises has increased dramatically over the past 50 years (Brown et al, 1997).  Correlated with this increase in fertilizer usage has been a dramatic increase in agricultural production.  While part of this increase in production can be attributed to improvements in crop breeding techniques, much also appears to be due to the use of fertilizers.  This increase in production has not been without cost, both in damage to the environment due to agricultural runoff and energy consumption due to fertilizer production (Miller, 1982).  Energy consumption is a particular concern in the production of nitrogen fertilizers (Morrison and Boyd, 1983), the most commonly used fertilizer component in industrialized countries.  As the impact of fertilizer use has been felt, a need to re-examine application techniques to ensure that they are used with maximum benefit and minimum impact has developed. 

The purpose of research outlined in this proposal is to address the question of when fertilizers can be most effectively applied, either before or following germination.  As the first stage of seed germination is the process of imbibation, in which the seed absorbs water and activates enzymes before beginning the shoot and root growth process (Campbell, 1996), it seems reasonable to suppose that nutrients contained in fertilizers dissolved in the water may also be absorbed and provide an abundance of needed nutrients to the growing embryo that would not be otherwise available.   Thus it seems reasonable to hypothesize that fertilizer will improve the growth of seedlings over the week following fertilization.

Proposed Methods

This project will be divided into three phases: 1) Setup, 2) Data Collection and 3) Data Analysis.

·        Setup – Two groups of three disposable cups will be used.  The first set of cups will serve as a control.  Into each of the three, two crumpled paper towels will be placed, then the towels will be moistened with 20 ml of distilled water.  20 ____________ seeds will then be placed on top of the moist towels with an approximately even amount of space between each seed.  The ____________ seeds will then be covered with two more crumpled paper towels which will be moistened with another 20 ml of water.  Seeds germinating in these containers will provide a negative control, giving a baseline set of data for seeds germinated in the absence of fertilizer.

A second set of three cups will be set up in a way identical to the first set of three, the only variable being that instead of water, a solution of Miracle Grow plant food will be used with 5 g of plant food per liter of solution.  This will be the experimental set and by comparing the seedlings from this set of cups with those of the control set of cups, it should be possible to determine if the fertilizer had an effect on germination and seedling growth.

All cups will be clearly marked, then loosely covered to retard evaporation while allowing some air exchange for respiration.  They will then be set aside for a week at room temperature.  Figure 1 shows the basic setup for a single disposable cup.

Figure 1

Setup for Both Experimental and Control Cups

Each disposable cup will contain 20 seeds sitting on top of two paper towels and overlain with an additional two paper towels.  Paper towels will be moistened with either water or a fertilizer containing solution.