Biology 171L – General Biology Lab I Lab 4: Biological Molecules

Introduction

There are four major classes of organic compounds (macromolecules) which comprise the bulk of living matter: carbohydrates, proteins, lipids, and nucleic acids.

In the first part of this laboratory you will conduct some simple chemical tests which will demonstrate the presence of one or more of these molecules in food you eat. We will focus primarily on carbohydrates, lipids and proteins, leaving nucleic acids for a later lab activity.

In the second part of this lab you will extract, purify and quantify DNA from a colony of bacteria.

Learning Outcomes

After completing this lab you will be able to:

1. Identify the four basic biological compounds

found in most living organisms. 2. Describe the characteristics and biological

function of these four basic compounds. 3. Test for the presence of the major biological

compounds in each of the four basic food groups brought in from home.

4. Sample a colony of bacteria for obtaining a pure sample of its DNA.

Pre-lab Preparation

1. Read Chapters 4 and 5 in Campbell Biology

(9th Ed.). 2. Formulate hypotheses BEFORE LAB

regarding the biochemical composition of the common foods that you will test in lab.

3. Bring four different types of food to lab for testing.

4. Bring personal protective gear: lab coat and closed-toe shoes.

Identification of Biological Molecules Carbohydrates

Carbohydrates comprise a wide variety of monomers, dimers, and polymers of saccharides (sugars) in living organisms. Sugars, the smallest carbohydrates, serve as fuel and carbon sources, as you will see later when we discuss cellular metabolism. Polysaccharides, the polymers of sugar, have storage and structural roles in cells and organisms.

You will conduct two tests specific for two important types of carbohydrates, reducing sugars and polysaccharides based upon the presence of the carbonyl group found in sugars. These structures are also components of aldehydes and ketones.or a ketone (Fig. 1).

Figure 1. Aldehyde and ketone functional

groups.

The reagent you will use is Benedict’s solution, a mixture of sodium citrate, sodium carbonate, and copper sulfate. Sugars that contain a carbonyl group are referred to as reducing sugars because other compounds or elements, such as the Cu+2 in Benedict’s solution, accept the electrons released by the oxygen in the carbonyl group. When reacted with a small quantity of reducing sugar, the blue-colored Benedict’s solution turns green; a test solution which contains a large amount of sugar turns the solution red-orange. The color change is due to the Cu+2 ion being reduced to Cu+ and precipitating as cuprous oxide. Benedict’s solution will not react with the oxygen if it is involved in a bond (glycosidic) between monosaccharide units.

Many polysaccharides (storage or structural polymers of sugar) react with Lugol’s reagent (iodine potassium iodide). Two such polymers, amylose and amylopectin (plant starch), react with Lugol’s reagent and turn to a blue-black color; glycogen (animal starch) turns red-brown; cellulose (the major component of

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plant cell walls) turns violet-brown to red-brown. The color change is the result of the iodine molecule reacting with the helical structure of the polysaccharide.

Keep in mind that these tests are only qualitative in nature and are used only to confirm the presence of a particular macromolecule. In more advanced classes you will be shown how to do qualitative testing for these molecules. Lipids

Two important lipids are fats (triacylglycerols) and phospholipids. Fats store large amounts of energy, whereas phospholipids are major components of cell membranes. Chemically, the two are very similar, as both are made from the same two subunits: glycerol and fatty acid.

Figure 2. The basic structure of a

triacylglycerol.

Glycerol, the backbone of a lipid, is a three carbon molecule and is attached to three fatty acid molecules (at the -OH group) to make a triacylglycerol (Fig. 2). The long, nonpolar hydrocarbon tails of fats make them insoluble in water (you’re probably already aware of this if you like vinegar and oil salad dressing), and this insolubility itself is a good test for the presence of fats. Another test utilizes a lipid-soluble dye called Sudan III. Sudan III dissolves in nonpolar substances such as fats and oils, staining them orange-red. Proteins

Proteins are large molecules composed of many amino acids linked together to form a long chain of amino acids called a polypeptide. These amino acids are joined together through a peptide bond (Fig. 3). A peptide bond is formed

by a condensation reaction between the carboxyl group (-COOH) of one amino acid and the amino group (-NH2) of a second amino acid to form a carbamyl group (-CONH). To test for the presence of this carbamyl group you will use Biuret’s Reagent (sodium hydroxide and copper sulfate). Substances containing two (or more) carbamyl groups, such as proteins, react positively with Biuret’s reagent), turning from a blue to pinkish-violet color.

Figure 3. A dipeptide molecule formed by two amino acids joined through a peptide bond.

Nucleic Acids

Nucleic acids are constructed of long chains of subunits called nucleotides. A nucleotide is composed three smaller units: a nitrogenous nucleotide base, a five-carbon monosaccharide, and a phosphate group. The nucleotides are joined together through their monosaccharides and phosphate: the phosphate group of one nucleotide joins to the monosaccharide of the adjacent nucleotide. The nucleotide bases, joined to the monosaccharides, stick out from what is essentially the sugar-phosphate “backbone” of the nucleic acid.

Nucleotide bases are ring structures involving carbon and nitrogen atoms. There are two classes of nucleotide bases typically found in nucleic acids: purines and pyrimidines. Purines (e.g., adenine and guanine) are double structures, whereas pyrimidines (e.g., thymine, cytosine, and uracil) are single six-sided rings.

The two major types of nucleic acids in living organisms are ribonucleic acid (RNA) and deoxyribonucleic acid (DNA). In RNA the five-carbon monosaccharide is ribose. But in DNA the monosaccharide is deoxyribose, which means this monosaccharide has one less oxygen atom in it than does ribose. In addition, RNA is single-stranded, while DNA consists of

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two nucleic acid strands arranged parallel (anti-parallel actually) to each other (Fig. 8).

DNA may be extracted from tissues by precipitation in alcohol from homogenized tissues or cell culture samples. However, other biological molecules (e.g., protein) may also precipitate in alcohol. So if we destroy these other molecules (via enzyme degradation) before alcoholic precipitation, the only precipitable material would be DNA. We only need to clean up the precipitate and re-suspend it to obtain a clean DNA solution. Then we can subject this purified DNA solution to subsequent treatment and analyses. One of these analyses may involve using a special type of instrument called a spectrophotometer to quantify the concentration of DNA in solution by quantifying the absorption of ultraviolet light at 260 nm.

We will be extracting and purifying DNA from colonies of bacteria prepared prior to the lab activity. The extraction, purification and resuspension will done using the Qiagen DNAeasy kit. Then we will quantify our yields using a Nanodrop system.

You will work in pairs. But each student is responsible to submit his/her own lab summary.

Procedures and Assignment I. TESTING FOR THE PRESENCE

OF BIOLOGICAL COMPOUNDS IN FOOD

A generalized procedure for each

biochemical test is provided below. You will also have the following compounds as positive controls for comparison to your four foods: • 1% glucose (a reducing sugar), • 1% egg albumin (a protein), • 1% soluble plant starch (a polysaccharide), • corn oil (a lipid).

These controls should be run in experiments in exactly the same way you test your food samples.

You are to use the food products you brought to this lab and apply each of the following tests on that food. You should also observe the tests conducted by your bench partners and note the food and color changes they obtained when conducting these chemical tests.

I.A. BENEDICT’S TEST FOR REDUCING SUGARS

1. Add 1 mL of the test food solution to a

clean, dry test tube. (If you are using a solid food substance, you will need to take a small amount and mix with water (about 1 ml) to get it into solution).

2. Add 1 mL of Benedict’s reagent to the test tube, mix well, and heat the tube in a dry bath at 95-99°C for 4-5 minutes.

3. Carefully remove the tube and allow it to cool.

4. Compare the resulting color with your positive and negative controls. Record the color change in your data chart as + or -.

I.B. LUGOL’S TEST FOR

POLYSACCHARIDES 1. Add 1 mL of your test solution to a clean,

dry test tube. 2. Add 1-2 drops of Lugol’s solution to the

tube and mix well. 3. Compare the color change with your

positive and negative control. Record the resulting color change as + or -.

I.C. SUDAN III TEST FOR LIPIDS 1. With a pencil, write the identity of the test

solution on a piece of filter paper located at your bench top.

2. With a clean plastic bulb pipette, transfer a VERY SMALL drop of the test solution to the filter paper. The solution will spread out very rapidly so try to just touch the pipette to the paper.

3. Allow the spot to dry completely (4-5 minutes). Then, using forceps, submerge the filter paper in a dish of Sudan III solution for exactly 1 minute.

4. Transfer the filter paper to a dish of distilled water for 1 minute to wash away the excess dye.

5. Compare the color change with your positive and negative controls. Record the color change as + or -.

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I.D. BIURET TEST FOR PROTEIN 1. Add 1 mL of your food solution to a clean,

dry test tube.

The following step uses NaOH, a STRONG alkali. Be sure to use all your personal protective gear (lab coat, safety goggles, and gloves) when working with this material. 2. Add 4 drops of Biuret reagent to the tube

and mix well. Compare the color change against your positive and negative controls. Record the resulting changes as + or -.

II. DNA Extraction, Purification and

Quantification II.A. DNA Extraction and Purification

We will use the Qiagen DNAeasy kit to

extract DNA from bacteria samples from culture plates provided to you in lab. But before you get started be sure you are familiar with the materials you will be working with. They are listed below. Before starting the lab spend some time recognizing and finding these items. 1. four micropipetters (0.5-10 µL, 5-50 µL, 20-

200 µL, 100-1000 µL volumes) 2. clear pipette tips (for 0.5-200 µL volumes) 3. blue pipette tips (for 100-1000 µL volumes) 4. container for disposal of used pipette tips

(pipette tips only!) 5. biohazard disposal bags (for used gloves,

bacteria transfer loops, paper wipes, agar plates, etc. – no pipette tips, nor sharp waste should be disposed of in these bags)

6. 56°C water bath 7. vortex apparatus (for mixing contents of

tubes) 8. gloves 9. boxes of Kimwipes 10. 5-10 bacteria transfer loops per pair of

students 11. beaker of sterile microfuge tubes

12. two DNeasy Minispin columns, each in a 2 mL tube (individually packaged), per pair of students

13. chemical reagents (most set up in a microfuge tube holder) per pair of students a. 15 mL tube of sterile water b. two 1.5 mL microfuge tubes containing

180 µL ATL buffer (labeled “ATL”) c. one 1.5 mL microfuge tube containing

proteinase K (labeled “Pro K”) d. one 1.5 mL microfuge tube containing

AL buffer (labeled “AL”) e. one 1.5 mL microfuge tube containing

100% ethanol (labeled “EtOH”) f. one 15 mL tube containing AW1 buffer

(labeled "AW1") g. one 15 mL tube containing AW2 buffer

(labeled "AW2") h. one 1.5 mL microfuge tube containing

AE buffer (labeled “AE”) 14. four 2 mL collection tubes (these are round-

bottomed tubes) per pair of students. The DNA extractions steps are listed below (see also Figure 4). Be sure you read and understand each step before doing it. I suggest you highlight areas of importance (ex: amounts, time). 1. Using a bacteria transfer loop transfer a

small colony of bacteria into a microcentrifuge tube containing 180 µL ATL buffer (labeled “ATL”). Repeat this step with the second sample into a separate tube. Be sure to record the source, dilution, growth medium, and characteristics of this bacteria colony. Also be sure you label your tubes to keep track of them.

2. Add 20 µL proteinase K (tube labeled “Pro

K”) to each tube. Cap and mix this tube by vortexing a few seconds. Use the minifuge to spin the liquid to the bottom of the tube. Place the tube in the 56°C water bath for 1 hour.

While the incubation is taking place, carry out some of the other lab activities. 3. Vortex for 15 seconds. Add 200 µL of AL

buffer (labeled “AL”) to each tube, then mix immediately to yield a homogeneous solution.

4. Add 200 µL ethanol (in tube labeled “EtOH”)

to each tube. Vortex again.

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5. Do the following for each of your bacteria preparations from step 5: using a pipetter, carefully transfer the mixture into a DNeasy Mini spin column placed in a 2 mL collection tube (packaged together). Centrifuge at 8000 rpm for 1 minute (we’ll do this step together as a class). Discard the flow-through liquid and collection tube. Save the column! The DNA should be found in the column (though you won’t be able to see it).

6. Place DNeasy Mini spin columns into new 2

mL collection tubes. Add 500 µL AW1 buffer (labeled “AW1") to each. Centrifuge again for 1 minute at 8000 rpm. Discard the flow-through and collection tubes. Save the columns! This step is a wash step that removes contaminants in the column. The DNA should remain in the columns.

7. Once again, place the DNeasy Mini spin

columns into new 2 mL collection tubes. Add 500 µL AW2 buffer (labeled “AW2") to each. Centrifuge at 14,000 rpm for 3 minutes to remove all residue. Discard the flow-through and collection tubes. This step is also a wash step. The DNA should remain in the columns.

8. Place the DNeasy Mini spin columns into 1.5

mL microcentrifuge tubes. Carefully detach the column caps from the column.s

9. Carefully add 100 µL AE buffer (labeled

“AE”) into each column (directly over the filter disk in the column – don't touch the disk) and incubate at room temperature for 1 minute. Then centrifuge for 1 minute at 8000 rpm. This time DO NOT DISCARD THE FLOW-THROUGH. It contains your DNA!

10. Without removing the 1.5 mL

microcentrifuge tube used to collect the flow-through, add another 100 µL AE buffer and repeat the centrifugation. Thus the flow-through will be about 200 µL in volume. Again DO NOT DISCARD THIS FLOW-THROUGH. It contains your DNA!

Your total flow-through liquid in each collecting tube should contain all of the extracted DNA. You may throw away the columns and carefully close the microcentrifuge tubes and save for any DNA anaylsis. Be sure all tubes are properly labeled so that you will be able to identify and

use your samples in a later lab activity. Congrats!

Figure 4. Summary of DNA extraction protocol. II.B. DNA Quantitation We will measure the DNA concentrations in our extracts using a Nanodrop spectrometer. The procedure will involve dispensing 1 µL of your DNA extract onto the sample platform of the instrument and measuring the absorbance of your this sample at 260 and 280 nm. Your instructor will explain and demonstrate the use of this instrument. Record the values and units of the instrument read-out. III. CLEAN UP Clean your test tubes with detergent and return them (inverted to dry) to their test tube racks, Please return all re-usable supplies to their original location. Finally, be sure to disinfect your workstation.

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Summary of Assignment to be Turned In

1. Descriptive title for assignment. 2. Brief Introduction describing the purpose of

this lab activity and its objectives. 3. A Results and Discussion section that

includes the following components. Be sure to follow the rules for presenting figures and tables! a. Table (Table I) that presents the results

of your tests (controls and food samples).

b. Written analysis for the chemical compositions of your four food types. Be sure to explain how you arrived at your conclusion.

c. DNA quantitation results. 4. A Conclusion paragraph summarizing what

was learned and could be concluded from the data.

Assignment must be typed. Written text must utilize correct spelling, complete sentences, and correct grammar.