University of New England

School of Science and Technology

BCHM210/410

Introductory Molecular Biology and Biochemistry

Practical Manual

Unit Coordinator: Dr. Sinead Henderson

© University of New England

CRICOS Provider No: 00003G
 All students must submit two practical reports:
Practical 3 - β-galactosidase activity
Practical 4 - Separation of Macromolecules

See the Assessment information online for the weight and

submission dates of your practical reports.

Introductory molecular biology and biochemistry ________________________________________________________ iii

iv ________________________________________________________ Introductory molecular biology and biochemistry
 TABLE OF CONTENTS
INTRODUCTION………………………………………………………………………………….vi
SAFETY PRECAUTIONS AND ACCIDENT PROCEDURES ........................................... vi
LABORATORY RULES AND PROCEDURES .............................................................. vii
INSTRUCTIONS FOR THE USE OF CENTRIFUGES ......................................... viii
CONTROL OF ACCURACY ............................................................................ ix
GENERAL LITERATURE ........................................................................................... x
CALCULATIONS IN BIOCHEMISTRY ....................................................................... xi

PRACTICAL 1 - SPECTROPHOTOMETRY ....................................................................................... 1

INTRODUCTION .................................................................................................... 1
PART I: CORRECT USE OF PIPETTES ....................................................................... 1
PART II: SPECTROPHOTOMETRY ............................................................................ 4

PRACTICAL 2 – DNA PURIFICATION AND MOLECULAR CLONING ................................... 13

PART I - DNA PURIFICATION ................................................................................ 13
INTRODUCTION ......................................................................................... 13
EXPERIMENTAL PROCEDURE ....................................................................... 16
PART II – MOLECULAR CLONING (“DRY LAB”) ....................................................... 17
BACKGROUND ........................................................................................... 17
AIM ........................................................................................................ 17
PROCEDURE ............................................................................................... 17
Role 1: Technician .................................................................................... 17
Role 2: Scientist ........................................................................................ 18
Role 3: Scientist ........................................................................................ 18
Role 4: Restriction enzyme SmaI .............................................................. 18
Role 5: DNA ligase ..................................................................................... 18
Role 6: mars- bacterial cell......................................................................... 19

PRACTICAL 3 - THE INDUCED SYNTHESIS OF ß-GALACTOSIDASE IN E. COLI .............26

INTRODUCTION ......................................................................................... 26
EXPERIMENT .............................................................................................. 30
PROCEDURE ............................................................................................... 31

PRACTICAL 4 - SEPARATION OF MACROMOLECULES ........................................................... 38

INTRODUCTION ......................................................................................... 38
EXPERIMENT .............................................................................................. 41

PRACTICAL 5 - DIGESTION OF STARCH AND CELLULOSE ................................................... 50

PART I: EFFECT OF ENZYME CONCENTRATION ON ENZYME ACTIVITY .................52
PART II: EFFECT OF CHLORIDE ION ON THE ACTIVITY OF AMYLASE .....................53
PART III: DEMONSTRATION EXPERIMENT ............................................................. 55

Introductory molecular biology and biochemistry _________________________________________________________ v

 INTRODUCTION

We selected the practicals in this biochemistry unit to cover as wide a range of

biological compounds and processes as possible and to provide you with experience in
a range of techniques used in biochemical research. We wanted to provide you with
training and skills in laboratory procedures, report writing and introduce an analytical
approach to the evaluation of experimental results. The general skills learnt in the
practical course are relevant to all vocations, not just science and biochemistry.
By the end of this course it is expected that you will be able to:

A. By data gathering and processing

1. accurately record data from experiments in a suitable form;
2. process recorded data using appropriate mathematical techniques;
3. present processed data in graphs, tables or other forms suitable for inclusion in a
report;
4. interpret data and reach valid scientific conclusions;
5. produce reports in a clear and concise manner in a format similar to that of most
scientific reports;

B. By performing experiments
6. explain the theoretical basis behind individual practicals;
7. explain the importance of the techniques learnt in practicals;
8. formulate plans for performing experiments;
9. arrange the appropriate amount and type of equipment for performing a particular
practical;
10. use and understand the operation of a limited range of laboratory equipment;
11. explain the rationale behind any steps in the practicals and
12. behave in a safe and responsible manner in accordance to instructions given by
your demonstrators and the general laboratory rules.

SAFETY PRECAUTIONS AND ACCIDENT PROCEDURES

• All accidents, including fire or injury, however trivial, must be reported to a

demonstrator at once.
• Experiments should not be left unattended especially if materials of a flammable nature
are being used. Never evaporate a flammable solvent within range of a naked flame -
always use the fume cupboard.
• Become familiar with the position and operation of fire extinguishers and safety
shower. Do not use water to put out solvent or electrical fires - in the former case, water
will merely spread the fire.
• First aid materials are in the laboratory. Make sure you know where they are.

vi ________________________________________________________ Introductory molecular biology and biochemistry

 LABORATORY RULES AND PROCEDURES
YOU ARE EXPECTED TO BRING THE FOLLOWING TO CLASS:
Lab coat, covered shoes, safety glasses, laboratory manual, permanent
marker pen, notebook, calculator, ruler, USB memory stick or laptop
computer
• Failure to wear a laboratory coat and covered shoes will mean your immediate exclusion
from the practical session and a mark of zero for that practical. Safety glasses are
provided but you will usually be more comfortable if you bring your own.
• You are expected to read practical notes and plan work before coming to class.

• For most classes you will be expected to work along or in pairs, working in larger groups
unless directed by your demonstrator in not permitted.
• Do not, under any circumstances pipette solutions by mouth. Any possibility of acute
poisoning must be reported immediately - delay could be fatal. Be careful not to inhale
undue amounts of solvent vapours. Chronic poisoning is a real risk.
• Eating, drinking and smoking are not permitted in the laboratory.

• You are not permitted in the laboratory until demonstrators arrive. Surplus materials and
clothing must not be brought into the laboratory.
• No practical work whatsoever may be commenced before the pre-laboratory talk. Work
outside normal class hours may be done only with the express permission of the
lecturer/demonstrator in charge.
• The rules for using spectrophotometers and centrifuges must be strictly observed. If
there is any doubt regarding instructions or procedures a demonstrator should be
consulted.
• Apparatus issued for a particular section of the work must be returned in a clean
condition at the earliest opportunity.
• Always use a tile under Bunsens to avoid burning the bench.
• Reagent bottles must not be removed from the front bench. Dispense the reagent into a
labelled container and do not take more than is actually required. Many reagents are
expensive, or are difficult to prepare and supplies may be limited. If you waste reagents,
you may be refused further materials and be unable to complete the practical.
• Do not dip pipettes into reagent bottles. Pour the approximate amounts required into
separate, clean, dry, labelled containers and then pipette what is needed from these.
Discard the remainder. In some cases a pipette or dropper will be provided with the
reagent bottle. Do not use this pipette or dropper for any purpose other than that for
which it has been provided.
• At all times during work in the laboratory, vessels containing substances or reagents
should be adequately labelled.
• Be careful not to spill chemicals, but mop up any accidental spillage at once.

• Glassware should be washed after use with detergent and warm water applied with
vigorous brushing, then rinsed thoroughly. Glass pipettes should be rinsed out several

Introductory molecular biology and biochemistry ________________________________________________________ vii

 times with water immediately after they have been used for fluids such as blood or
protein solutions. If a pipette retains droplets or forms rivulets on its walls during
delivery, it is not clean. Return glassware to allocated locker.
• Complete each day's work by washing-up and cleaning the bench top.

A breach of laboratory safety can result in your exclusion from the practical class
and a mark of zero recorded for that practical.

INSTRUCTIONS FOR THE USE OF CENTRIFUGES

For general use, the required rotational speed of centrifuges can be obtained from the
demonstrators in terms of the appropriate rheostat setting. Note however that
centrifugal force is the only consistent way of specifying centrifugation conditions.

Centrifugal Force (g) can be calculated from the formula:

r.n 2
R.C.F. =
89,000
and Centrifugal Force = R.C.F. x weight

where, R.C.F. is Relative Centrifugal Force,

r is average radius (in cm). [12.6 cm for the Clements, 8.8 cm for the MSE,

and 7.5 cm for the Martin Christ 10 ml tubes].

n is revolutions per minute.

1. For effective and safe centrifugation it is essential that the complete assembly
head be in the centrifuge head, accurately balanced. Pre-balance two glass beakers on
a swing balance. Balance the two systems by adding water in the space around the
tubes, or where the volume of the contents is not important, by removing some of the
contents. The weight variation for any pair must not exceed ± 0.25 g. When the pair
is adequately balanced the tubes should be transferred to opposite positions in the
head.
2. A preliminary check should be made by running the machine up to approx.
500 r.p.m. If there is no significant vibration the speed control knob may be turned
clockwise slowly, until the desired speed is obtained. If there is significant vibration
the centrifuge must be stopped, each pair checked and adjustments made to the
balancing.
3. Timing should begin once the centrifuge has reached the desired speed.
When the required time has elapsed the speed control knob should be turned
anticlockwise to the "off" or "zero" position, and the centrifuge allowed to run down
unassisted.
4. When the centrifuge has stopped remove the tubes, ensuring that the head
and bucket are left clean.

viii ______________________________________________________ Introductory molecular biology and biochemistry

 CONTROL OF ACCURACY

In laboratory work which involves some form of measurement, potential sources of

error should be appreciated.
Determinate, or constant errors, such as result from wrongly calibrated pipettes,
wavelength errors in spectrophotometers or poor quality chemicals must be avoided,
minimised or taken into account. Such errors can be minimised by the calibration of
instruments (eg. weights, flasks, burettes, pipettes) and the application of the
appropriate factors to the original measurements; by performing the analysis in an
alternative manner and checking the agreement with the method in use; or by
techniques such as standard addition or isotopic dilution. In these practicals, errors
will be most often minimised by the running of a blank determination, containing no
sample, under exactly the same conditions as are employed in the analysis of the
sample; by running a series of standards, containing known amounts of the sample
under analysis, under, as nearly as possible, identical experimental conditions to those
of the sample, and by the running of parallel determinations (i.e. duplicates or
replicates) and calculating the average value. The running of parallel determinations
also allows correction for random or accidental errors, which are otherwise
indeterminate. Variable and random errors occur due to factors such as incomplete
mixing of reacting solutions. For the purposes of these practicals, duplicate
estimations are sufficient provided there is good agreement (precision) between
them. Good agreement between duplicate determinations, however, does not justify
the conclusion that the result is correct; a constant error may be present. The
agreement merely shows that the accidental errors, or variations of the determinate
errors, are the same, or nearly the same, in the parallel determinations.
An additional error to be considered when working with material derived from living
matter is biological variation, the extent of the normal range depending upon the
constituent being measured.

Significant Figures and Computations

The final result of any analysis should indicate the accuracy of the measurement.
The term digit denotes any one of the ten numerals, including the zero. A significant
figure is a digit which denotes the amount of the quantity in the place in which it
stands. The digit zero is a significant figure except when it is the first figure in a
number.
Thus in the quantities 1.2680 g and 1.0062 g the zeros are significant, but in the
quantity 0.0025 kg the zeros are not significant figures; they serve only to locate the
decimal point and can be omitted by proper choice of units, eg. 2.5 g instead of
0.0025 kg. The first two numbers contain five significant figures, but 0.0025 contains
only two significant figures.
There are a number of rules for computations with which the student should be
familiar:

Introductory molecular biology and biochemistry ________________________________________________________ ix

 1. Retain as many significant figures in a result or in any data as will give
only one uncertain figure. Thus a volume which is known to be between 20.5 ml and
20.7 ml should be written as 20.6 ml, but not as 20.60 ml, since the latter would
indicate that the value lies between 20.59 ml and 20.61 ml. Also, if a weight, to the
nearest 0.1 mg, is 5.2600 g, it should not be written as 5.260 g or 5.26 g, since in the
latter case an accuracy of 0.01 gram is indicated and in the former a milligram.
2. In rounding off quantities to the correct number of significant figures, add
one to the last figure retained if the following figure (which has been rejected) is 5 or
over. Thus the average of 0.2628, 0.2623, and 0.2626 is 0.2626 (0.26257).

3. The uncertainty in a sum or difference is the sum of the uncertainties.

If m1 = 100 ± 1 g and m2 = 50 ± 1 g

then, m1 + m2 = 150 ± 2 g and m1 - m2 = 50 ± 2 g.

4. The percentage uncertainty in a product on quotient is the sum of the

individual percentage uncertainties. In the example in 3 above m1 has a percentage
uncertainty of 1%, and m2 a percentage uncertainty of 2% and so :

m1m2 = 5000 g2 with an uncertainty of 3% i.e. 5,000 ± 150 g2

m1/m2 = 2.0 with an uncertainty of 3% i.e. 2.0 ± 0.06.

GENERAL LITERATURE

You are encouraged to be aware of the nature of chemical substances which you have
not encountered before, and of the means by which they perform their function in the
experiment. For this purpose the following references are particularly useful. As an
exercise in professional development it is expected that you learn how to use these:

1. The "Merck Index".

A single volume reference containing brief descriptions of the nature of >10,000
chemical substances of agricultural, analytical, and pharmacological significance.

2. "Methods in Enzymology".
A series of 160+ volumes containing detailed information on every procedure used in
modern biochemical laboratories.

x ________________________________________________________ Introductory molecular biology and biochemistry

 CALCULATIONS IN BIOCHEMISTRY

During this practical course you will often be required to dilute a stock solution of a
compound to a particular concentration, or to take a specific amount of a compound
from a stock solution provided.
Since students often have difficulty in distinguishing between absolute amount (mass)
and concentration and in making the appropriate calculations, some information and
some sample calculations are given below.

Calculations involving proportional amounts

If two solutions or mixtures are proportional, equation 1 can be used to calculate an

unknown quantity:

C1V1=C2V2 (Equation 1)

C1V1= Concentration/amount (start) x Volume (start) of the same solution

C2V2 = Concentration/amount (final) x Volume (final) of the same solution

This equation is useful if you need to calculate:

1. How much ingredient is contained in a different volume of the same concentration

2. A conversion of one concentration to another
3. The quantity of a substance required to produce a different final concentration and
volume.

Ensure that the units are equivalent for C1 and C2 and for V1 and V2.

Example:

A postgraduate student has 2 g/L of solution A. How much of solution A does he need
to add to make 3L of a 1 g/L solution?

Calculation
Use C1V1=C2V2

Introductory molecular biology and biochemistry ________________________________________________________ xi

 C1 (2 g/L) x V1 (We don’t know this) = C2 (1 g/L) x V2 (3L)

Rearrange the equation to solve for V1 = (1 g/L x 3L)/ 2 g/L

= 1.5 L
Therefore the student will add 1.5L of solution A to make 3L of a 1 g/L solution.

The Unit of Concentration of a Substance in Solution is molar.

Molar: a molar solution of a substance contains one mole of that substance per litre of
the solution.

If C = concentration (in molar)

n = the amount (in mol) of a substance

V = volume (in L)

Then C = n/V (Equation 2)

Abbreviation: M = Molar (mol/L)

Prefixes: mM = Molar x 10-3,

µM = Molar x 10-6, etc.

Since most biochemistry is done on a semi-micro or micro scale, you will frequently
use amounts of the order of µ moles, concentrations of the order of mM and volumes
of less than 1 ml.

From equation 2: C = n/V

By simple rearrangement, n =CxV (Equation 3)

And: V = n/C (Equation 4)

If you always enter your data in the correct units (C in mol/L, A in mol, and V in
litres) into the above equations, these can be used to solve many common biochemical
problems.

xii _______________________________________________________ Introductory molecular biology and biochemistry

 Example 1:
How much compound X is in 5 ml of a 7.5 mM solution of X? (i.e. solve for n)

Calculation:
Use equation 3 : n=CxV
The amount of X in 5 ml (n) = 0.0075 (mol/L)(C) x 0.005 (L)(V) = 37.5 x 10-6 moles

= 37.5 mmol

Example 2:
Given a stock solution of 10 mM compound B, pipette out a volume which contains
exactly 3 µmol of compound B. (Put another way, this question is asking what volume
of the 10 mM solution of B contains 3 x 10-6 mol of B; i.e. solve for V).

Calculation:
Use equation 4: V = n/C
V (litres) = 3 x 10-6 (mol)(n) / 0.010 (mol/L)(C)

The volume required is 3 x 10-4 L (= 0.3 mL) of stock solution.

Calculation of unknown concentrations using an equation

The equation of a straight line in a graph is

𝒚𝒚 = 𝒎𝒎𝒎𝒎 + 𝒃𝒃
Where: 𝒚𝒚 is absorbance of a solution
𝒎𝒎 is the slope of the line
𝒙𝒙 is the concentration of that solution, and
𝒃𝒃 is the 𝒚𝒚 intercept. In many cases, 𝒃𝒃 = 𝟎𝟎 as you set the y intercept to 0
when making the graph
You can rearrange the equation to calculate the concentration of unknown solutions as
follows:

𝒚𝒚 − 𝒃𝒃 𝒚𝒚
𝒙𝒙 = 𝒐𝒐𝒐𝒐 𝒙𝒙 = (𝒊𝒊𝒊𝒊 𝒃𝒃 = 𝟎𝟎)
𝒎𝒎 𝒎𝒎

Use this rearranged equation to calculate undiluted concentrations of your unknowns.

Introductory molecular biology and biochemistry _______________________________________________________ xiii

 PRACTICAL 1 - SPECTROPHOTOMETRY

INTRODUCTION

This lab is designed to give you an introduction to two pieces of equipment you will
need to use in the majority of biochemical experiments, i.e. automatic pipettes and
spectrophotometers. You will discover that practical biochemistry is a very
quantitative subject where the quality of your results is dependent on your ability to
measure volumes accurately (pipettes) assay concentrations (spectrophotometer), as
well as use simple proportion and calibration graphs to calculate results. These are
skills which many, if not most of you, will need to use, not just in the remaining labs of
this subject, but in a wide range of biochemical assays used across the broad range of
biological sciences from environmental science to food science. We will therefore
spend this lab making sure our techniques are right before launching into the rest of
the subject.

PART I: CORRECT USE OF PIPETTES

In chemistry you learnt to use accurately glass pipettes. We assume you know how to
use these correctly. Remember:

- Distinguish whether the pipette is “blow out” or not and use appropriately

- wipe surplus liquid from the outside of the pipette

- use the most appropriate size of pipette for the volume required.

In biochemistry the repetitive measurement of identical volumes, the requirement to

measure out very small volumes, as well as the need sometimes to work with sterile
solutions means that automatic pipettes are commonly used, particularly for volumes
under 500 µL. As with many items of equipment these:

(a) are EXPENSIVE

(b) are BREAKABLE

(c) must be used correctly to obtain the desired volumes.

We have a set of automatic pipettes for volumes between 2 and 5000 µL. Instructions
for use are set out below. Please read carefully. You will also be given a
demonstration of autopipette use. The Gilson 'Pipetman' is a high quality instrument
which offers excellent reproducibility and, when calibrated, high accuracy.

Introductory molecular biology and biochemistry ________________________________________________________ 1

 It is important to choose the correct automatic pipette for the volume you wish to
dispense.

The autopipettes available are:

1000 - 5000 µl

200 - 1000 µL

20 - 200 µL
2 - 20 µL

CHECK the RANGE on the autopipette before you use it.

YOU CANNOT USE the autopipette OUTSIDE that RANGE. - IF IN DOUBT ASK.

Adjustment of Volume

The desired volume is set by simply turning the volume adjustment ring. When
increasing the volume setting it is necessary to go about one-third of a turn above the
volume, and then come back in decreasing value to the desired setting. When
decreasing the volume setting, the desired volume can be achieved directly. Read the
volume from top to bottom in microlitres for the P20, P100, P200 and in millilitres for
the P1000 and P5000.

DO NOT EXCEED the volume range of the pipette, i.e. if a 20 to 200 µL pipette is to be
used and you try to adjust it to 250 µL YOU WILL BREAK THE PIPETTE!

NOTE: Never leave pipette on the bench with liquid in the tip.

Always carry a filled pipette in the vertical position.

Never insert above the disposable tip of the pipette into liquid.

Practicing Pipetting a Sample

A sample solution will be provided for you to practice your pipetting skills.

1. Using a P200 automatic pipette, adjust the volume display to 050 (50µL).
2. Place a disposable tip on the shaft of the pipette. Press on firmly with a slight twisting
motion to ensure a positive, airtight seal.

3. Depress the push-button on the first positive stop.

4. While holding the pipette vertically immerse the tip 3 or 4 mm into the sample liquid.
5. Release the push-button SLOWLY to draw up the sample.

2 ________________________________________________________ Introductory molecular biology and biochemistry

6. Wait 1 or 2 seconds, and then withdraw the tip from the sample. Wipe any fluid from
the outside of the tip, taking care not to touch the orifice of the tip.
7. You can now return the liquid to the sample container. To dispense the sample, place
the tip end against the inside wall of the vessel and depress the push-button SLOWLY
to the first stop. Wait a couple of seconds and then depress the push-button
completely to expel any residual liquid.
8. With the push-button fully depressed, carefully withdraw the pipette with tip sliding
along the wall of the vessel.

9. Release the push-button.

10. Remove the used tip by depressing the tip ejector button or remove manually.
11. Repeat steps 1-9 until you are confident that you can accurately remove and return
50µL of sample.

12. Following the steps above, add 50µL of sample solution to a microfuge tube. Add 50µL
of sample solution to another microfuge tube.

13. Compare your two tubes. Do both tubes look as though they contain the same amount
of sample solution? If your answer is no, repeat this process with fresh tubes. When the
volume in both of your tubes looks the same, you are ready to progress to Part II.

Figure 1: Important elements of an automatic Pipette

Introductory molecular biology and biochemistry ________________________________________________________ 3

 PART II: SPECTROPHOTOMETRY

Spectrophotometry is one of the most widely used and versatile of all the analytical
tools in biochemical laboratories. It allows particular components in a solution to be
examined without interference from other molecules present, the sample examined is
not destroyed in the process, and the measurements are made quickly.

The selectivity of the method is achieved by examining a sample solution at a

particular wavelength which corresponds to the absorption of light by a chemical
grouping which is characteristic of the component to be analysed and not of other
components in the solution.

When spectrophotometry can be used, it can be the first choice because:

(1) It is generally a sensitive method and thus requires very little material.

(2) It generally does not damage or change the sample.

(3) It can be highly selective and identify chemical changes in real time.
The spectrophotometer is an instrument that measures the fraction of incident light of
a given wavelength transmitted by a solution. Readings are commonly recorded as
absorbance, equal to log10 (incident/transmitted light intensity), since this quantity is
proportional to the concentration of the light-absorbing compound (Beer’s Law). Both
the colorimeter and the spectrophotometer measure absorbance, but the latter has
greater advantages of accuracy, sensitivity, broad spectral range, and sharp selection
of wavelength.

In Biochemistry the spectrophotometer is used principally for three different types of

determinations. First, the concentration of a compound can be determined by
measurement of absorbance at one wavelength, under conditions where no changes
occur, provided that the molar absorptivity of the compound is known. Second, the
course of a reaction can be observed in a complete assay system by measurement of
the rate of production or disappearance of a light-absorbing compound. Commercially
available recording spectrophotometers that make a continuous plot of absorbance vs
time are most useful for these measurements. A third sort of measurement, valuable
for identification of compounds, requires the determination of absorbance as a
function of wavelength. Here again, instruments are available which trace such plots
automatically.
This practical class will introduce you to the concepts of the characteristic absorption
spectra of some compounds and the use of these spectra in quantitative measurement.

THEORY

If white light is passed through a solution containing a coloured compound, certain

wavelengths of light are selectively absorbed. The resultant colour observed is due to
the transmitted light. Classic natural coloured compounds include haemoglobin (the
red colour of blood) and chlorophyll (the green colour of leaves).

4 ________________________________________________________ Introductory molecular biology and biochemistry

 Quantitative photometric measurement is based on two formalised laws. The first,
Lambert's Law states that the proportion of incident light absorbed by a medium is
independent of its intensity, and that each successive unit layer of the medium absorbs
an equal fraction of the light passing through it. The second, Beer's Law states that
the light absorbed is proportional to the number of molecules of absorbing substance
through which the light passes. Thus, if the absorbing substance is dissolved in
transparent solvent, the absorption of the solution is proportional to its molar
concentration.
A combination of these two laws, often termed the Beer-Lambert Law is given by the
equation:
Io
A = log10 = ε.ℓ.c
I

where:

A = Absorbance (also called Optical Density, O.D.)

Io = intensity of incident light
I = intensity of transmitted light
ε = the absorption coefficient - a constant for a given solute at a particular
wavelength.
ℓ = breadth or thickness of the solution through which the light passes (cm).
c = concentration of the solute (g/l or moles/l)

Hence, provided the thickness of the solution (ℓ) remains constant, at any given
wavelength a linear relationship exists between Absorbance, A, and concentration, C.
At a concentration of 1 mol/L and a light path of 1 cm, the coefficient ε is called the
molar absorptivity or extinction coefficient; εcmM having the dimensions cm-1 M-1.
(εcm.mM has the dimensions cm-1mM-1).

Often the molar absorptivity is not known and a calibration graph relating
absorbance to concentration is drawn up instead. This line is then used to
determine the concentration of substance in an unknown sample. In today’s
practical, we will calculate a line relating absorbance to concentration and
determine the concentration of unknown samples.

Quantitative Measurement of a Commercial Food Dye by

Spectrophotometry

The food dye Green S (Sodium 4-[(4-dimethylaminophenyl)-(4-dimethylazaniumylidene-

1-cyclohexa-2,5-dienylidene)methyl]-3-hydroxynaphthalene-2,7-disulfonate) is a
synthetic coal tar triarylmethane dye used as a food colour. It has the additive number
of E142, and is often found in sauces, desserts and ice cream. It is approved for use in
the EU, Australia and New Zealand, although has been prohibited from use as a food
additive in Canada, Japan, Norway and the United States.

Introductory molecular biology and biochemistry ________________________________________________________ 5

 Figure 2: The food dye Green S.

Green S appears green to the eye because it absorbs light mostly in the orange and
violet regions, which leave both blue and yellow as the visible colours. The absorption
spectrum of Green S is shown below. When looking for a wavelength to measure the
absorption of a compound, the wavelength with the highest peak is usually chosen.

3.5

2.5
Absorbance

1.5

0.5

0
350 375 400 425 450 475 500 525 550 575 600 625 650 675 700 725 750
Wavelength (nm)

Figure 3: Absorption spectra of Sodium 4-[(4-dimethylaminophenyl)-(4-

dimethylazaniumylidene-1-cyclohexa-2,5-dienylidene)methyl]-3-hydroxynaphthalene-
2,7-disulfonate (Green S).

Question: Using the spectrum in Figure 3, which wavelength would be best to measure
the absorbance of Green S, and why?

In this practical, you will measure the absorbance of known concentrations of Green S,
to allow you to graph a standard curve. Preparation of a standard curve, also known as
a calibration curve, is a common technique used in biochemistry that allows
researchers to determine the concentration of unknown samples. Researchers can use

6 ________________________________________________________ Introductory molecular biology and biochemistry

 the relationship between samples where the concentration is known, to deduce details
about unknown samples.
To do this, a series of samples (standards) are prepared at known concentrations. The
absorbance of these samples is measured and the relationship between absorbance
and concentration is plotted. This should be a straight line through the origin and a
'line of best fit' should be drawn through the points. Samples where the concentration
is not known can then be measured and the absorbance and the standard curve can be
used to determine the concentration of these samples.

Method

Students should work individually for this experiment.

1. You have been given a 10mM stock solution of Green S. Make a 1mM working
solution of dye by mixing 500µL stock solution with 4.5mL RO water into a labelled
centrifuge tube. Invert 3-5 times to mix well.

2. Label six microfuge tubes from 1-6.

3. Complete Table 1 to determine the volumes of water and 1mM dye solution required
to create 6 standards for a standard curve (or calibration curve).

Table 1: Dilutions of Green S dye for Practical experimentation

Tube 1 2 3 4 5 6

Volume of 500 0
Working Dye
Solution
(1mM) µL

Volume of RO 0 500
Water (µL)

Total Volume 500 500 500 500 500 500

(µL)

Final Dye 1000 800 600 400 200 0

Concentration
(µM)

HINT: When filling out the table, note that the concentration of the dye solution is
presented in mM, however the final concentration needs to be written in µM. You will
need to convert these units. Remember that 1mM = 1000 µM.

Introductory molecular biology and biochemistry ________________________________________________________ 7

 HINT: You may also find equation 1 from the “Calculations in Biochemistry” section
helpful.

4. Add dye solution (1mM) and RO water to your 6 tubes as calculated in Table 1.

5. The absorbance of each sample will be read in a 96 well plate. Find one-two students
next to you to share a plate with.

6. Each student will read two sets of each standard so the average value may be
determined later. Using the plate map below, carefully add 200 µL of each standard
(Tubes 1-6) to the correct wells on the 96 well plate.

7. Using the plate map below, carefully add 200 µL of each unknown sample (tubes A-C)
to the correct wells on the 96 well plate.

Student 1 Student 2

Standards Unknowns Standards Unknowns

1 2 3 4 5 6 7 8 9 10 11 12

Tube 1 Tube 1 Tube Tube Tube 1 Tube 1 Tube Tube

A
(1000 µM) (1000 µM) A A (1000 µM) (1000 µM) A A

Tube 2 Tube 2 Tube Tube Tube 2 Tube 2 Tube Tube

B
(800 µM) (800 µM) B B (800 µM) (800 µM) B B

Tube 3 Tube 3 Tube Tube Tube 3 Tube 3 Tube Tube

C
(600 µM) (600 µM) C C (600 µM) (600 µM) C C

Tube 4 Tube 4 Tube 4 Tube 4

D
(400 µM) (400 µM) (400 µM) (400 µM)

Tube 5 Tube 5 Tube 5 Tube 5

E
(200 µM) (200 µM) (200 µM) (200 µM)

Tube 6 Tube 6 Tube 6 Tube 6

F
(0 µM) (0 µM) (0 µM) (0 µM)
G

8. Load your plate into a plate reader. Click the [P1 630nm] button on the SpectroStar
Nano analysis software, and wait for the plate to be read (it will take approximately
one minute).

9. Click on [Open Last Test Run], wait for MARS to open, and then click [Export table
to Excel]. Save your data onto a thumb drive and perform your data analysis on
another laptop. Remove your plate from the reader once it has finished.

10. Clean up your work space. Place all disposables in the bin, and wash all glassware.

8 ________________________________________________________ Introductory molecular biology and biochemistry

 Data Analysis

1. Load the excel spreadsheet.

2. Begin a new sheet to graph your data.

3. Column A will contain the concentrations of samples in your experiment. Label well
A1 as ‘concentration (µM)’. Include the concentrations of the standards in cells A2-A7
underneath (see example below).

4. Columns B and C will contain the absorbance results. Label cells B1 and C1 as
‘A630nm #1’ and ‘A630nm #2’. Add the correct absorbance values for each
concentration to columns B and C.

5. Create a new column (column D) for the average absorbance values. Type a formula
into each cell to calculate the average absorbance values.

HINT: If you are unsure how to use the formula function in excel, follow the example
formulas in wells D2 to D7 below. Type each formula into each cell and press enter to
calculate the value. (The absorbance values shown are examples only and will not
work for you! You must use your own data).

6. Create another column to subtract the average absorbance at 0 µM from other

average concentrations (column E). Calculate the values in the spreadsheet.

HINT: If you are unsure how to do this, copy the text written in column E. (This will
only work if your numbers are in the same cells as the numbers shown.)
The Average absorbance at a concentration of zero is subtracted to ensure that we
only measure the molecules of interest, not the media it is diluted in! This is called
blanking the sample.

A B C D E

A630nm A630nm
Concentration (µM) Average Average - Zero
1 #1 #2

2 1000 0.50 0.53 =Average(B2:C2) =D2-D7

3 800 0.42 0.41 =Average(B3:C3) =D3-D7

4 600 0.32 0.31 =Average(B4:C4) =D4-D7

5 400 0.22 0.20 =Average(B5:C5) =D5-D7

6 200 0. 11 0.12 =Average(B6:C6) =D6-D7

7 0 0.02 0.03 =Average(B7:C7) =D7-D7

7. Select the Concentration column and the Average-Zero column (NOTE: You may need
to hold the ‘ctrl’ key down to do this).

Introductory molecular biology and biochemistry ________________________________________________________ 9

 8. Click the ‘Insert’ tab at the top of the Excel window. The Toolbar should now show
the available types of charts. Select an XY Scatter plot. Choose the chart with data
points only (first option).

9. Click the ‘Design’ tab at the top of the Excel window. Insert axis labels using the ‘Add
Chart Element’ menu. These should be SPECIFIC e.g. “Green S Dye Concentration
(µM)”.

10. Tidy up the chart appropriate for a scientific paper. Clear gridlines and make sure the
background is white. Format the X axis so the maximum value is 1000µM (to do this,
double click a number on the X axis and the ‘format Axis’ menu will appear).

11. If a title has been automatically added above the figure, this should be deleted.
Remember that titles for figures in scientific papers are found below each figure.

12. Right click on data points -> choose Add Trendline.

13. Make sure that the Set Intercept, Display Equation on chart and Display R-squared
value on chart boxes are ticked in the Format Trendline pane.

14. Look at the equation displayed on your chart. You can now use this equation to
determine the concentrations of your unknown solutions. You will need to
determine the average absorbance of your duplicate samples and subtract the
zero BEFORE using the equation.

If you are unsure of how to use these values in the equation, please read the
information on ‘calculation of unknown concentrations using an equation’ at the
beginning of this manual. Please set out your calculations neatly and logically below.

NOTE: Remember to state the relevant units.

HINT: Note the label is on each axis. One axis should refer to the absorbance and the
other axis should refer to the concentration. If you know the absorbance of an
unknown solution, you can use the equation!

The concentration of unknown A:

The concentration of unknown B:

The concentration of unknown C:

10 _______________________________________________________ Introductory molecular biology and biochemistry

 Practical 1: Questions

Question 1: Why was the Absorbance at 0 µM subtracted from the other average
Absorbance values?

Question 2: You have repeated this experiment with different unknown samples. You
have found that the absorbance of one of the unknown samples is considerably higher
than the absorbance of your highest standard. Can you still determine the
concentration of this unknown sample? If not, why? If so, how?

Introductory molecular biology and biochemistry _______________________________________________________ 11

 Practical 1: Notes and Calculations

12 _______________________________________________________ Introductory molecular biology and biochemistry

 PRACTICAL 2 – DNA PURIFICATION AND
MOLECULAR CLONING
PART I - DNA PURIFICATION

INTRODUCTION
Revise the structure of DNA as given in your lectures and in your textbook: the main
points to consider as far as this experiment is concerned are (a) the size of individual
DNA molecules, (b) the spatial distribution of hydrophobic and hydrophilic groups in
DNA and (c) the organisation of DNA in chromatin.

(a) Size of DNA

DNA molecules are the largest molecules known. The E. coli bacterial chromosome,
for example, is a single (circular) molecule 1.4 mm long and with a molecular weight of
2.5 x 109. This corresponds to approximately 4,800,000 base pairs. By comparison,
the DNA in an average human chromosome, which is linear, has a molecular weight of
1011, and consists of approximately 1.3 x 108 base pairs. The total number of base
pairs in the DNA of human cells is approximately 3 x 109, distributed over 23
chromosomes. It is thought that each chromosome is composed of a single molecule
of DNA, which would make it several cm long!

Because of their very large sizes, DNA molecules can actually be broken by physical
means, eg. by stirring DNA solutions too vigorously. The breaking of covalent bonds
in DNA by physical means is termed shearing.

(b) Charge characteristics of DNA

The DNA double helix has a hydrophobic core and a highly negatively-charged surface.
The distribution of hydrophobic and hydrophilic groups is a result of the orientations
of the aromatic bases and the sugar and phosphate groups of the individual nucleotide
residues in the DNA molecule. The overall structure of a charged surface covering a
hydrophobic interior resembles the structure of globular proteins, and the same
thermodynamic considerations apply. As a result of the pK of the phosphate groups
(pK ~ 1) the surface of the DNA double helix is highly charged at physiological pH, and
the molecule is soluble in aqueous solvents at this pH.

Introductory molecular biology and biochemistry _______________________________________________________ 13

 (c) The Structure of Chromatin

The very high charge density on the surface of the DNA double helix at physiological
pH prevents the 'naked' molecule from adopting any tertiary structure and being folded
into a more compact shape. Given their size, the large DNA molecules in a eukaryotic
chromosome could not be packed into the relatively small volume of a nucleus. For
this reason, DNA molecules in eukaryotic cells exist in the form of a DNA-protein
complex called chromatin or deoxyribonucleoprotein (DNP). The proteins that bind to
DNA in chromatin are usually very basic and bind to the negatively charged surface of
the double helix by non-covalent electrostatic interactions. As a result the charge on
the DNA molecule is effectively neutralised, allowing the chromatin to be condensed
very tightly into a relatively small volume. Intact chromatin is insoluble at
physiological ionic strength (0.16M salt) in aqueous solvents but is relatively soluble at
very low ionic strengths. At very high ionic strengths (eg. 2M salt), chromatin
dissociates as the electrostatic interactions between the DNA and proteins are broken.

(d) Uses of purified DNA

Modern techniques of gene analysis now allow the identification and isolation of
individual genes, and determination of the complete nucleotide sequences. In
addition, it is possible to map the positions of genes in chromosomes. All of these
techniques use, as their starting point, purified DNA. A variety of methods have been
developed for the purification of DNA from different organisms, including both
prokaryotes and eukaryotes. The procedures differ for different cells, particularly
where the cells have cell walls e.g. bacterial, fungal and plant cells.

Isolation of DNA from Yeasts

In this practical session you will extract DNA from the yeast Saccharomyces cerevisiae
(also called Baker’s yeast or Brewer’s yeast). In the following protocol, you will collect
cells and lyse the plasma membrane by adding lithium acetate (weakens the cell walls)
in 1% SDS (disturbs the cell membrane and denatures proteins). The proteins are then
removed by extraction with chloroform/amyl alcohol. The highly charged 'naked' DNA
molecules will partition into the aqueous phase during extraction.
For a successful preparation, it is important to remove as much chromatin protein as
possible by chloroform/amyl alcohol extraction: the less protein contamination there
is, the more soluble the final DNA preparation will be and the higher the eventual yield.
You will then use ice-cold ethanol to precipitate the DNA before re-suspending it is a
small amount of Tris-EDTA (TE) buffer (this way you will concentrate the DNA in the
solution).

14 _______________________________________________________ Introductory molecular biology and biochemistry

 Spectrophotometric Determination of DNA Purity

DNA absorbs light in the ultraviolet region of the electromagnetic spectrum due to the
absorption of its constituent purine and pyrimidine bases. The four bases (adenine,
cytosine, thymine and guanine) that are found in DNA have different absorption
spectra and maxima (λmax). The spectrum of whole DNA is, to a first approximation,
a composite of these individual spectra and will depend on the exact base composition
of the DNA. In fact the spectrum of the whole DNA has an approximate λmax=
260nm with an ε1%,1cm, 260nm of about 200. This means that the absorbance of a 1%
solution of DNA (1 g per 100 ml), using a 1 cm light path, at 260 nm, is 200 OR a
solution of 50µg/mL has an absorbance of 1. (See notes for practical 1,
Spectrophotometry).
This apparent extinction co-efficient is not simply the sum of the contributions due to
the individual nucleotides, since when bases are stacked (as in the native structure of
the DNA double helix) their extinction co-efficients are reduced - this effect is called
hypochromicity.

The concentration of pure DNA is usually estimated from the absorbance of the
solution at 260 nm and assuming an absorbance of 1 for a 50µg/mL solution.
The ratio of the absorbance of the solution at 260 nm compared to 280 nm is used to
assess the purity of the DNA solution. Pure DNA has an A260/A280 ratio of 1.8 (the
ratio of its extinction coefficients at these two wavelengths). If the solution is
contaminated with protein this ratio will be less than 1.8, since λmax for proteins is
280 nm and they will have a lower relative absorbance at 260 nm compared to 280
nm.

Estimation of DNA concentration using Diphenylamine

The concentration of DNA can also be determined by a variety of chemical methods.

Under strongly acidic conditions DNA is converted to a mixture of its constituent
bases, deoxyribose and phosphate by hydrolysis of both the phosphodiester bonds
between sugar and phosphate residues and the N-glycosidic bonds between the sugars
and bases. The ring of deoxyribose is opened under these conditions to form δ-OH
levulinic aldehyde (see Figure 1). This is reacted with diphenylamine to form a blue
complex which can be quantitated spectrophotometrically at 595nm. You will not
perform this assay during the practical.

Figure 1: Formation of δ-OH levulinic aldehyde from deoxyribose

Introductory molecular biology and biochemistry _______________________________________________________ 15

 EXPERIMENTAL PROCEDURE

1. Collect a centrifuge tube containing 2mL overnight culture of S. cerevisiae in YPD

broth which has been centrifuged at 3000rpm for 3 minutes to pellet the cells.

2. Label the tube with your name, and carefully pour off the supernatant into the waste
bottle on your bench.

3. Resuspend cells in 2mL of 200mM lithium acetate in 1% SDS. Vortex for 30 seconds.

4. Incubate at room temperature for 5 minutes.

5. In the fume hood, add 2mL of 24:1 chloroform:amyl alcohol to the tube.

6. Vortex for 1 minute. Note: the amount of protein precipitated in this step is directly
related to the amount of mixing between the aqueous and organic phases.

7. Centrifuge tube at 3000rpm for 10 minutes.

8. The denatured protein will form a thick film between the organic (lower) phase and
the aqueous (upper) phase containing the DNA. In the fume hood, carefully transfer
the aqueous phase to a clean labelled centrifuge tube, taking care to not disturb the
protein layer. Discard the organic phase into the chloroform waste bottle in the fume
hood. DO NOT POUR DOWN THE SINK.

9. Gently add 3mL ice cold ethanol to the tube. (The ethanol will be pre-chilled. You do
not need to place your tube on ice).

10. Allow to stand for 3 minutes – afterwards mix gently.

11. Centrifuge tube at 3000rpm for 10 minutes

12. Pour off supernatant, and invert onto tissue for 5 minutes.

13. Resuspend pellet in 2.5 mL TE buffer.

14. Using ultraviolet spectroscopy, scan the absorbance over the range 220-300nm
(against a TE buffer blank).

16 _______________________________________________________ Introductory molecular biology and biochemistry

 PART II – MOLECULAR CLONING (“DRY LAB”)

Cloning the mars gene

BACKGROUND

A group of scientists studying spoilage in a marsTM bar factory has isolated two
populations of bacteria. Bacteria from one of these populations (group 1) are able to
grow and thrive when grown on mars bars, while bacteria from the second population
(group 2) are unable to grow, and die when provided with mars bars as their nutrient
source. Genetic experiments using large numbers of bacteria and mars bars have shown
that the defect in bacteria from group 2 is due to mutations in a single gene, which has
been called mars. Thus, bacteria from group one have a functional (wild type) copy of
the mars gene and are able to grow on mars bars, while bacteria from group 2 have
mutations in the mars gene, so the product of the mars gene is non-functional, and they
are killed when grown on mars bars. In genetic terms, bacteria from group 1 are mars+
-
and bacteria from group 2 are mars .

AIM

The aim of this exercise is to use a model to simulate the preparation of a library of
genomic DNA from mars+ bacteria, and to use this library to isolate the mars gene.

PROCEDURE

During this exercise, you will play a variety of different roles in different steps. You
should carry out this exercise individually.

Role 1: Technician

Prepare eight circular molecules of the plasmid pUC18 by joining together with
sticky tape the ends of the linear molecules provided in the pUC18 DNA preparation
at the front of the laboratory. Make sure that the bases are on the outside, not the
inside, of the molecule when you join the ends together.

Note: pUC18 is 2,700 bp long. To make the exercise easier, we are using a much
shorter DNA molecule.

Introductory molecular biology and biochemistry _______________________________________________________ 17

 Role 2: Scientist
Take a mars+ bacterial cell from the culture provided (at the front of the laboratory) and
extract the genomic DNA from the cell (open the envelope and remove the DNA
model).

Note: during the preparation of a genomic library, you would normally start with billions
of cells, so you would have billions of DNA molecules. A variety of methods are used to
isolate DNA. In the accompanying practical, you are isolating genomic DNA from yeast
cells. The DNA which you prepare from yeast cells could be used to prepare a yeast
genomic library.

Role 3: Scientist

Use physical methods to break the DNA into shorter pieces (cut the DNA molecule
into 8 pieces of similar size).

Note: DNA can be broken into smaller pieces by sonication – exposing it to high
frequency sound waves. The DNA molecule in your model is only about 200 bp long,
and you are breaking it into 8 pieces, which are each about 25 bp long. If you were
preparing a genomic library from E. coli, the chromosome would be about 5,000,000 bp
(5000 kb) long, and you would break each chromosome into about 1000 pieces. As
noted above, you would start with billions of molecules of genomic DNA.

Role 4: Restriction enzyme SmaI

Take your eight molecules of plasmid and cut each molecule at the SmaI site. SmaI
recognises the sequence CCCGGG, and cuts between the C and the G. (use scissors to
cut the plasmids at the SmaI site).

Note: if the plasmid is cut with SmaI, the ends will be compatible with the ends of the
fragments you have prepared from genomic DNA.

Role 5: DNA ligase

Join each fragment of the genomic DNA to two ends of the pUC18, to make a circular
plasmid (use sticky tape).

Note: the plasmids which you have prepared are part of the genomic DNA library of
mars+ bacteria. A library would normally contain millions of plasmids like this.

Add your plasmids to the library at the front of the laboratory.

18 _______________________________________________________ Introductory molecular biology and biochemistry

 Role 6: mars- bacterial cell
-
In order to clone the mars gene, bacterial cells from group 2 (mars ) are transformed
with plasmids from the library. During this process, bacterial cells take up plasmids
from the library.

Take a plasmid from the library.

-
As you are a mars cell, you would not normally be able to survive on a diet of mars bars.
If, however, the plasmid which you have taken from the library contains the wild-type
mars gene, then you will now be able to thrive on mars bars.

To determine whether the plasmid that you have taken up contains the mars gene,
you need to translate the codons from the DNA sequence. You will only be translating
the codons in the piece of DNA from the mars+ bacterium (white paper), not the codons
in the pUC18 DNA (Green paper). Protein-coding regions in the DNA must begin with a
start codon, ATG. There are two DNA strands. The top DNA strand is written from the
5’ to 3’ direction, so you should translate this from left to right. The bottom DNA strand
is complementary to the top strand and antiparallel, so it is written from 3’ to 5’, and
you need to read it from right to left.

Look for start codons in both strands of your DNA sequence.

Note that the codons in the table are written for an RNA molecule. The DNA sequence
of the coding strand is the same as the sequence of the mRNA, except that there is a T
in the DNA sequence where there is a U in the RNA sequence.

If you find a start codon (ATG), write out on the results sheet (look ahead in the
practical manual) the DNA sequence, beginning at the start codon. Then use the
genetic code table to translate the DNA sequence into an amino acid sequence. You
should stop when you reach a stop codon, or when you reach the end of the white
paper. If there are 1 or 2 bases left at the end of the sequence, ignore them. Write
out the amino acid sequence using the three letter abbreviations for amino acids,
then use the table of single letter abbreviations to change your amino acid
sequence to single letters. If you think that you have cloned the mars gene, take
your plasmid form the library and your results sheet to your demonstrator. If you
have not been successful on your first attempt, you can try again.

Note: protein sequences usually contain 50 – 500 amino acids. The sequences in this
exercise have been shortened to make it easier.

Introductory molecular biology and biochemistry _______________________________________________________ 19

 THE STANDARD GENETIC CODE

UUU Phe UCU Ser UAU Tyr UGU Cys

UUC Phe UCC Ser UAC Tyr UGC Cys

UUA Leu UCA Ser UAA Stop UGA Stop

UUG Leu UCG Ser UAG Stop UGG Trp

CUU Leu CCU Pro CAU His CGU Arg

CUC Leu CCC Pro CAC His CGC Arg

CUA Leu CCA Pro CAA Gln CGA Arg

CUG Leu CCG Pro CAG Gln CGG Arg

AUU Ile ACU Thr AAU Asn AGU Ser

AUC Ile ACC Thr AAC Asn AGC Ser

AUA Ile ACA Thr AAA Lys AGA Arg

AUG Met ACG Thr AAG Lys AGG Arg

GUU Val GCU Ala GAU Asp GGU Gly

GUC Val GCC Ala GAC Asp GGC Gly

GUA Val GCA Ala GAA Glu GGA Gly

GUG Val GCG Ala GAG Glu GGG Gly

20 _______________________________________________________ Introductory molecular biology and biochemistry

 SYMBOLS FOR AMINO ACIDS

A Ala Alanine

B Asx Asparagine or Aspartic acid

C Cys Cysteine
D Asp Aspartic acid

E Glu Glutamic acid

F Phe Phenylalanine
G Gly Glycine

H His Histidine

I Ile Isoleucine
K Lys Lysine

L Leu Leucine

M Met Methionine
N Asn Asparagine

P Pro Proline

Q Gln Glutamine
R Arg Arginine

S Ser Serine

T Thr Threonine

V Val Valine

W Trp Tryptophan

Y Tyr Tyrosine
Z Glx Glutamine or Glutamic acid

Introductory molecular biology and biochemistry _______________________________________________________ 21

 RESULTS SHEET – CLONING THE MARS GENE

Attempt 1:

DNA sequence:

Protein sequence (3 letter abbreviations)

Protein sequence (1 letter abbreviations)

Attempt 2:

DNA sequence:

Protein sequence (3 letter abbreviations)

Protein sequence (1 letter abbreviations)

Attempt 3:

DNA sequence:

Protein sequence (3 letter abbreviations)

Protein sequence (1 letter abbreviations)

22 _______________________________________________________ Introductory molecular biology and biochemistry

 Attempt 4:

DNA sequence:

Protein sequence (3 letter abbreviations)

Protein sequence (1 letter abbreviations)

Attempt 5:

DNA sequence:

Protein sequence (3 letter abbreviations)

Protein sequence (1 letter abbreviations)

Attempt 6:

DNA sequence:

Protein sequence (3 letter abbreviations)

Protein sequence (1 letter abbreviations)

Introductory molecular biology and biochemistry _______________________________________________________ 23

 Practical 2: Questions

Question 1: A 50µg/mL solution of DNA has an absorbance of 1. Use this relationship

to calculate the concentration of your DNA sample in mg/ml. Show your calculations
below:

Question 2: Calculate the A260/A280 ratio of your sample.

Question 3: Based on the A260/A280 ratio, comment on the purity of your DNA
preparation.

Question 4: Explain why the SmaI restriction enzyme was used to cut the pUC18
plasmid, but not the bacterial DNA in this simulation.

24 _______________________________________________________ Introductory molecular biology and biochemistry

 Practical 2: Notes and Calculations

Introductory molecular biology and biochemistry _______________________________________________________ 25

 PRACTICAL 3 - THE INDUCED SYNTHESIS OF ß-
GALACTOSIDASE IN E. COLI

INTRODUCTION

Knowledge on the control of protein synthesis in bacteria has largely emerged from the
studies of Francois Jacob & Perse Monod on the induced synthesis of ß-galactosidase in
E. coli. Studies of this system led to the discovery of regulatory events in the
transcription of DNA and the grouping of functionally related genes into clusters called
operons. Jacob and Monod were awarded the Nobel Prize in 1961. The experiment
you will perform is part of one of their classic experiments that are still in practice in
current times.
Specific mechanisms exist to regulate the levels of gene expression in a cell. This
regulation may be at the level of transcription, translation or messenger RNA stability.
In all cases the genetic potential of a cell is regulated with respect to the synthesis of a
given protein. In bacterial cells this may mean that a protein is synthesised only under
certain circumstances or that its level changes depending upon growth conditions. In
multicellular organisms such mechanisms may also regulate tissue specificity of gene
expression.

In this experiment we will investigate the regulation of transcription of bacterial genes.

Inducible and Repressible Enzyme Systems

In bacteria, the levels of the enzymes in some systems respond to changes in the
environment. These are called adaptive enzyme systems, in contrast to constitutive
systems in which enzyme levels show little variation with changes in the environment.
We can subdivide adaptive enzyme systems into two broad categories, the inducible
and the repressible enzyme systems.

Inducible enzymes appear when an inducer is present in the environment. These

systems are usually catabolic (being involved in the breakdown of complex molecules)
and are often involved in carbohydrate metabolism. The inducer is usually the
molecule broken down by the enzyme system.
For example, lactose induces synthesis of the proteins ß-galactosidase, galactose
permease and thiogalactoside transacetylase in E. coli. Galactoside permease
transports galactosides, such as lactose, into cells. β-galactosidase hydrolyses β-
galactosides, such as lactose, which is the first step in their metabolism. The
physiological role of thiogalactoside transacetylase is unknown. The genes for β-
galactosidase, galactoside permease, and thiogalactoside transacetylase are called
lacZ, lacY and lacA, respectively. They are found in a cluster, which is known as the
lac operon, shown in Figure 1, below.

26 _______________________________________________________ Introductory molecular biology and biochemistry

 Figure 1. The arrangement of genes in the lac operon of E. coli.

Pi is the promoter for the lacI gene, which encodes the Lac repressor protein. Plac is
the promoter for the lacZ, lacY and lacA genes. The operator region is located
between the Plac and the start of the lacZ gene (there are three operators: O1, O2 and
O3). When the Lac repressor protein is bound to the operator region, transcription of
lacZ, lacY, and lacA is prevented.

The lacZ, lacY and lacA genes are transcribed from a single promoter to give a
polycistronic mRNA, which is translated to give the three enzymes. Transcription of
the lacZ, lacY and lacA genes is controlled by the Lac repressor protein, which binds to
operator sequences, located between the promoter for the lacZ, lacY and lacA genes,
and the start of the coding region for the lacZ gene. The gene for the repressor is
called the lacI gene.

When there is no lactose, or other β-galactoside, in the cell, the repressor is bound to
the operator region, and transcription of the lacZ, lacY and lacA genes is almost
completely prevented, so the levels of ß-galactosidase, galactoside permease, and
thiogalactoside acetylase are very low.

ß-Galactosidase

The E. coli enzyme ß-galactosidase is an example of an inducible enzyme. It catalyses

the hydrolysis of a wide variety of ß-galactosides, such as lactose (see Fig. 2).

Introductory molecular biology and biochemistry _______________________________________________________ 27

 Figure 2. Hydrolysis of a ß-galactoside by ß-galactosidase.

When E. coli is grown in media free of lactose, or any other ß-galactoside, the content
of ß-galactosidase in the cell is very low (<10 molecules per cell). When an inducer
such as lactose is added to the medium, the level of enzyme activity in the cell is
greatly increased due to the synthesis of new enzyme in the cells (rather than the
activation of pre-existing inactive enzyme precursors).
The rate of synthesis of ß-galactosidase molecules is 1000-fold higher in the presence
of lactose than in its absence. Note that lactose is both a substrate of the enzyme
and an inducer of lactose operon!

The Measurement of Enzymatic Activity

Lactose is not the only substrate of β-galactosidase. β-Galactosidase activity assay is

based on the ability of the enzyme to hydrolyse the β-galactoside bond of the synthetic
substrate o-nitrophenol-β-D-galactoside (ONPG), to yield a yellow product, ortho-
nitrophenol (ONP-), which can be quantified using absorption spectrometry. The
enzyme content of a sample (e.g. E. coli culture) may be conveniently assayed in vitro
by measuring the rate of hydrolysis of ONPG to yield ONP- (see Fig. 3).

Figure 3. Hydrolysis of ONPG by ß-galactosidase.

28 _______________________________________________________ Introductory molecular biology and biochemistry

 The products of this hydrolysis are galactose and the o-nitrophenolate (ONP-) anion.
The yellow product ONP- has strong absorbance at 420 nm whereas ONPG does not.
Therefore the rate of the reaction may be followed spectrophotometrically at 420 nm
by measuring the rate of appearance on ONP-.
ONPG does not readily cross the E. coli cell membrane, therefore to measure the
enzyme content of a suspension of intact cells the bacteria have to be made permeable
to the substrate. This is conveniently achieved by adding an aliquot of the cell
suspension to a small amount of toluene in ethanol. This treatment has the advantage
of not only permeabilizing the cells by disrupting their cell membranes, but also of
stopping cell growth and allowing the determination of enzymatic activity at accurately
determined times after induction.

The Mechanism of Induction of ß-Galactosidase

As mentioned above, the synthesis of ß-galactosidase is induced in E. coli upon

addition of lactose to the growth medium. What is the mechanism?

Some lactose enters the cell due to the activity of a small amount of lactose permease
(product of lacY), present in non-induced E. coli cells. The lactose is metabolised to
allolactose, which then binds with high affinity to the Lac repressor protein.

In the absence of allolactose, the Lac repressor binds to the operator region of the lac
operon, blocking the transcription of the three genes in this operon by RNA
polymerase, ie. lacZ, the gene coding for ß-galactosidase; lacY, the gene coding for
galactose permease; and lacA, the gene coding for thiogalactoside transacetylase.
However, the allolactose/Lac repressor complex has a relatively low affinity for the
operator compared with that of the uncomplexed repressor. In the presence of
lactose, therefore, the repressor protein dissociates from the operator, allowing RNA
polymerase to transcribe the three genes of the lac operon.
For transcription to occur at reasonable levels, a Catabolite Activator Protein (CAP)
must also bind to the DNA. Binding of CAP stimulates the binding of RNA polymerase
to the lac promoter. The CAP has to be activated to bind the promoter region by
creating a complex with a molecule of cAMP.
cAMP levels are low when glucose is available and high when glucose is absent in the
growth medium. Therefore, it is important to use a medium low in glucose for the lac
operon to be transcribed in the presence of lactose.

REFERENCES

1. Jacob, F. and Monod, J. (1961) Genetic regulatory mechanisms in the synthesis

of proteins. J. Mol. Biol. 3: 318-356. (Original experiments)
2. TEXTBOOK – Nelson and Cox (2013) pp 1158-1160; 1165-1167.

Introductory molecular biology and biochemistry _______________________________________________________ 29

 EXPERIMENT

Overview

There are a few stages to this experiment:

1. Growing E. coli cell culture in a medium, which keeps the lac operon repressed to
suppress synthesis of ß-galactosidase.

2. Inoculating fresh medium with the grown culture and dividing into two portions:
one keeping un-induced (control) and the other inducing with lactose.

3. Sampling at different times after lactose addition and measuring ß-galactosidase

activity in the induced and un-induced cultures.

4. To measure ß-galactosidase activity, we remove a sub-sample and add to it the

substrate ONPG. We then measure the appearance of the product ONP-.

5. We then calculate the enzyme activity per gram of E. coli cells and conclude from it
regarding the induction of the lac operon and enzyme synthesis.

You will work in groups – each group will measure the enzyme specific activity at a
different time after induction with lactose. The class results from all groups will
then be combined.

Growth of Organisms (done for you in advance)

Cells are grown in a defined medium with succinate as a carbon and energy source, so
that the lac operon is repressed. Transcription and resultant enzyme (protein)
synthesis can be induced by the addition of lactose to the culture medium. The time
course of this induction will be assayed by following ß-galactosidase activity.
A loop of E. coli K12 maintained on agar slopes is transferred aseptically into 20 ml of
nutrient broth and allowed to grow aerobically overnight at 37oC with shaking. On the
morning of the practical, 1 ml of the overnight culture is added aseptically to 39 ml of
fresh succinate medium and divided into two 20 ml portions in sterile flasks. Growth
is continued at 37oC in a shaking water bath until the inducer is added.

30 _______________________________________________________ Introductory molecular biology and biochemistry

 PROCEDURE

This experiment will compare E coli samples that have or have not been stimulated
with Lactose. The E coli samples will be examined over time, from 0 minutes to 210
minutes.
Designated groups of students share a control (C), and a lactose (L) flask. Each group
should do the part for Group I or II or III or IV (as indicated on your benches). Class
results will be obtained by combining results from these parts.
To save time, each group will only sample the E. coli flasks at two time points. This
means that the lactose inducer has already been added to each of the lactose flasks,
EXCEPT for the time 0 flask.

Therefore, ONLY Group I (‘0’ and ‘30’ times group) must add lactose to their L
flask with the help of a demonstrator. For other groups the lactose has already
been added into the L flask only.

Perform only the part allocated to your group!!! You will be told
specific times to collect your samples. You must be ready!

Figure 4: Representation of the sampling time for the experiment. The lactose flasks of
groups II, III and IV were stimulated with lactose prior to the practical session. Group I
will add the lactose inducer to the lactose flask. Each group collects data at two time
points, and class data is combined for analysis.

Introductory molecular biology and biochemistry _______________________________________________________ 31

 Group I. 'O' and ‘30’ min times:
Together with the demonstrator, add Lactose, the inducer:
At your designated time, add 1.0 ml of sterile water to a 20ml E. coli aliquot. Mark the
flask as 'C'.
To the other 20 ml aliquot - add 1.0 ml of lactose, mix and mark as 'L'.
SAMPLE IMMEDIATELY.

Sampling times correspond to 0 and 30 min respectively.

Group II. '60' and ‘90’min times:

The inducer was already added 60 minutes before the first sampling time point (to the
L flask only). Thus, sampling will correspond to 60 and 90 min from induction,
respectively.

Group III. '120'and ‘150’ min times:

The inducer was already added 120 minutes before the first sampling time point (to
the L flask only). Thus, sampling will correspond to 120 and 150 min from induction,
respectively.

Group IV. '180' and ‘210’min times:

The inducer was already added 180 minutes before the first sampling time point (to
the L flask only). Thus, sampling will correspond to 180 and 210 min from induction,
respectively.

SAMPLING – All groups

1. Label two plastic centrifuge tubes as L and C.

2. At the appropriate times, remove 5 ml of culture from the appropriate control (C)
flask and add to the centrifuge tube labelled (C). Immediately remove a 5 ml aliquot
from the lactose (L) culture and add to the L centrifuge tube.

It is important that lactose does not enter the control flask. Separate pipettes should
be used to make additions to, or to sample from flasks.

3. Using cell culture medium as a blank, read the absorbance of each sample at 600 nm
as a measure of cell growth (density). RETURN YOUR SAMPLE TO THE CORRECT
CENTRIFUGE TUBE AFTER READING THE ABSORBANCE. Write your values in Table 1
below.

4. Chill the samples for 2 minutes.

5. Centrifuge at 3,500 rpm for 10 min to pellet the cells. You need to keep the cell
pellet (solid phase in the bottom of the tube)!
Make sure that your tubes are clearly labelled and easily identified. Label the side of
the tube, AND THE LID.

32 _______________________________________________________ Introductory molecular biology and biochemistry

6. Carefully pour off and discard the supernatant into the waste bottle provided (NOT
the sink).
7. Add 2.0 ml of Tris HCl/KCl buffer (0.05 M Tris HCl, 0.05 M KCl, pH7.0) onto the
pellet.
8. Using a Pasteur pipette, resuspend the pellet by pipetting up and down until you have
a uniform suspension.

9. Take a 1 ml aliquot of this bacterial suspension into a fresh labelled glass test tube.
10. Add 2 drops of toluene and vortex vigorously for thirty seconds.

11. Place in a water bath for at least 30 min (minimum time) to liberate the enzyme.
(These tubes will remain in the water bath until all the tubes for the
β-galactosidase assay are ready).

When ALL the tubes are ready:

12. Note the time, then add 0.5 ml of ONPG substrate solution (0.005 M) to each tube
and incubate at 37oC (in a water bath) for EXACTLY 15 min (after the substrate
addition).
13. Stop the reaction by adding 5.0 ml of triethanolamine-acetate to each tube (1.0 M, pH
8.8).
14. Pour the entire contents of each tube into separate (clearly labelled) plastic centrifuge
tubes. Centrifuge at 3,500 rpm for 10 min. This time you need to keep the
supernatant (liquid phase).
15. Add 200 µl of each supernatant to a microplate. Record the location of your samples
below:

1 2 3 4 5 6 7 8 9 10 11 12

16. Read the absorbance of the supernatant at 420 nm (measuring yellow colour) against
a distilled water blank. Record your results in Table 2.

Introductory molecular biology and biochemistry _______________________________________________________ 33

 Calculations

The enzyme activity is presented as ß-galactosidase activity units (sometimes also

called Miller units) per mg of cells per min.

Therefore you first need to calculate the cell density – i.e. how many mg of cells were
in each mL of E. coli culture used. You calculate this in Part A below (Cell growth).
You then have to convert the A420 readings of the yellow colour produced from the
enzyme activity to activity-units/min. This is shown in Part B below (ß-galactosidase
activity).
Finally, you need to plot a graph of the activity units/mg cells/min against time (to find
out the time it took to induce the lac operon). For this you will need the results from
the entire class.

A. Cell growth.

The density of bacteria in the culture (mg cells/ml) may be estimated by measuring the
absorbance at 600 nm. This is actually a measure of the scattering of light by the
particulate E. coli cells. The cell density (D) may be obtained from the relationship:
D (mg cells/ml) = 0.940 x A600
Set up a spreadsheet on a laboratory computer with the columns below.
Enter the A600 values. Enter appropriate formulae into the spreadsheet to calculate the
cell density values. Ask the demonstrators for help!

Fill in the Table 1 below.

Table 1. Cell Growth – Own results

Cell Density
A600
Time mg cells/ml

(min) Control Induced Control Induced

(C) (L) (C) (L)

34 _______________________________________________________ Introductory molecular biology and biochemistry

 B. ß Galactosidase Activity.

One unit of enzyme activity is defined as producing 1 nmol of ONP- per min at 37oC, pH
7.0. The A420 of 1 nmol of ONP- under the assay conditions is 0.006 (this value is
known from past experiments – you did not have to calculate it).
Therefore, to convert your A420 readings to nmol of ONP-, you need to divide by 0.006.
This gives you the number of nmol of ONP- produced in your assay tube in 15 min.

To convert to nmol per min, divide by 15.

You took a 5 ml sample of the bacterial culture, resuspended it in 2 ml, then assayed
one ml. This means that the activity which you measured is the activity in 2.5 ml of
culture. Therefore, to find the nmol/min/ml of culture, you must divide by 2.5.

Hence, the calculation is:

Activity = A420 / (0.006 x 15 x 2.5)

Set up a new worksheet on your spreadsheet with a table as shown below.

Enter A420 values into your spreadsheet.

Enter appropriate formulae into the spreadsheet to calculate the enzyme activity
values. Ask the demonstrators for help!

Fill in Table 2 below.

Table 2. ß-Galactosidase Activity – Own results

Activity Specific activity

A420
Time (Unit/ml/min) (Unit/mg cells/min)

(min) Control Induced Control Induced Control Induced

(C) (L) (C) (L) (C) (L)

C. Specific activity

Specific activity is obtained by dividing your values for enzyme activity by the
appropriate values for cell density from Table 1. You will enter appropriate formulae
into your spreadsheet (Table 2) to do this.
When you have finished your calculations, add your values to the class spreadsheet.

Introductory molecular biology and biochemistry _______________________________________________________ 35

 Practical 3: Questions
Question 1: Why was experimentation at each time point conducted by different
groups? If this experiment was repeated in another laboratory, would this be
necessary?

Question 2: Why was cell culture medium used as a blank?

Question 3: Explain the use of ONPG in this experiment

Question 4: What can this experiment show us about the regulation of gene
expression in the lac operon?

36 _______________________________________________________ Introductory molecular biology and biochemistry

 Practical report - β-galactosidase

Once the entire class has entered data, the spreadsheet of specific activity of β-
galactosidase will be posted on the unit Moodle page for you (after the intensive
school).
Use this information to graph the specific activity of β-galactosidase i.e. the enzyme
activity per mg of bacteria per min using your spreadsheet. Make sure you check all
the values from each group compiled in the spreadsheet.
There should be several values for each time point (from the different groups that
carried out the same sampling times). Use the spreadsheet to find the mean of the
values for each time point, then draw a graph showing the enzyme activity rate ( β-
galactosidase activity/mg cells/min) against time for control (C) and induced (L)
cultures (on the same graph).

This practical must be written up as a full report. See Moodle for further instructions.

Practical 3: Notes and Calculations

Introductory molecular biology and biochemistry _______________________________________________________ 37

 PRACTICAL 4 - SEPARATION OF
MACROMOLECULES BY SEPHADEX GEL
FILTRATION

INTRODUCTION

The study of the detailed mechanisms of cellular processes requires that individual
components of the cellular "machinery" can be isolated in pure form. These
components are mostly macromolecules with molecular weights exceeding 10,000
Daltons. They may be protein, nucleic acid or polysaccharide in nature. Strategies for
the purification of macromolecules usually involve multiple stages, and exploit physical
properties such as charge, size or shape, or biological properties such as the ability to
bind to specific structures.
Exploitation of the "charge carried" for separation of macromolecules, is seen in the
methods of electrophoresis and ion exchange chromatography. Separations based on
molecular weight may involve ultracentrifugation or, more commonly, gel filtration (or
molecular exclusion chromatography). The laboratory exercise described here
demonstrates a very simple separation of coloured molecules based on differences in
their molecular weight. Coloured molecules were chosen only because the separation
is more readily followed.

The technique of gel-filtration is based on the use of a column packed with particles of
a porous gel. The two most common products used are "Sephadex", which is a trade
name for a polysaccharide gel and "Bio-Gel", which is a polyacrylamide gel. The size of
the pores in the tiny bead particles are of the same order as the size of the
macromolecules to be separated; the pore size is controlled during the manufacture
of both gel types by altering the extent to which the polysaccharide or polyacrylamide
chains are chemically cross-linked.
One could visualise each small bead particle as a tiny sponge with pores of various
sizes, the largest pores being able to accept macromolecules. Because of the
controlled degree of cross-linking there is a set maximum pore-size throughout the gel
particle. If the particle is completely permeated with solvent, two "domains" of solvent
exist, that inside the particle, and that surrounding the particle. It is obvious that a
molecule which is larger than the pore size of the particle cannot enter the solvent
domain within the particle and has available to it only the solvent external to the
particle. A molecule which is much smaller than the pore size (eg. water, Ca2+,
sucrose), has available to it both domains of solvent. Molecules of size intermediate
between these two extremes have partial accessibility to the internal solvent domain,
the degree of accessibility being determined by the size of the molecule, and the
fraction of the total pore volume which is larger than that molecule.

38 _______________________________________________________ Introductory molecular biology and biochemistry

 Figure 1: A diagram of Gel filtration chromatography: Nelson & Cox, 2013; fig 3-17

On the macroscopic scale, when we pack a column of Sephadex gel particles, all the
domains of solvent within gel particles add up to a measurable volume called the
internal volume (Vi) of the column, i.e. the volume of solvent which is present wholly
within gel particles. The solvent domain external to the gel particles adds up to a
measurable volume called the external volume or void volume (Vo). The final volume
which needs to be considered is the volume of gel matrix (Vg) (i.e. the volume of the
solid polymer substance itself). All these volumes when added together constitute the
bed volume (Vt) of the column which can be expressed as:

Vt = Vo + Vi + Vg

Vt is determined from the column dimensions. Vo and Vi can be determined

experimentally, and so Vg can be found.

Introductory molecular biology and biochemistry _______________________________________________________ 39

 The elution volume, Ve, of a molecular species is the volume of solvent required to
elute that molecular species. The lower limit to a value for Ve is Vo, the void volume,
since that is the elution volume of a molecule so large as to be completely excluded
from the gel. If the molecule is small enough that it can partially or completely enter
the gel, then it has a larger volume available to it than the void volume, and so will
appear later in an elution profile (the plot of concentration of material versus volume
of eluate).

A suitable equation to express this fact is:

Ve = Vo + Kd. Vi

Kd is the distribution coefficient.

This coefficient indicates the fraction of the inner volume which is accessible to a
particular molecular species, i.e.

Ve - V0
Kd = Vi

If a molecule is completely excluded from the interior of the gel particles, then Kd = 0
and Ve = Vo, a fact already intuitively deduced.
If a molecule has complete accessibility to the internal volume of the gel, then Kd = 1
and Ve = Vo + Vi.
Molecules therefore normally have Kd values between 0 and 1. If Kd is greater than 1,
then adsorption effects are disrupting normal molecular exclusion. This most
commonly occurs with aromatic compounds.
Determination of Kd is the first step in establishing the molecular weight of an
unknown protein. The gel filtration column can be calibrated with a series of proteins
of known molecular weight, which are run through one by one and the elution volume
of each measured. Next, Kd for each is calculated. Then a calibration plot of Kd,
versus Log10 molecular weight is constructed (like Fig.2). This allows the molecular
weight of the unknown protein to be read directly from the calibration plot.

REFERENCES
TEXTBOOK - Nelson and Cox (2013). “Lehninger Principles of Biochemistry”, p. 89-92

Andrews, P. (1964). "Estimation of the molecular weights of proteins by Sephadex gel-

filtration." Biochemical Journal 91: 222-234.

40 _______________________________________________________ Introductory molecular biology and biochemistry

 EXPERIMENT

Columns of Sephadex G-100 (which has large pores capable of holding

macromolecules of about 100 kilo Daltons) are provided. The Sephadex was swollen in
0.1 M phosphate buffer and fine particles removed by decantation prior to packing.

Table 1. Exclusion limits, water regain values and bed volumes/g dry gel for various
grades of commercially available Sephadex

Type Exclusion limit Water regain (Wr) Bed volume

(MW+)* g H2O/g dry gel ml/g dry gel
G10 700 1.0 2.5
G15 1,500 1.5 3.0
G25 5,000 2.5 5.0
G50 10,000 5.0 10.0
G75 50,000 7.5 13.5
G100 100,000 10.0 17.5
G150 150,000 15.0 25.0
G200 200,000 20.0 35.0

*The exclusion limits given were determined using soluble neutral dextrans of known
molecular weight. These figures can differ somewhat for proteins since elution volume
of proteins is determined to some extent on shape and charge as well as molecular
size.

Separation Procedure

The mixture provided consists of haemoglobin, cytochrome c and blue dextran. Blue
dextran is a high molecular weight dextran to which a bright blue chromophore has
been attached covalently so as to render it visible in solution. Because of the high
molecular weight of blue dextran it is completely excluded on G-100 and emerges at
the void volume of the column. Haemoglobin and cytochrome c, having molecular
weights less than the exclusion limit of the column will emerge at elution volumes
greater than the void volume.

1. Have ready a 10 ml measuring cylinder, and 20 numbered centrifuge tubes

calibrated to indicate a 1.0 ml volume.

2. Adjust the height of the column outlet and reservoir to set a SLOW FLOW RATE
of approx 0.5mL.min (about 1 drop per 4s).

Introductory molecular biology and biochemistry _______________________________________________________ 41

 3. Allow the buffer on top of the column to drain down to the level of the gel until
the meniscus just disappears. THROUGHOUT THE PROCEDURE THE BUFFER
LEVEL MUST NOT BE ALLOWED TO FALL ANY LOWER THAN THIS. Discard the
liquid which has been drained off.

4. Pipette 0.3 to 0.4 ml of the mixture onto the top of the gel taking great care
not to disturb the gel underneath.

5. Allow this solution to run into the gel.

6. Using a disposable pipette, wash the top of the gel with a few drops of buffer
allowing the buffer to run just into the top of the gel bed (to minimise
spreading of the bands by back-diffusion into the elution buffer when added).
BEGIN COLLECTION OF ELUATE INTO THE 10 ML MEASURING CYLINDER FROM
THIS POINT Repeat the washing until the coloured band has just moved from
the top of the gel bed.

7. Very carefully fill the column above the gel bed with phosphate buffer, so as
not to disturb the gel surface. Continue to run the column with a flow rate of
about 1 drop per 4s.

8. After the first 10 ml of eluate has been collected in the measuring cylinder
(keep this for later), switch to a calibrated centrifuge-tube (tube 1). Collect 1.0
ml of eluate in tube 1, then switch to tube 2, and continue collecting the 1.0 ml
fractions until all visible colour has emerged from the column. Number and
calibrate additional tubes if required.

9. While you are waiting for your column to run, construct a table In Excel with the
following columns to record your data:

Elution A540 A650 Colour Intensity (0-

volume (mL) 5)

10

11

12

13

↓ etc

42 _______________________________________________________ Introductory molecular biology and biochemistry

 10. Record visually the colour and intensity of each fraction by recording an
intensity value between 0 and 5. The most intensely coloured fractions should
be labelled as 5, and lower values should be recorded for fractions with less
colour.

11. Add 200ul of each sample to a 96 well microplate. Ensure that you include a
sample of the initial 10 ml and each 1 ml fraction thereafter. Record the
location of your samples on the plate map:

1 2 3 4 5 6 7 8 9 10 11 12

12. Read these fractions on the Spectrophotometer at 540 nm and 650 nm against
a distilled water blank. The absorption maximum for cytochrome c is 550 nm
that for haemoglobin is 540 nm, whilst the absorption maximum for blue
dextran is 650 nm.

When all data is entered continue below.

Preparing a Figure

1. First, create a clustered column graph of your absorbance values:

Select Absorbance data (including the A540 and A650 column titles). Do NOT
select the elution volumes. Click the ‘insert’ tab in Excel  in the Charts toolbar
‘Insert Column or Bar Chart’ icon  choose clustered columns (first option).

Introductory molecular biology and biochemistry _______________________________________________________ 43

 2. Next, adjust the X axis labels:

Right click on chart then choose “select data” from menu. The select data source
dialogue box will open.

Click ‘Edit’ under Horizontal Axis Labels

Edit the Axis label range by selecting data in the elution volume column (do not
select the title).  OK  OK.

3. Enter appropriate labels for X and Y axes:

Select the ‘Design’ tab in Excel  Add chart Element.

44 _______________________________________________________ Introductory molecular biology and biochemistry

 4. Tidy up your chart for scientific presentation:
Clear background, gridlines, and chart title (the title should be replaced by an
informative figure legend below the graph in your report).
5. Remove any gaps between the columns:
Right click the graph and select ‘Format Data Series’  Set gap width to zero.

6. Modify the units displayed on the Y axis:

Right click on the Y axis and select ‘Format Axis’. Change the major unit to = 0.1

7. Your chart should now look like this (without the arrows and their labels):

0.4
Ve blue dextran
0.3
Ve Hb
Ve Cyt c
0.2
Absorbance

A650
A540
0.1

0
10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32
Elution volume (mL)

8. You can now read your graph to obtain the elution volumes of each absorbance
“peak”. These correspond to Ve first for blue dextran, then haemoglobin and finally
cytochrome c (see above).

CALCULATION OF RESULTS

The purpose of the calculations is to determine the Distribution Coefficient (Kd) for
Haemoglobin and Cytochrome c and to use this to obtain an estimate of the molecular
weight of each compound, which you will compare with literature values (see text
book)

Introductory molecular biology and biochemistry _______________________________________________________ 45

 1. Determine the elution volumes for each component (see above)
Ve Blue dextran __________mL

Ve haemoglobin __________mL

Ve cytochrome c __________mL

2. Determine the void volume (Vo)of the column

As blue dextran molecules are so large as to be completely excluded from the gel
particles

Ve (blue dextran) = Vo the void volume = __________mL.

3. Determine the total volume of the column Vt

The Total Volume Vt = πr2h =____________ =____________mL

(The internal diameter of the column is 1.6cm; you need to measure the height of
the gel (in cm) before parting with your column.

4. Calculate the mass of dry gel "a" (grams) required to prepare the column from;

a = Vt/ bed volume (ml/g dry gel -see table 1) = / = g

5. Determine the internal volume (Vi) of the gel bed as follows:

Vi = aWr = x = _______________mL

“Wr' ml/g is the water regain i.e. the volume of water just required to swell the gel
particles without leaving any excess water external to the particles, see Table 1.

6. Determine the Kd for Haemoglobin and Cytochrome c

Use the formula provided in the Introduction to calculate the values of the
distribution coefficients using the elution volumes for each compound and the
values calculated above for Vo and Vi.

Kd haemoglobin =
Kd Cytochrome c =

7. Calculate the Log Molecular weight of Haemoglobin and Cytochrome c

The graph below shows the relationship between Log MWt vs Kd for a number of
proteins of known molecular weight:

46 _______________________________________________________ Introductory molecular biology and biochemistry

 4.9
4.7
y = -1.8499x + 5.0876
4.5
R² = 0.999

Log MWt
4.3
4.1
3.9
3.7
3.5
0 0.5 1
Kd

Figure 2: Calibration curve of Log molecular weight versus Kd

Use the equation for this relationship to calculate Log MWt for each protein from
Kd.
Log MWt Haemoglobin:

Log MWt Cytochrome c:

8. Find the molecular weight of each component from the Log MWt.

Estimated MWt Haemoglobin:

Estimated MWt Cytochrome c:

9. Search the available literature to find the actual molecular weight of Haemoglobin
and Cytochrome C. Note where you found this information as you will need to
reference it in your practical report.

Actual MWt Haemoglobin (literature):

Source:

Actual MWt Cytochrome C (literature):

Source:

Introductory molecular biology and biochemistry _______________________________________________________ 47

 Practical 4: Questions

Question 1: Why was the buffer drained to the level of the gel prior to the addition of
the protein mixture?

Question 2: Why was it necessary to add additional buffer after the protein mixture
was added to the column?

Question 3: If the following mix of molecules were purified using size exclusion
chromatography, what would be the order in which the molecules leave the column?
Mixture: haemoglobin, 65,000 daltons; myoglobin, 17,000 daltons; myosin, 180,000
daltons.

Question 4: The above solution is modified to include a fourth protein with a

molecular weight of 16,000 daltons. Would this technique be suitable to separate this
mixture?

48 _______________________________________________________ Introductory molecular biology and biochemistry

 Practical report:
Please see detailed instructions for the preparation of this practical report on Moodle.

Practical 4: Notes and Calculations

Introductory molecular biology and biochemistry _______________________________________________________ 49

 PRACTICAL 5 - DIGESTION OF STARCH AND
CELLULOSE

INTRODUCTION

There are many aspects to the dependence of animal metabolism on plants. Just one
dramatic example is the supply of energy and organic carbon for maintenance and
growth principally from glucose, the most important sugar produced by
photosynthesis. Most of this glucose in plants is locked up in the polymers, starch and
cellulose. Starch is the polymer stored by plants in plastids to provide sugar for
metabolism when photosynthesis is not active. Cellulose is a polymer used to build cell
walls. Both polymers are composed of monomers of glucose, but there the similarity
ends. Animals have evolved different lifestyles to utilise these sources of glucose.
Perhaps this is so because cellulose is more difficult to break down than starch at the
molecular level. The practical session aims to demonstrate these differences.

Starch

Starch is a large component of specialized storage tissues such as tubers and the
endosperm of seeds, but is also present in all photosynthetic tissues. Starch consists
of two polymers, amylose and amylopectin. Amylose is about 20% of starch and
consists of 50 to 500 glucose residues linked by α1→ 4 covalent bonds. This
bonding twists the polymer into an open helix. Amylopectin has mostly the same
bonding between residues, but differs in size (up to 5,000 glucose residues) and in the
presence of branch points where some residues have an extra covalent link, α 1→ 6)
forming branch chains. This extra link about every 30 residues produces an open,
globular polymer. Thus, starch can absorb water and disperses in solution.

Cellulose

Almost all plant cell walls contain a large fraction of their dry weight as cellulose. The
polymer consists of 5,000 to 10,000 glucose residues linked in a chain by β 1→ 4
covalent bonds. The β -linkage enables the residues to rotate by 180 degrees to each
other to form an extended linear chain. About 50 to 70 of these chains are synthesised
together by special complexes in the plasmalemma membrane, which enables the
chains to pack closely together, often in a crystalline array. There are many hydrogen
bonds between the chains and a fibre is formed that excludes most water from its
interior. Thus, an insoluble, flexible, and strong fibre is formed. Notice how the
configuration of bonds between monomers can dramatically change the properties of
polymers.

50 _______________________________________________________ Introductory molecular biology and biochemistry

 Structures of starch and cellulose are illustrated in your textbook.

Digestion

It is the physical differences between starch and cellulose that govern the ability of
enzymes to degrade them. There is no significant difference in the strength of the
chemical bonds linking the monomers in starch and cellulose. The energy barriers to
hydrolysis are similar, thus the rate of hydrolysis of the linkages in isolated chains
should be similar by similar enzymes. However, starch has a structure that an enzyme
can readily approach and form a complex for catalysis. Amylopectin also has many
chain ends per polymer that can be attacked simultaneously. Cellulose has on the
average a very low fraction of hydrated structure that an enzyme can attack. Cell walls
are more difficult to degrade than individual cellulose fibres because the cellulose is
also chemically linked to other components of the wall.

BEFORE COMING TO THE LAB: In the second experiment today you will be looking
at the effect of chloride ions on amylase activity. Find out why and how chloride
affects the enzyme.

Starch is degraded by two enzymes, amylase and dextranase. Amylases hydrolyse the
α1→ 4 linkages anywhere along the polymer chains. Dextranases hydrolyse only the
α1→ 6 linkages. Thus, starch can be completely degraded by these enzymes to
glucose. Amylases have been extensively studied, and are now being used in large
scale production of corn syrup (a major challenge to the sugar industry).

Pure cellulose is degraded by a minimum of two enzymes, an endoglucanase and an

exoglucanase. The endoglucanases can hydrolyse β1→ 4 linkages anywhere along the
chains that solvent has exposed. The exoglucanases hydrolyse the same bond, but
only by cleaving glucose from the reducing end of chains. The latter activity is
probably important because the polymer chains in cellulose tend to separate at the
ends, allowing the enzyme access. Among animals, only the Gastropods (snails and
slugs) are capable of digesting cellulose. However, a wide range of microorganisms
degrade cellulose. The ecology of microbial/animal interactions in the digestion of
cellulose is extremely important to agriculture.

EXPERIMENTS

Introductory molecular biology and biochemistry _______________________________________________________ 51

 The below two experiments are designed to demonstrate the speed and efficiency of
starch degradation in the digestive tract, and illustrate the dependence of many
enzymes on specific solvent components for catalysis.

Assay for amylases

There is a simple and unique way to measure the rate of decomposition of starch.
Elemental iodine specifically forms a complex with amylose that has a blue colour.
This complex is dependent upon the intact helical conformation of the polymer.
Chains of iodine atoms are held inside the helix. As the polymer is degraded by
amylase, the number of complexes that can form with iodine decrease. The
fragmented polymers give first a blue to red change, and finally, the colour fades
completely. The reddish colours are due to the interactions of iodine with amylopectin.
The point of total loss of iodine colour is called the achromatic point. It is not
possible to say what is the exact degree of degradation of the starch polymers at this
point.

PART I: EFFECT OF ENZYME CONCENTRATION ON

ENZYME ACTIVITY

1. Prepare the salivary amylase enzyme by collecting a saliva sample:

Rinse your mouth with 30 ml of the water, moving it around in your mouth several
times. Empty your mouth into a clean beaker. This solution of saliva should not
be opalescent and viscous. If it is, dilute with an equal volume of distilled water.

2. Filter the saliva solution through filter paper. It is now ready for use. Use the
preparation soon after, as bacteria from your mouth will be degrading the proteins
present with time.

3. Set up 5 large test tubes. To each tube, add 5 ml of 1% starch, 2 ml of 0.2 M NaCl,
and 2 ml phosphate buffer (pH 6.8). Mix the contents gently. These tubes are your
STARCH tubes (NOTE: These tubes are identical and do not require labelling. Only
initial the tubes to distinguish between samples from different groups).

4. Place the tubes in a water bath for around 5 minutes to equilibrate to 37oC

5. Prepare another set of 5 test tubes and label 1 to 5. Prepare as shown in the table
below:

Tube 1 2 3 4 5

52 _______________________________________________________ Introductory molecular biology and biochemistry

 Enzyme prep. (ml) 1 2 3 4 5

Water (ml) 4 3 2 1 0

6. Mix the content gently and then equilibrate the tubes to 37oC in the same bath for
about 5 mins. These tubes are your ENZYME tubes

7. Place a disposable plastic pipette in each of the enzyme tubes.

8. Perform the reactions one tube at a time. ONCE EACH REACTION HAS
COMMENCED, THE REACTION MAY PROCEED QUICKLY AND YOU WILL NEED TO
BE READY TO SAMPLE IMMEDIATELY. Read the steps 9-12 prior to commencing.

9. Start each reaction by adding the contents of a starch tube to an enzyme tube, mix
thoroughly, and keep in the bath at 37oC. Write down the time each reaction
commenced.

HINT: Due to the variability of saliva preparations it is best to start with the sample
with the highest concentration of saliva i.e. tube 5. If you do not reach the
achromatic point for this tube within about 5-6 mins you should think about
making a new preparation as more dilute samples will react VERY SLOWLY.

10. To sample each reaction mixture, pipette 2 drops of the sample into a well on the
plate provided, and add 1 drop of 0.02 N iodine.

11. Repeat this sampling procedure at frequent intervals (15, 30 or 60s depending on
the speed of reaction), using a new well for each sampling time point.

12. Watch the colours and continue sampling until the achromatic point is reached or
until some common intermediate shade is reached, which you can identify. The
sample should change from dark blue to the colour of iodine. Write down the time
of this end point for each reaction.

13. The reciprocal of the time taken to reach the end point in each reaction is a
measure of the rate of the reaction due to amylase.

PART II: EFFECT OF CHLORIDE ION ON THE ACTIVITY

OF AMYLASE

Your saliva conveniently provides the correct conditions for the action of amylase.
Note that in the experiment above, your amylase was provided with a buffered solution
of NaCl for action (see the starch tube preparation section). To test the influence of Cl-
on the enzyme, it was necessary to remove all chloride from the enzyme solution. This

Introductory molecular biology and biochemistry _______________________________________________________ 53

 had to be done in advance of the class by using dialysis. You are provided with a pure
preparation of amylase from animal pancreas for this experiment.

1. Set up five labelled test tubes (labelling the tube and adding the specified amount
of NaCl and water to each tube) as follows:

Tube 1 2 3 4 5

NaCl (0.01 M), ml 0 0.2 0.5 1.0 2.0

Water, ml 2.0 1.8 1.5 1.0 0

Final chloride
concentration
µmol/mL*

* the total volume of the solution = Vol NaCl + Vol H2O + Vol phosphate buffer + Vol 1% starch + Vol pancreatic enzyme

2. To each tube add 2 ml of phosphate buffer and 5 ml of 1% starch (located on the

front desk!).

3. Incubate the tubes in the water bath at 37oC for 10 minutes

4. Perform the reactions one tube at a time. ONCE EACH REACTION HAS
COMMENCED, THE REACTION MAY PROCEED QUICKLY AND YOU WILL NEED TO
BE READY TO SAMPLE IMMEDIATELY. Start each reaction by adding 3 ml of
dialysed pancreatic enzyme (also at 37oC ) to each tube..

5. To sample each reaction mixture, pipette 2 drops of the sample into a well on the
plate provided, and add 1 drop of 0.02 N iodine.

6. Repeat this sampling procedure at frequent intervals (15, 30 or 60s depending on

the speed of reaction), using a new well for each sampling time point.

7. Watch the colours and continue sampling until the achromatic point is reached or
until some common intermediate shade is reached, which you can identify. The
sample should change from dark blue to the colour of iodine. Write down the time
of this end point for each reaction.

8. Calculate the reciprocal of time to give a relative rate for each reaction.

54 _______________________________________________________ Introductory molecular biology and biochemistry

 PART III: DEMONSTRATION EXPERIMENT

This experiment has done by our helpful technician, you need to observe the
consequences of the reaction and read the below text, then answer Q7.

Since it takes a long time to hydrolyse even pure cellulose fibres, this section of the
class has been organised as a demonstration. To illustrate the process, the bacterium,
Ruminococcus albus*, an organisms found commonly in the rumen of large herbivores,
is used in this practical. In the rumen, life is working in the total absence of oxygen.
Thus, these microbes must be grown anaerobically - in an atmosphere of nitrogen and
carbon dioxide. For this demonstration, bacteria were grown on cellulose fibres in a
liquid culture and then the bacteria were removed from the medium. The medium only
was transferred to new tubes containing filter paper strips. You should note the
changes to the filter paper with time.

*Ruminococcus albus is one of the most actively cellulolytic of the rumen bacteria, producing a
number of extracellular cellulases which completely digest the cellulose surrounding colonies in
cellulose agar roll tubes. It can also digest the cellulose and hemicellulose in alfalfa and grass
hays.

PLOTTING YOUR DATA

A. Effect of enzyme concentration on enzyme activity
1. Open a new work book in excel and set up the following columns:
Amount of enzyme (mL)
Time to reach endpoint (min)
Reaction rate = 1/t (1/min)

2. Select ONLY the columns for amount of enzyme (this is proportional to [E] in
tube) and reaction rate (1/t)

3. Click the ‘Insert’ tab  Insert XY scatter (choose chart with data points only and
select the first option)

4. Insert appropriate labels for the X and Y axes using the ‘Design’ tab and the ‘Add
Chart Element’ menu. These labels should be SPECIFIC and must have correct
units.

5. Tidy up chart appropriate for scientific paper. Clear gridlines and any coloured
background to chart. Choose appropriate spacing for major units on axes.

Introductory molecular biology and biochemistry _______________________________________________________ 55

 6. Insert trendline.
To do this first THINK about the relationship expected between enzyme
concentration and reaction rate?
What is the expected relationship? Linear or other?
Should your trend line go through the origin or not?

NOW, right click on data points  choose ‘Add Trend Line’  Choose the correct
type of trendline. Decide whether to tick the ‘Set Intercept’ box (insert correct
intercept if relevant)  Select ‘Display R Squared Value on Chart’.

B. Effect of chloride ions on the activity of amylase

1. Open a new work book in excel and set up the following columns:

Volume of 0.01 M chloride

Chloride concentration in (µmol/mL)

Time to reach endpoint (min)
Reaction rate = 1/t (1/min)

2. Select ONLY the columns for Chloride concentration and reaction rate (1/t)

3. Insert => XY scatter => choose chart with data points connected by smoothed line
(second option) – you do not know what relationship to expect so inserting a
trendline is not really appropriate.

4. Insert appropriate titles for chart, X and Y axes using the layout menu. These
should be SPECIFIC and must have correct units.

5. Tidy up chart appropriate for scientific paper. Clear gridlines and any coloured
background to chart. Choose appropriate spacing for major units on axes.

56 _______________________________________________________ Introductory molecular biology and biochemistry

 Practical 5: Questions
Question 1: Graph the reaction rate (as 1/time taken to reach the achromatic point)
against amount of salivary amylase preparation used (mL). Print, email or copy below.

Question 2: Graph the reaction rate (as 1/time taken to reach the achromatic point)
against chloride ion concentration (µmol/mL). Print, email or copy below.

Question 3: What relationship do you expect between reaction rate and enzyme
concentration? Does your graph support your hypothesis? Explain in one sentence.

Question 4: Compare your results for Experiment 1 with results for two other pairs of
students; is your enzyme more or less active? Comment on possible reasons for
differences.

Introductory molecular biology and biochemistry _______________________________________________________ 57

 Question 5: Briefly discuss the shape of your graph of chloride conc. versus reaction
rate.

Question 6: Comment on the relative rates of starch digestions by amylase and the
cellulose digestion in the demonstration.

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 Practical 5: Notes and Calculations

Introductory molecular biology and biochemistry _______________________________________________________ 59

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