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BIOSCI106
Foundations of Biochemistry

Lab Manual 2024

 BIOSCI 106 Foundations of Biochemistry
Laboratory Guide, 2024 Edition

This material is protected by copyright and has been copied by and solely for the
educational purposes of the University or Auckland under licence. You may not
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to any other person. Where provided to you in electronic format, you may only
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terms of this warning may expose you to legal action for copyright infringement
and/or disciplinary action by the University of Auckland.

Note: Copyright protection also extends to audio recordings of lecture and

laboratory classes. Please seek the permission of the Course Coordinator BEFORE
The cover
making an of thisrecording
audio laboratory
forguide
your was
owndesigned
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file available from www.Shutterstock.com (file no 226274737).
 Laboratory Health & Safety:

Please ensure you have completed the on-line health and safety quiz
before your first laboratory. You will not be able to work in the lab until
you have passed this quiz.

GENERAL RULES

1. Lab coats, gloves, safety glasses, 2. Do not wear your lab coat, safety
and closed-toe shoes must be glasses or gloves outside the
worn laboratory

3. Long hair must be tied back 4. No food, drink, smoking/vaping,

or phone/device use in the lab

5. Wash your hands during and 6. Keep aisles clear, work in a tidy
after laboratory work and careful way that does not
disrupt or endanger others

7. Use chemicals safely 8. Report any accidents or near

misses immediately
 EMERGENCY PROCEDURES - EVACUATION:

Please familiarise yourself with the floor layout, exits and stairs for this
building to prepare for an emergency evacuation. In case of a fire or
other emergency, you should know the location of the nearest exits from
the laboratory.

EXIT
If the building alarm sounds continuously, please evacuate the
building:

EXIT Walk briskly and do not run. Exit down the stairs to the ground floor
and leave the building. Do not use the elevators. All emergency
evacuation routes are indicated by green ceiling signs – follow them
to the exit.

EXIT
Follow the instructions of the building wardens, who will be wearing
yellow vests, or floor wardens, who will be wearing orange vests.

Once outside the building, move to the assembly point, which is the
lawn behind Old Choral Hall and in front of the Albert Wall next to
the General Library.

Stay out of the building while the alarm is sounding and return only
after a building warden tells you that it is safe to go back inside.
 EMERGENCY PROCEDURES – INJURY & EMERGENCY
SERVICES:

First Aid for Minor Injury:

If you sustain a minor injury, please report this immediately to the staff
member or tutor running the lab. The first aid cabinets contain stocks
of basic medical supplies and the Lab Manager is a certified First Aid
person. Specialist emergency equipment is present in the lab; eyewash
stations for cleansing eyes, and showers for chemical spills. This
equipment is for immediate use by any person when required. Do not
wait for approval.

First Aid for More Serious Injury:

If you sustain a more serious injury, a staff member will accompany
you to the University Health Service.

If the injury is serious, dial 111 and ask for an ambulance.

111 In the case of any accident, the student will be required to fill out an
Accident Form as part of the University’s accident reporting policy.
This form is available from the Laboratory Technicians.

Emergency Services:
If you need to call emergency services:
• Dial 111 and ask for fire, ambulance, or police.
111 • Tell them the building name; Building 106, Old Biology
Building.
• Give them the building’s street address; 5 Symonds Street,
Central Auckland.
• Explain the nature of the emergency.
 CLEAN UP AND WASTE DISPOSAL:

You are responsible for keeping your bench and work area tidy. This includes
cleaning up at the end of each laboratory exercise. Each laboratory includes
specific instructions for bench and workstation clean-up. These can be found in
your laboratory guide and above the sink or ask your demonstrator.

flammable explosive biological toxic

Common hazard symbols from the laboratory. Please be familiar with these, as you may see
them in the laboratory and should know what they mean, and how you should treat those items.

DISPOSAL OF HAZARDOUS WASTE. The yellow ‘Medi-

safe’ buckets are for sharps (glass, needles, razor and scalpel
blades, etc.). Yellow bags (inside bins) are for gloves, and other
experimental consumables as per the lab clean up procedures. Do
NOT put any laboratory waste in the general rubbish bins.

Hand washing.
The hand washing rules are not simply for your benefit. The labs
are used by several courses and the bench surfaces can be
contaminated with a variety of biological pathogens and
chemicals which can persist even after routine clean-ups. You
must wash your hands after each lab to ensure that you do not
carry pathogens outside the lab.
 REGULATIONS FOR WORKING WITH GMOs:

The use of Genetically Modified Organisms has approval from the

Environmental Protection Authority (EPA) under the Hazardous Substances
and New Organisms (HSNO) Act 1996.
EPA approval has been given on the following conditions: That all approved
GMOs are developed within an MPI approved Containment Facility
complying with MPI/EPA Standard 154.03.02 Containment Facilities for
Microorganisms.
Work practices specified under Physical Containment Level 1 (PC1)
specified in AS/NZS 2243.3:2010 "Safety in Laboratories Part 3:
Microbiology" are followed.

Work practices specifically required for PC1 containment are to be followed

throughout the laboratory sessions:

1. No eating or drinking in the 2. No food or drink to be stored in

laboratory. the laboratory

3. Laboratory coats, eye protection 4. Laboratory coats must be

and closed shoes must be worn removed and hands are to be
during laboratory sessions washed before leaving the
laboratory areas
5. All cultures are to be clearly 6. Work surfaces are to be
labelled, dated and decontaminated regularly and
appropriately stored after spills

7. Spills and accidents must be 8. Laboratory waste is to be

reported immediately to the decontaminated before disposal
staff teaching the laboratory – solid waste to be autoclaved
and liquid waste to be
autoclaved or chemically
disinfected

9. Special care must be taken to prevent chemical/biological

contamination of reading/writing materials. Do not use mobile phones
or other devices in the laboratory.
 EPA/HSNO INFORMATION:
Course Number BIOSCI 106
Laboratory Number(s) Laboratories 2 and 6
Course Coordinator Julie McIntosh
Relevant ERMA GM010/UA008
approval
Teaching Laboratory 106-301, 106-307, School of Biological
Sciences Containment Facility
Laboratory
Guide
__________________________________________
LABORATORY ONE
Introduction to BIOSCI 106 labs
Before the Lab:

Please read the health and safety requirements under the “Laboratory Information” section
and complete the health and safety quiz.

https://canvas.auckland.ac.nz/courses/103628/quizzes/126406

The quiz will test your understanding of the health and safety requirements of the labs.
To access the quiz, please:

Go to CANVAS → Click on QUIZZES → Click on the SBS Health and Safety Quiz and
Declaration
Completion of the quiz is worth 0.5% of your grade. There is no time limit for the quiz but remember
that it has to be completed before your first lab.

My recommendation is that you read the Laboratory Information section of your lab
BEFORE viewing the actual questions.
Make sure you complete the pre-lab quiz for this laboratory on Canvas.

Learning Objectives for Laboratory 1:

In this laboratory you should:

• get to know the names of your peers and to know the teaching staff.

• work in a collaborative manner.

• become familiar with the lab environment

• practice using lab equipment and software that will be relevant for subsequent labs in
BIOSCI 106 (including micropipettes, the spectrophotometer, UV torches and Excel)
 LABORATORY RISK ASSESSMENT – LABORATORY 1 BIOSCI 106

Please take note of the following materials or procedures that we have

identified as a potential risk in this experiment. Risk minimisation controls are
listed. Full safety data sheets (SDS) for the listed chemicals are available
within the CANVAS Laboratory module/pages.
Note: other risks exist in the laboratory; the list below is not extensive nor complete.

HAZARD IDENTIFICATION – MATERIALS

Hazardous substance:
Hazard Classification: (tick as appropriate)
None

Toxic

Acute
Health
Hazard

Chronic
Health
Hazard

Flammable

Oxidising

Corrosive

Other
 MINIMIZING RISK - MATERIALS

Hazard Materials Risk Controls:

No hazardous chemicals have been identified in this experiment. Take
None standard precautions and use personal protective equipment as normal.

HAZARD IDENTIFICATION – EQUIPMENT / PROCEDURES

Hazard Description and risk controls:

UV Torches Do not shine the torches in your own eyes or anyone else’s eyes. Only
shine the torch at the samples as indicated in the experiment.
 Introduction
You find yourself in the BIOSCI 106 labs with a group of people – some strangers,
some friends…. Who are they?
Will you escape BIOSCI 106 labs successfully at the end of semester?
A logbook with tips on how to escape is conveniently found at your bench… Let the
challenges begin!

Tube Concentration (mg/L) Absorbance

1

Unknown (sports drink)

 LAB 1 CLEAN UP INSTRUCTIONS

• Pay attention to your tutor for clean-up information.

 LABORATORY TWO
Protein Folding & Equilibrium

Before the Lab:

Watch the introductory videos, read through the laboratory, and information on bovine serum
albumin (BSA) and the equilibrium between folded and unfolded forms of proteins. To save you
time in the laboratory session, you may like to pre-cut the templates for the paper models you will
build in Part 2 of the laboratory exercise.
Make sure you complete the pre-lab quiz for this laboratory on Canvas.

Learning Objectives for Laboratory 2:

In this laboratory you should:

• apply your theoretical knowledge of protein folding and equilibrium processes to your
experimental system and then analyse your experimental results.

• develop key skills for accurately performing a scientific experiment.

• learn to accurately record experimental results and observations.

• develop skills to visualise protein structural motifs in 3-dimensions.

 LABORATORY RISK ASSESSMENT – LABORATORY 2 BIOSCI 106

Please take note of the following materials or procedures that we have

identified as a potential risk in this experiment. Risk minimisation controls are
listed. Full safety data sheets (SDS) for the listed chemicals are available
within the CANVAS Laboratory module/pages.
Note: other risks exist in the laboratory; the list below is not extensive nor complete.

HAZARD IDENTIFICATION – MATERIALS

Hazardous substance:
Hazard Classification: (tick as appropriate)
NaOH
(250 mM)

Toxic

Acute
Health
Hazard

Chronic
Health
Hazard

Flammable

Oxidising

Corrosive

Causes severe skin

Other burns and eye
damage. Harmful to
aquatic life.
 MINIMIZING RISK - MATERIALS

Hazard Materials Risk Controls:

NaOH Pipette carefully and gently at arm’s length. Wear lab coat, gloves, and eye
(250 mM) protection to avoid skin and eye contact. Do not ingest. Please read the
SDS available in the CANVAS Laboratories module/pages.

HAZARD IDENTIFICATION – EQUIPMENT / PROCEDURES

Hazard Description and risk controls:

UV Torches Do not shine the torches in your own eyes or anyone else’s eyes. Only
shine the torch at the samples as indicated in the experiment.
 Introduction
This laboratory is carried out in two parts. In Part 1 you will determine whether a protein
(bovine serum albumin or BSA) is unfolded or folded in a range of buffer conditions. You
will also determine if the transition from folded to unfolded states is a reversible, equilibrium
process. In Part 2 you will visualise β-sheet and α-helical protein structures to understand
their stability.

Background – Folding/Unfolding Equilibrium:

Proteins within our cells exist in folded and unfolded forms in equilibrium. What does this
mean? We would normally think about a protein being made at the ribosome then folding up
and staying in this folded form until it is recycled. But in reality, proteins will be present in
both folded and unfolded forms:

Figure 1. An equilibrium between unfolded and folded forms of a protein.

(Imagery; C. Squire and J. Squire)

In a reversible equilibrium process, the two forms interconvert with the forward (folding)
rate, kf, equal to the backward (unfolding) rate, ku. This is not to say that the predominant
form of the protein is not the folded state – at equilibrium there may be many, many more
copies of the folded protein than unfolded:

Figure 2. A conceptual diagram showing a population of proteins in a cell. The proportions of

folded and unfolded protein are indicated. At equilibrium, if one folded protein unfolds, another
unfolded protein will fold up – forward and reverse reactions happen at the same rate at
equilibrium by definition. (Imagery; C. Squire and J. Squire)
 In our cells, the proportion of unfolded protein is very likely much smaller than this figure
suggests for most proteins – but it is simply too difficult to illustrate. What the equilibrium
process tells us is that when one folded protein in this population unfolds, statistically another
unfolded protein will fold up – the forward and backward reactions happen at the same rate.

The proportions of folded and unfolded protein can be influenced by the environment by
factors including the pH, and the presence of stabilising or destabilising chemicals – this is
what you will investigate in the laboratory using the protein bovine serum albumin (BSA)
which exists in folded and unfolded forms in equilibrium.

Background – BSA protein and dye binding as a reporter of folding:

Bovine serum albumin is derived from the blood plasma of cows and can be used as a protein
standard in laboratories. Serum albumins like BSA are the most abundant proteins in
vertebrate blood and are critical in the maintenance of blood pressure. One very well studied
property of these proteins is their ability to bind and carry hydrophobic steroid hormones,
fatty acids, and thyroid hormones. BSA can also bind the fluorescent dye called 8-
anilinonaphthalene-1-sulfonic acid (ANS).

8-anilinonaphthalene-1-sulfonic acid (ANS)

(Imagery: C. Squire)

ANS acts as a reporter of BSA folding – it only binds to the folded form of BSA and only
fluoresces once bound within a pocket of the protein:

Figure 3. Equilibrium between unfolded and folded forms of bovine serum albumin, a blood plasma
protein. When the reporter molecule ANS binds to folded BSA, fluorescence is observed.
(Imagery; C. Squire and J. Squire)
In this laboratory, you are provided with a solution of BSA dissolved in water. To this you
will add various concentrated buffer solutions ranging between pH 5 and pH 12, and observe
the effect of pH on the folding/unfolding equilibrium. You will also be shown a sample of the
BSA solution that has been heated to over 80 °C to investigate a related process.

The structure of BSA comprises numerous α-helical secondary structure elements that folded
into the correct 3D form provide exceptional stability to the protein.

BSA

(Imagery; C. Squire)

In the final part of the laboratory you will use protein paper folding models to explore the
properties of proteins rich in either α-helical or β-sheet secondary structure.

The paper folding templates and instructions have been sourced from the RCSB Protein Data
Bank. These simple models can be cut from paper, folded and stuck together with sticky tape
to reproduce the structures of green fluorescent protein (GFP) and a G-protein coupled
receptor (GPCR). While these models are made of paper and are of a scale some 100 million
times larger than the molecular scale of the real proteins, the properties of the models in
terms of their “stiffness” or “floppiness” or flexibility provide real insight to molecular level
properties. As part of this conceptual exercise you will all contribute to a class model of an
amyloid fibre on the largest scale we can manage using paper!
 GFP

GPCR

Figure 4. Molecular structures of GFP and the membrane segment of the opioid GPCR
alongside their respective paper models. A model of an amyloid beta fibril from an
Alzheimer’s patient is shown to the right.

(Imagery; C. Squire and the RCSB PDB)

 Part 1: Visualising folding/unfolding of BSA

Follow the instructions below carefully.

You have been provided with a 10 mL sample labelled bovine serum albumin (BSA) in water.
You will be mixing this protein sample with various buffers to determine the effect of pH.
You will add the fluorescent dye 8-anilinonaphthalene-1-sulfonic acid (ANS) to the protein
as a reporter; folded protein will induce ANS fluorescence.

Equipment and reagents:

1. Pre-prepared BSA solution (15 μM BSA in water)
2. ANS solution (10 mM in water)
3. Buffer solutions:
• MES, pH 5 (1 M)
• HEPES, pH 7 (1 M)
• Tris.HCl, pH 9 (1 M)
• NaOH, pH 12 (250 mM)
4. 1.5 mL Eppendorf tubes
5. Black light torch (shared)

Caution: Please consider all chemicals used in this laboratory as toxic and make sure you are
using labcoats, safety glasses, and gloves to avoid any contact.
In particular, take care using the solution containing sodium hydroxide.
Follow ALL instructions provided in this guide and within the laboratory by teaching staff
and demonstrators.
EXPERIMENT 1. Control experiment – checking for BSA/ANS fluorescence:

1. Ensure you are wearing the correct personal protective gear, including a lab coat, safety
glasses and gloves.
2. Pipette 1 mL of your BSA solution into an Eppendorf tube and add 20 μL of ANS reagent.
Mix the dye into the protein GENTLY by inversion. Label this tube “1. BSA/ANS”.
3. In a second Eppendorf tube, pipette 1 mL of your water and add 20 μL of ANS reagent. Mix
gently by inversion. Label this tube “2. water/ANS”.
4. In a third Eppendorf tube, pipette 1 mL of BSA solution. Label this tube “3. BSA”.
5. Visualise all three samples using the black light torch provided.
6. Record your observations about the fluorescence of each sample below:

Label each sample appropriately. Indicate in the drawing below, the relative fluorescence of
each sample. Record more detailed observations describing the overall outcome of the
experiment.

CONTROL SAMPLES:

1. 2. 3.

Observations:
Do you see fluorescence in each tube?

What does this experiment prove you need in the tube to see fluorescence?
EXPERIMENT 2. Effect of pH on BSA:

1. Label four tubes with: “4. pH 5”, “5. pH 7”, “6. pH 9”, and “7. pH 12”.

2. Pipette 0.9 mL of BSA solution into each tube.

3. Add 100 μL of the appropriate buffer solution to each tube to match the
labels e.g. add 100 μL MES, pH 5 buffer to the pH 5 tube.

4. Add 20 μL of ANS reagent to each sample and mix GENTLY by inversion.

Remember to change your pipette tip each time to avoid cross contamination.

5. Image the samples as previously using the black light torch. Use the “1.
BSA/ANS” sample from instruction 1 above as a positive control for
fluorescence.

Record your observations about the fluorescence of each sample on the sheet provided in this
guide:
pH-modified SAMPLES:

1. 4. 5. 6. 7.
Observations:
Do all the tubes fluoresce? Why/why not?

1. To understand the reversibility of the folded/unfolded equilibrium take your pH 5 and pH

12 samples and to each add an additional 200 μL of pH 5 buffer. The “4. pH 5” sample
should remain at pH 5. The pH in your modified “7. pH 12” sample should now be
somewhere between 6 and 7.

2. Revisualise these two samples (“4.* pH 5” and “7.* pH 12”) using the black light torch –
use the “BSA/ANS” sample tube 1 as a positive control for fluorescence if you need to.

3. Record your observations about the revisualised samples. Does this experiment prove that
the effect of pH on BSA fluorescence is reversible?

pH-modified SAMPLES - revisualised:

1. 4.* 7.*
Observations:
What change can you see in the tubes?
EXPERIMENT 3. Demonstration - Effect of heating BSA to 80 °C:

A sample of BSA solution that has been heated will be passed around the laboratory.
Record your observations. Do you think the effect of heating BSA to 80 °C is reversible?

Observations:

LAB 2 CLEAN UP INSTRUCTIONS

(complete this before beginning Part 2)

• Pay attention to your tutor for clean-up information.

Safety Check: do not remove any samples or reagents from the laboratory
 Part 2: Folding Paper Models of Proteins and amyloid
fibrils

Follow the instructions below to build a paper model of a protein. You may like to pre-cut
your paper templates before attending – this will save you time in the laboratory session.

You have been provided with a template to build either a model of green fluorescent
protein (GFP) or a G-protein coupled receptor (GPCR). These have been downloaded
from the RCSB Protein Data Bank as an educational resource. This exercise is to
show you how a long protein chain folds up into a compact 3D protein structure,
how the secondary structure elements in a protein structure (α-helices and β-
strands) interact together, and how the different properties of protein secondary
structure determine the final physical properties of the protein. We would also like
you to contribute to the class model of an amyloid fibril that will be about 2 metres
tall! Pay close attention to how your small part of this fibril (a single short, misfolded
segment of protein) contributes to the formation of these giant fibril structures in
disease.

Instructions

1. Choose one of the models to fold, either the GFP or the GPCR. Your laboratory partner
should make the alternative model.

2. Follow the detailed instructions provided to fold up your model. Use scissors to cut
the model parts out and sticky tape to join them together. The process you use to
fold the model mimics (more or less) how the protein really folds in nature.

3. Compare and contrast the two models (one a β-barrel, one an α-helical bundle) to
answer the questions associated with this part of the laboratory exercise.

4. Using your newly acquired paper protein folding skills, assemble the two-stranded
amyloid sequence (template again provided below) and attach it to the scaffold
provided in the laboratory as directed by a demonstrator or tutor – join your
classmates in building a giant amyloid fibril! Have a good look at how the amyloid
model is assembled – this mimics real fibril assembly processes – and answer the
questions associated with this part of the exercise.
GFP folding
GFP Template
GPCR folding instructions
GPCR Template Page 1
GPCR Template Page 2
Amyloid Fibril Template
 LABORATORY THREE
Enzyme Action and Inhibition
Before the Lab:
Thoroughly read through the laboratory and go through all resources on the Canvas page associated
with this laboratory.
Make sure you complete the pre-lab quiz for this laboratory on Canvas.

Learning Objectives for Laboratory 3:

At the end of this laboratory you should:

• apply your theoretical knowledge of enzyme kinetics to determine the KM and Vmax in your
experimental system

• develop skills to create graphs in Excel; you will be producing graphs including Lineweaver
Burke and Michalis-Menten Graphs

• apply your knowledge of enzyme inhibitors to determine the type of inhibition observed in
your experimental system.

• develop key skills for scientific note-taking including to accurately record and transcribe
data.
 LABORATORY RISK ASSESSMENT – LABORATORY 3 BIOSCI 106

Please take note of the following materials or procedures that we have

identified as a potential risk in this experiment. Risk minimisation controls are
listed. Full safety data sheets (SDS) for the listed chemicals are available
within the CANVAS Laboratory module/pages.
Note: other risks exist in the laboratory; the list below is not extensive nor complete.

HAZARD IDENTIFICATION – MATERIALS

Hazardous substance:
Hazard Classification: (tick as appropriate)
nitrocefin

Toxic

Acute
Health
Hazard

Chronic
Health
Hazard

Flammable

Oxidising

Corrosive

Other
 MINIMIZING RISK - MATERIALS
Hazard Materials Risk Controls:

Harmful if swallowed or inhaled. May cause skin and eye irritation.

Nitrocefin Pipette carefully and gently at arm’s length. Wear lab coat, gloves, and eye
protection to avoid skin and eye contact. Do not ingest.

HAZARD IDENTIFICATION – EQUIPMENT / PROCEDURES

Hazard Description and risk controls:
Spectrophotometer Please follow all instructions from your lab tutor and demonstrators when
working with the spectrophotometer equipment.
 Introduction
Mycobacterium tuberculosis (M. tuberculosis) is a pathogenic bacterium that is the causative
agent of tuberculosis (TB). It is thought that one in three people in the world are infected with
M. tuberculosis, and it is estimated that around 1.8 million people per year die from TB.
Worryingly, multiple-drug resistant (MDR) strains of the bacterium have emerged in recent
years, leading to a crucial need to develop new antibiotics that are effective against these
MDR strains.

Antibacterial drugs, or antibiotics, function in one of two ways; they can either kill the
bacteria directly (known as bactericidal) or inhibit the reproduction of bacteria (known as
bacteriostatic). To date, a number of antibiotics have been developed which will be discussed
in more detail in the “Nutrition and Antibiotics” section later in the semester. This laboratory
focuses on one class of antibiotics called the β–lactam antibiotics. The β–lactam class of
antibiotics, which include penicillin, inhibit the enzyme family transpeptidases (also known
as penicillin-binding proteins) that catalyse a step in the synthesis of bacterial cell walls.
Bacteria can acquire resistance to this group of antibiotics by expressing a class of enzymes
called β–lactamases.

β–lactamase
β–lactamase is an enzyme expressed by some bacteria that inhibits the function of β–lactam
antibiotics by cleaving the four-atom ring known as a β–lactam, see the reaction diagram below:

https://commons.wikimedia.org/wiki/File:Lactamase_Application_V.1.svg

The β-lactamase enzyme has been considered as a target for therapeutic intervention against
M. tuberculosis. It has been demonstrated that M. tuberculosis expresses β-lactamase.
Further, there is some evidence that administration of β-lactam antibiotics together with β-
lactamase inhibitors can be effective in the treatment of mice and humans infected with M.
tuberculosis. A number of β-lactamase inhibitors have been developed, including clavulanate
(or clavulanic acid). Clavulanate binds stably to β-lactamase and irreversibly inactivate the
enzyme.

Treatment with both the antibiotic and an inhibitor of the β-lactamase enzyme (for example
clavulanate) would allow β-lactam antibiotics to inhibit bacterial cell wall synthesis. This
would be bacteriocidal, preventing bacterial reproduction and therefore halting the spread of
disease.
 To inhibit an enzyme, it is important to first know the kinetics of the enzyme. To study the
kinetics of β–lactamase, we can use the chromogenic substrate such as nitrocefin. These
substrates contains a β–lactam ring (shown in the figure below) but does not have any known
antimicrobial properties. Hydrolysis of the β–lactam ring in nitrocefin results in a colour
change from yellow to red, which can be measured as a change in light absorbance.

Figure 2: Structure of nitrocefin (https://en.wikipedia.org/wiki/Nitrocefin)

The Measurement of Enzyme Action and Inhibition

As part of the wider research program, you are able to both undertake your own research, and
collaborate with other researchers. Your role in this research project will be to investigate the
KM and Vmax values for β–lactamase. You will then use data from other researchers (given to
you by your tutor) to determine the Ki value for the inhibition of the reaction by the inhibitor
clavulanate /clavulanic acid

Part A: The determination of KM and Vmax for the

enzyme β–lactamase

In part A you will:

1. Determine the rate of the enzyme reaction under different conditions of substrate
concentration;

2. Plot these rates in a Lineweaver–Burk plot;

3. Determine the values of KM and Vmax for the enzyme.

Each measurement will involve adding enzyme (β–lactamase) to a solution of the nitrocefin
substrate and observing the hydrolysis of substrate β–lactam ring at the wavelength of
486nm:

For this experiment, one student will perform the experiment and the other record the
absorbance readings and complete Table 1.
Method:

1. Turn on your spectrophotometer and set the wavelength to 486 nm.

2. Set up test tubes as per the table below.

Tube No 1 2 3 4 5 6

Nitrocefin conc (M) 0 25 50 75 100 125

MES buffer (L) 950 825 700 575 450 325

Nitrocefin 200 M (L) 0 125 250 375 500 625

Total Volume 950 L 950 L 950 L 950 L 950 L 950 L

3. Work with one tube at a time. First put Tube 1 into the spectrometer.

4. Zero the spectrometer.

Steps 5-9 requires you to work as quickly as possible.

5. Remove the cuvette from the spectrophotometer.

6. Add 50 μl of the enzyme (β–lactamase ).

7. Cover the cuvette with a piece of parafilm.

8. Invert the cuvette to ensure complete mixing of the enzyme and sample.

9. Place the cuvette back in the spectrophotometer and record the absorbance values at 486nm at
the times specified in Table 1. See figure B.

10. After the allotted time, remove the cell and repeat the experimental steps 3-10 as above
 Table 1: Time course data to determine reaction rate for the β-lactamase enzyme.

Time Tube 1 Abs Tube 2 Abs Tube 3 Abs Tube 4 Abs Tube 5 Abs Tube 6 Abs
(sec)
0
15
30
45
60
75
90
105
120

To determine the rate of the enzyme reaction for each experiment:

1. Open the excel spreadsheet in Canvas called “Enzyme kinetics Part A”. This can be found
under: BIOSCI106=>Modules=> Lab 3 => Excel Spreadsheets => “Enzyme kinetics Part A”

2. Save the spreadsheet onto your desktop and rename it specifically for your own group (do
NOT work directly from the server).

3. Enter the data from Table 1 and plot a graph of Abs vs time for each experiment. this is your
“Beta-lactamase Graph 1”

4. Using Beta-lactamase Graph 1, determine the slope (gradient) of the graph in the initial linear
portion of the graph, e.g. the first 30-45 seconds. This can be done by entering the time
period used, the absorbance at the start of the period and the absorbance at the end of the
period into the Excel spreadsheet. The spreadsheet will automatically calculate the Velocity
rate (Abs/min.)

5. Remembering Beer’s Law, you next need to convert the Velocity (rate Abs/min) to the rate of
product formation ie. V (nmol prod/min).

The extinction coefficient for nitrocefin is 20,500 M-1 cm-1.

Enter the extinction coefficient for the product and the volume of the tube into the Excel
spreadsheet. The rate V (nmol prod/min) will automatically be calculated for you. Under the
‘Michaelis-Menten Graph’ tab, you will find that V (nmol prod/min) is plotted against the
Substrate concentration (μM).
Determining KM and Vmax

You will now use your rate data (Table 2) to determine the KM and Vmax, using a Lineweaver-
Burk

plot (see lecture notes if you are unsure what this is).

Use the Excel file provided in Canvas to:

1. Plot a graph of 1/rate vs 1/[S] using the data from Table 2.

2. Remember that in your Lineweaver-Burk plot, the intercept on the y axis is 1/Vmax and the
intercept on the x axis is -1/KM. You can calculate the following values using the equation on
your graph.

3. Record these values below.

Vmax= Km= _________________________

Save your Lineweaver-Burk graph as a PDF, give it an appropriate caption and labels, then
upload it into your assignment quiz.

Part B: Determination of Ki for clavulanic acid

inhibition of β–lactamase .

In part B you will be given a set of results from your tutor. Use this data to:

1. Determine the rate of the enzyme reaction under different conditions of substrate
concentration and inhibitor concentration;

2. Plot these rates in Lineweaver–Burk plots to determine the nature of the inhibition and the
apparent KM and Vmax values at different inhibitor concentrations;

3. Use this data in a secondary plot to determine the Ki value for the inhibitor, clavulanic acid.
To determine the inhibition kinetics of β–lactamase:

1. Open the excel spreadsheet in Canvas called “Enzyme Kinetics Part B”. This can be found
under: BIOSCI106=>Modules=> Lab 3 => Excel Spreadsheets => “Enzyme kinetics Part B”.

Where tables or section numbers are indicated in bold, these refer to sections in the
spreadsheet.

2. Note that each tube contains differing concentrations of substrate and inhibitor clavulanic
acid.

3. Enter the data from your tutor into the table and plot a graph of Abs vs time for each
experiment. Your graph can be viewed under the β–lactamase Graph tab (remember to
enter the title and axes labels requested in 3).

4. Determine the rate of the reaction by calculating the gradient of each one of the lines. This
can be done by entering the time period used, the absorbance at the start of the period and the
absorbance at the end of the period into the Excel spreadsheet. The spreadsheet will
automatically calculate the Velocity rate (Abs/min.)

5. Remembering Beer’s Law, you next need to convert the Velocity (rate Abs/min) to the rate of
product formation ie. V (Δmol prod/min). The extinction coefficient for the product is 20,500
M-1 cm-1. Enter the extinction coefficient for the product and the volume of the tube into the
Excel spreadsheet. The rate V (nmol prod/min) will automatically be calculated for you.

6. The values for 1/[S] and 1/v should calculate automatically. These values will then be used to
draw Lineweaver Burk plot in the “Lineweaver-Burk plot” tab.

7. In the spreadsheet, enter the [I] and slope values into the blue boxes in 9.

8. Click on the “Secondary Plot” tab and save the graph as a PDF (remember to enter the
information requested in 10). Hand this in as an upload to your assignment quiz.

LAB 3 CLEAN UP INSTRUCTIONS

• Pay attention to your tutor for clean-up information.

• Please complete the assignment sheet and hand it in at the back of the lab.
 LABORATORY FOUR
How do we design liposomes for drug delivery?

Before the Lab:

Thoroughly read through the laboratory and go through all resources on the Canvas page associated
with this laboratory.
Make sure you complete the pre-lab quiz for this laboratory on Canvas.

Learning Objectives for Laboratory 4:

In this laboratory you will:

1. apply your theoretical knowledge of lipid structure and assembly to predict experimental
results

2. accurately perform experiments to investigate conditions affecting liposome structural

stability and critically assess the experimental results.

3. develop key skills for accurately performing a scientific experiment.

4. learn to accurately record experimental results and observations.

5. apply your knowledge of liposomal structure and potential modifications to design a drug
delivery vehicle for different types of drugs.
 LABORATORY RISK ASSESSMENT – LABORATORY 4 BIOSCI 106
Please take note of the following materials or procedures that we have
identified as a potential risk in this experiment. Risk minimisation controls are
listed. Full safety data sheets (SDS) for the listed chemicals are available
within the CANVAS Laboratory module/pages.
Note: other risks exist in the laboratory; the list below is not extensive nor complete.

HAZARD IDENTIFICATION – MATERIALS

Hazard Materials Risk Controls:

No hazardous chemicals have been identified in this experiment. Take
None standard precautions and use personal protective equipment as normal.

HAZARD IDENTIFICATION – EQUIPMENT / PROCEDURES

Hazard Description and risk controls:

UV Torches Do not shine the torches in your own eyes or anyone else’s eyes. Only
shine the torch at the samples as indicated in the experiment.
 Introduction
Lipids are critical biological molecules that are features, in particular, of biological
compartmentalisation in all living organisms. They form lipid bilayers that separate cells
from each other and separate organelles within cells. They can also spontaneously assemble
in aqueous buffer to form closed bilayer vesicles called liposomes. Since their discovery,
liposomes have been used as drug-delivery vehicles because they can transport both
lipophilic and hydrophilic compounds into cells. Hydrophobic molecules associate with the
lipid tails in the bilayer while hydrophilic drugs are loaded into the interior of the vesicle.
There are many liposomal-based therapeutics in current use and more in clinical
development. These include drugs to treat pain management (morphine sulfate), meningitis
(Cytarabine), and cancers, including leukaemia, multiple myeloma, breast cancer, and lung
cancer.

You may well have been treated recently with a liposome! The RNA-based COVID-19
vaccines are composed of a modified liposomal structure that delivered the SARS-CoV-2
spike protein-encoding RNA inside your cells! More on this later…

Figure 1: Microscopy of liposomal preparations (100x) a) uncoloured liposomes,

b) liposomes coloured by hydrophilic dye, and c) liposomes coloured by hydrophobic dye. A
conceptualised liposome diagram is included top right. Adapted from source: Abu-Much R,
Najami N, Hugerat S, et al. Simple Method for the Demonstration of Drug-Loaded Nano-
Liposomes. Int J Chem Sci. 2017;15(4):209.

In this laboratory we will investigate the stability of liposomes in a range of conditions (Part
A) and apply our knowledge of liposomal characteristics to conceptually design vehicles for
drug delivery (Part B).
Lipid preparation

Liposomes range from 20 to ~1000 nm in diameter, with some control of the size during
production. The method used in this laboratory for liposome preparation is shown in the
schematic below:

Source: Liu et al, (2021) Targeted liposomal drug delivery: a nanoscience and biophysical
perspective. Nanoscale Horiz., 6, 78

Once the liposomes are formed, the liposome solution undergoes extrusion to produce a
uniform size. In this technique, the liposome suspension is passed through a membrane filter
of a defined pore size. The equipment for this is shown below:

Source: Abu-Much Ret al. Simple Method for the Demonstration of Drug-Loaded

Nano-Liposomes. Int J Chem Sci. 2017;15(4):209

 Part A: Liposomal structure and stability
Liposomes are composed of at least one type of
phospholipid, like phophatidylcholine, and are often
stabilised by other components, for example
cholesterol. We have used a soy phospholipid extract to
make our liposomes – these lipids have a high
proportion of unsaturated lipid tails making the liposomes overly fluid and
a little leaky. Stable lipid bilayers are fluid, but not too fluid! We have
added ~20% cholesterol to help stabilise the structure and retain the
fluorescent dye. Cholesterol can be thought of as a buffering molecule in
the membranes of animal cells that prevents abrupt changes in membrane
fluidity over a range of temperatures. Our liposomes prepared for this
laboratory are exposed to a range of temperatures from 4-25 °C. Liposomal
stability can be affected by the lipid composition and environmental
conditions including the surrounding salinity and the presence/absence of detergent or other
disrupting agents. To ensure the utility of liposome as drug-delivery vehicles, conditions
Why do
they kink?
Figure. The impact of unsaturated and
saturated phospholipid tails on bilayer
fluidity. Cholesterol (bottom) stabilises
bilayer structure and helps retain dyes
and drugs inside liposomes. Rank the
phosphatidylcholine lipids below in
order from 1-3 for their fluidity-
inducing effect. Which of the lipids is
pictured top left?

during preparation must be optimised for their stability.

How do we study liposome stability?

To study the stability of liposomes, we need a method to detect when liposomes are intact
and when their structure has been disrupted. To do this, a fluorescent dye (5,6-
carboxyfluorescein) is added to the hydration step of the synthesis. During the spontaneous
assembly of the liposomes, the dye is sequestered (entrapped) in the interior of the liposomes.
Excess dye (that is not sequestered inside the liposomes) is purified away using size
exclusion chromatography (this is the first experiment in this laboratory).

Following size exclusion chromatography, intact liposomes will exhibit no fluorescence (due
to dye quenching); if the membrane is disrupted, the dye will leak out of the liposomes will
dilute, and then the solution will fluoresce. The amount of fluorescence indicates the relative
amount of disruption of the lipid membranes – see the image below:
Increasing membrane
disruption
Intact Completely disrupted
Liposomes liposomes

Source: Seu, K.J. (2015) J. Chem. Educ. 2015, 92, 1522−1525

Experiment 1: Purification of the liposomes by size
exclusion chromatography (Demonstration)
Prior to the lab, the liposomes were assembled up to the extrusion step in the purification
schematic above. Your lab tutor will demonstrate the purification of formed liposomes away
from the excess dye using the method of size exclusion chromatography. You can help by
telling your tutor when to collect the yellow/orange-coloured liposomes as they exit the
size exclusion column.* Don't collect any of the dye or the experiment is ruined!

Method:

1. First, place a size exclusion column in a retort stand and run ~20 mL of buffer through the
column to equilibrate it.
2. Gently pipette the liposome solution on the top of the column and allow it to move into the
size exclusion beads – buffer will drip from the column.
3. Add buffer to the top of the column to elute the liposomes. You should see the liposomes
separate from the excess dye.
4. The eluent is collected in a tube taking care not to include any dye. This is the liposome
sample you will use in your experiments.

As the sample moves through the column, record your observations.

Observations:
What colour was the solution when first added to the column? What colour(s) were
present when the sample started to move through the column?

What species elutes first? Why is this? What colour is the eluent?
Why do you see different colours in the column?

5. The eluent is collected in a tube taking care not to include any dye. This is the liposome
sample you will use in your experiment.
6. Following the demonstration, the column will be cleaned and prepared for the next
purification (the technical team will do this part). Take note of the colour of concentrated,
diluted 5,6-carboxyfluoresceine dye.

7. One member of the group will then take an empty Eppendorf tube to the to the tutor to collect
an aliquot of the purified liposomal solution, the volume of the liposomal solution is:

--------------------------- µL
Experiment 2: The effect of osmotic pressure on
liposomal structure

In this experiment, you will determine the effect of increasing osmotic pressure on the
liposomal structure and stability. The liposomes will be exposed to a high salt solution (an
isotonic solution because your liposomes were made in high salt buffer) and a low salt
solution (hypotonic solution). You will then determine whether the concentration of salt
within the environment of the liposome has an effect on the liposomal structure.

1. Pipette 200 µL of high salt buffer into a test tube – this is your high salt buffer negative
control (Tube 1 – make sure you label your tubes).

2. Pipette 200 µL of low salt buffer into another test tube – a second negative control (Tube 2).

3. Pipette 180 µL of high salt buffer into a test tube and add to this 20 µL of liposomes (Tube
3).

4. Pipette 180 µL of low salt buffer into a test tube and add to this 20 µL of liposomes (Tube 4).

5. Pipette 180 µL of high salt buffer into a test tube and add to this 20 µL of liposomes and then
20 µL of Triton-X100 detergent. This is your fully disrupted positive control (Tube 5).

6. Using your blacklight torch, shine the light through the bottom of the test tubes. Compare the
relative fluorescence intensities of each tube – record your observations below. Hint: it is
easiest to compare two samples together and swapping the light back and forth. Take your
samples into the dark room and use the lamps there to view the samples – one person in your
group can first take off their gloves, wash their hands, and then use a cell phone to visualise
the fluorescence – it works better than the naked eye.

Observations:
Do all the tubes show the same fluorescence intensity? Why/why not?
Experiment 3: The effect of detergents on liposomal
structure

In this experiment, you will determine the effect of hand washes (detergent) and hand
sanitisers (alcohol) on the liposome structure. You have already seen that a highly pure, lab-
grade detergent (Trion-X100) can disrupt liposomes and spill the fluorescent dye – will
consumer hand washes and sanitisers do the same job?

1. Keep your negative and positive control solutions from your previous experiment for
comparison.

2. There are a number of common household detergents at the back of the laboratory in small
jars and one sample of hand sanitiser. These detergents have been diluted to 50% in high salt
buffer so you can pipette them easily. The hand sanitiser is undiluted. As a group, choose two
samples to test within your group – share you results and enter them in the table below.

Names of common household detergent or sanitiser your group will test:

#1_______________________________

#2_______________________________

Look at the ingredient list, can you identify the detergent molecules in your sample?

Chemical name of detergent #1: ___________________________________________

Chemical name of detergent #2: ___________________________________________

 Use the table below to predict what the results will be?

3. Among the groups in the lab, there will be a number of different detergents tested; how
effective will each detergent be? How effective is the detergent that you use at home (if it is
present)? Use the table below to predict what the results will be?

Group Detergent Prediction:

Number: used:

Tutor Untreated No detergent was added; I predict the liposomes will

remain intact and no florescence will be observed.

Tutor Triton X- A purified/concentrated detergent was added; I

100 predict the liposomes will all lyse/burst/explode(!)
and the maximum florescence will be observed.
(10%
solution)

9
1. Pipette 180 µL of high salt buffer into a test tube and add to this 20 µL of liposomes. Finally
add 20 µL of either hand wash or sanitiser to this and swirl to mix. Visualise your results
again using the blacklight torches or lamps and record your observations. Share results (and
discuss them) within your group – and with other groups if you wish – we’ll get you to report
back.

Observations:
Do all the tubes fluoresce? Why/why not?

Which detergent has produced the most fluorescence? Did you predict this?
 Part B: Liposomes as drug
delivery vehicles
We have looked at the stability of liposomes in various solutions in the previous experiments
under laboratory conditions that could be used during the preparation of liposomes. However,
if liposomes are used as drug carriers in the body, how stable are they in physiological
conditions?

In vivo, conventional liposomes are rapidly cleared from the plasma through the actions of
the immune system, which limits their capability to deliver drugs to target tissues. Our
understanding of lipid biochemistry has allowed scientists to purposefully design liposomes
with improved characteristics.* These modified liposomes are more accurately termed lipid
nanoparticles (LNPs). These novel LNP can be engineered by:

• the addition of extra components including PEG (polyethylene glycol) molecules as a coating
on the outside of the LNP. This allows the LNP to evade the immune system and so extends
the serum half-life of these so-called “stealth liposomes” .

• including protein ligands in the liposome membrane to ensure site-specific delivery. These
ligands bind to receptors on the target tissue, for example, cancer cells, for directed delivery
of the drug cargo.

• addition of certain cationic lipids to incorporate, sequester, and stabilise nucleic acids, like
mRNA, and improve their resistance to enzymatic degradation. These LNPs also promotes
fusion of the liposome with the cell membrane to ensure delivery of the nucleic acid to the
cytoplasm.

• the inclusion of molecules that stabilise the lipids in the LNP structure, for example,
cholesterol.

*Once we understand the biochemistry, we can engineer more advanced drugs/drug delivery
systems to improve clinical outcomes. This will apply to the delivery of antibiotics which you
will learn in upcoming lectures.
 The diagram below indicates some modifications that have been introduced to liposomes. In
their simplest form, liposomes (shown in A) are composed of a simple lipid bilayer of
amphipathic molecules. These liposomes can transport both lipophilic and hydrophilic drugs
by trapping them in different parts of the liposome (B). Modifications such as the addition of
a ligand (C) or PEG (D) can improve delivery outcomes – these more complex delivery
vehicles are then termed LNPs.

Source: Tenchov et al ACS Nano 2021, 15, 11, 16982–17015. Publication Date:June 28,
2021. https://doi.org/10.1021/acsnano.1c04996

When tasked with designing a liposomal delivery system for humans, there are a few key
questions to ask:
• Where will the drug be included into the liposome, the lipophilic membrane or the

hydrophilic core?

• Does the lipid membrane need modification to capture the drug load?

• How can you stabilise the LNP to maximise the half-life in the body?

• Will the drug need to be targeted to a specific tissue or cell type?

Use these prompts to help you design LNPs for your assignment questions.

LAB FOUR CLEAN UP

INSTRUCTIONS

• Follow clean-up instructions given by your tutor.

• Please complete the assignment sheet and hand it in at the back of the lab.

Safety Check: do not remove any samples or reagents from the laboratory
 LABORATORY FIVE
Signal Transduction
The format of this laboratory will be different to those you have attended previously. The staff (tutor
and demonstrators) will be there to assist you but the discussions about the research in the references
you have been given and the results in the report will be initiated and continued by you, the students.
You are encouraged to work with your fellow students to complete the tasks.

To be involved in the lab, it is crucial that you read the laboratory guide thoroughly.
This lab is run as a tutorial; no hazardous equipment is used.
Please complete the prelab quiz for this laboratory on Canvas.

Learning Outcomes:

At the end of this laboratory you should be able to:

• apply your knowledge of signal transduction, protein structure and lipid metabolism to your
experimental system/results.

• critically assess and integrate results from multiple experimental data sets to draw conclusive
results.

• further develop your team-work skills by working collaboratively and effectively

communicating with your peers

• critically assess information sources for scientific research

• critically assess and interpret published scientific findings

In the laboratory session, you will be given information and data to analyse, which relies on
careful reading of all of the given information. Use your understanding of the experiments
and the results of the experiments as well as the group discussions to answer the assignment
questions.
 Introduction
Obesity is the result of an imbalance between energy intake and energy expenditure;
worldwide it is a rapidly growing public health problem. In New Zealand, almost one in three
adults are obese whilst one in nine children (defined as being younger than 14) are obese.
This corresponds to 1.24 million adults who are obese (Annual Update of Key Results
2019/20: New Zealand Health Survey). Obesity is a complex disorder that involves both
genetic and environmental factors. The increased burden of obesity on the health sector has
prompted researchers to search for signalling molecules or proteins involved in metabolism
that may be actively pursued as drug targets.

Mutations in melanocortin-4 receptor (MC4R) are the most common form of monogenic
obesity and can contribute to polygenic obesity. MC4R plays an important role in the control
of energy homeostasis in a variety of species from fishes to humans. The receptor is a seven-
transmembrane G-protein coupled receptor that is primarily expressed in the brain including
the hypothalamus. MC4R is activated by endogenous ligand α-melanocyte-stimulating
hormone (α -MSH) and once activated, MC4R couples to the Gs protein and activates
adenylate cyclase. The activation of adenylate cyclase results in increased cAMP production
which then increases the activity of protein kinase A (PKA), see figure 1. The result of
activation of the MC4R receptor is a decrease of food intake and an increase in energy
expenditure.

More than 150 mutations have been identified in patient cohorts, the effect of these mutations
are described below. But first, we will review the nature of genetic mutations.

Figure 1: MC4R activation by -MSH (adapted from Berg et al., Biochemistry,

8th Edition, WH Freeman and Co, Figure 14.6).

Mutations in DNA sequence – a reminder

A change in the primary sequence of a protein typically results from a mutation in the DNA
sequence which encodes that protein. A change in, insertion of or deletion of a single
nucleotide or a number of nucleotides in the DNA sequence will result in a change of the
amino acid sequence of the protein. Depending on the number of nucleotides that have
changed, this can result in the change of a single amino acid or a number of amino acids in
the protein sequence.

Even the change of a single amino acid in the protein sequence can have significant results on
protein structure and function. A change in the amino acid sequence can alter the way in
which the protein folds to adopt its secondary or tertiary structure; a change in the 3D-shape
of a protein can substantially affect the function of the protein. Specifically, if the mutation
results in a change in amino acid that has different properties to the wildtype amino acid (for
example, a hydrophobic amino acid changes to a hydrophilic amino acid), this will change
the final structure of that protein.

Alternatively, even if the protein structure does not change, a point mutation can alter the
function of the protein if the point mutation occurs in a functional region of the protein – that
is, if the point mutation occurs in for example the site where the ligand binds to the protein,
the ability of the receptor may lose its ability to bind to its ligand which will then prevent
signalling in the protein. Mutations in the DNA may also result in a truncated (or incomplete)
protein being produced if a point mutation in the DNA sequence results in the introduction of
a premature stop codon in the transcribed RNA. The truncated protein will typically be non-
functional and thus the mutation has resulted in a non-functional protein and effectively
stopped signal transduction through this pathway. This means that the hormone is less able to
signal and the clinical outcome mimics a hormone deficiency. If you require further revision
about genetic mutations, please see ref Campbell Biology – Global Edition, 10 edition Reece
et al Chapter 17 p411-413.
Outcomes of the mutations in the MC4R receptor protein

There are four different known effects that a mutation in the MC4R gene may have on the
function of the MC4R protein:

• Effect 1: Lack of or a decrease in the expression of MC4R protein: The mutation

may result in defective transcription and/or translation, or increased rate of protein
degradation. This means that the quantity of MC4R protein expressed or manufactured in the
cell will be decreased. This would result in a lower quantity of MC4R proteins able to
function at the cell surface (referred to as a lower expression level).

• Effect 2: MC4R is retained in the ER: mutations in the MC4R may result in the
protein being incorrectly folded (misfolded). The misfolded protein is detected by the cell and
retained in the ER and therefore are not targeted to the plasma membrane.

• Effect 3: Binding defective mutants: mutations in MC4R may occur in the part of the
protein where ligands would ordinarily bind. This type of mutant MC4R protein will be
targeted to the plasma membrane but once there, it is unable to bind to the ligand and
therefore unable to induce cell signalling. The mutation may result in a decreased affinity for
the ligand or the mutation may abolish binding of the ligand completely.

• Effect 4: Signalling defective mutants: mutations in MC4R may occur in the part of
the protein where signal transduction protein (or G protein) would ordinarily interact with the
protein. These mutants are targeted to the plasma membrane and are able to bind to the ligand
but are unable to induce signalling in the cell.

For a summary of these outcomes, please refer to the diagram below:

Figure 2: Schematic illustrating the effects of mutations on MC4R function

How to determine what the effect of a mutation is MC4R protein function

The presence of a mutation in MC4R is not always the cause of obesity in a patient, there are
many factors that can contribute to the development of the condition. However, once a
mutation is discovered in the genome of an individual, there are a number of experiments that
should be performed in order to determine whether the patient’s mutant MC4R protein has an
impaired function. If a series of experiments is performed, it is possible to determine the
exact effect of the mutation on the protein’s function.

Ligand binding typically results in the activation of MC4R. The activated MC4R receptor
then interacts with the Gs which leads to an increase in intracellular cAMP levels. An assay
that measures the levels of intracellular cAMP can be performed. This assay would compare
to the amount of cAMP produced by activation of the mutant MC4R to that of the wildtype
MC4R and determine whether there is any difference in the amount of cAMP levels
produced. This assay would indicate whether the mutation in MC4R has affected its ability to
signal relative to the wildtype receptor.

If the level of signalling is different compared to the wild type MC4R receptor signalling,
there are additional experiments required to determine the cause of this difference. Ligand-
binding experiments can determine whether the mutation has affected the ability of the ligand
to bind. If there is weakened binding of the ligand to the receptor, this would explain the
observed change in cAMP levels.

If there is no signalling and no receptor binding, a possible reason could be that the protein is
not targeted to the cell surface. To study this, MC4R fused to GFP (green florescent protein)
can be analysed to determine the location of the protein within a cell. By comparing the
location of the protein with known cellular locations (the cytoplasm, ER or plasma
membrane), it is possible to determine whether the protein has been correctly targeted to the
plasma membrane or if it is retained in the ER where it cannot bind to its ligand

Setting the scene:

You work for Causam Genetics, a private institute that undertakes both clinical and research
testing for genetic mutations. You will be given further information in your lab. Please use
your understanding of lecture content in across multiple topics, and the power of analysis to
answer all of the assessed questions.
 Results tables (for use in the lab):

Refer to the results in Refer to the results in Refer to the results in

Section B: Section C: Section D:
What is the cellular What is the relative ligand- What is the relative signal
location of the GFP binding efficiency of the transduction of the mutant
mutant protein? mutant compared to wt? compared to wt?

Patient
1

Patient
2

Patient
3

Table 1: Overall analysis of patient samples

Patient Effect type of the Explanation

Sample mutation on receptor
function

Patient 1

Patient 2

Patient 3

Table 2: Summary of patient MC4R mutation effects

LAB 5 CLEAN UP INSTRUCTIONS

• Pay attention to your tutor for clean-up information.

• Please complete the assignment sheet and hand it in at the back of the lab.
 LABORATORY SIX
Antibiotic Resistance in E. coli
Before the Lab:
Thoroughly read through the laboratory, read Appendix 3 and go through all resources on the Canvas
page associated with this laboratory.
Make sure you complete the pre-lab quiz for this laboratory on Canvas.

The design of the experiments in this laboratory practical and the methods used are devised
to simulate antibiotic resistance in bacteria and consequently eliminate risk for the individual.

This laboratory will be conducted under PC1 containment. The conditions associated with
PC1 containment are clearly Laboratory Health and Safety Information at the beginning of
this laboratory guide and will be reinforced in the introductory talk of the laboratory session
given by supervising staff member. This laboratory session has received approval from the
Environmental Risk Management Authority (ERMA) under the Hazardous Substances and
New Organisms Act 1996 (ERMA Approval: GM0010-UA008).

Learning Outcomes:

At the end of this laboratory you should be able to:

• analyse DNA samples using the technique of agarose gel electrophoresis..

• analyse antibiotic resistance using the Kirby-Bauer Technique.

• critically assess and integrate results from multiple experimental data sets to draw conclusive
results.

• research traditional methods to provide context and history of the method and current
scientific knowledge/research of the benefits of the traditional medicine.
 LABORATORY RISK ASSESSMENT – LABORATORY 6 BIOSCI 106

Please take note of the following materials or procedures that we have

identified as a potential risk in this experiment. Risk minimisation controls are
listed. Full safety data sheets (SDS) for the listed chemicals are available
within the CANVAS Laboratory module/pages.
Note: other risks exist in the laboratory; the list below is not extensive nor complete.

HAZARD IDENTIFICATION – MATERIALS

Hazard Materials Risk Controls:

No hazardous chemicals have been identified in this experiment. Take
None standard precautions and use personal protective equipment as normal.

HAZARD IDENTIFICATION – EQUIPMENT / PROCEDURES

Hazard Description and risk controls:

Please follow all instructions from your lab tutor and demonstrators when
Electrophoresis working with the electrophoresis equipment
equipment

Please follow all instructions from your lab tutor and demonstrators when
Agar plates working with agar plates; ensure the plates are kept closed at all times and
are left on the laboratory bench when not used for laboratory purposes.
 Introduction

Antibiotic resistance

The widespread use of antibiotics has resulted in adaptation/evolution by the bacteria to resist
the effects of the drugs thus rendering the drugs unable to stop microbial growth – i.e. the
development of antibiotic resistance. Antibiotic-resistant infections require alternative
medicines or higher doses of current drugs – both approaches increase the toxicity of the
drugs and are more costly, putting huge strain on the medical community resources. Of
particular concern is the development of multi-resistant strains which exhibit resistance to
more than one type of antibiotic. Certain bacterial strains are resistant to nearly all available
antibiotics. This scenario- based laboratory will investigate whether bacterial samples taken
from three (hypothetical) patients are resistant to two antibiotics: penicillin and
chloramphenicol.

β-lactamase and resistance to penicillin

The β–lactam antibiotic group includes all antibiotics that contain a β–lactam ring in their
molecular structure. These antibiotics include penicillin and structural analogues such as
ampicillin, which is used in this laboratory. Penicillin inhibits bacterial cell wall biosynthesis
(for more detail, please refer to your lecture notes from the M&A section of your course
guide).

Figure 1: The structure of penicillin indicating the active site of the -lactam ring (Berg et al.,
Biochemistry, 8th Edition, WH Freeman and Co, Figure 8.26)
 Resistance to ampicillin and other β-lactam antibiotics is mainly due to inactivation by
β-lactamase enzymes. Members of this large enzyme family cleave the amide bond in the β–
lactam ring rendering the β–lactam antibiotics harmless to these bacteria. β–lactamases are
encoded by genes located on a plasmid in the cytoplasm of the bacterial cell. β-lactamases are
~286 amino acids in length and are secreted into the periplasmic space of the bacteria where
they inactivate β–lactam antibiotics.

Figure 2: β -lactamase cleaves the amide bond of penicillin

(https://commons.wikimedia.org/wiki/File:Lactamase_Application_V.1.svg).

Chloramphenicol acetyltransferase and resistance to chloramphenical

Chloramphenicol is an antibiotic used in the treatment of bacterial infections such as

meningitis, cholera and ocular infections. It is a broad spectrum antibiotic that inhibits
bacterial protein synthesis by inhibiting the peptidyl transferase activity of the (bacterial)
ribosome. The use of chloramphenicol is generally only prescribed when other antibiotics
are ineffective as it has numerous side-effects including nausea, diarrhoea and blood
dyscrasia (bone marrow suppression).

Figure 3: Structure of Chloramphenicol

(https:// upload.wikimedia.org/wikipedia/commons/7/7d/ Chloramphenicol-2D-skeletal.png).

 Resistance to chloramphenicol is mediated by the bacterial enzyme chloramphenicol
acetyltransferase (CAT). CAT transfers an acetyl group from acetyl-CoA to chloramphenicol.
The covalently attached acetyl group prevents chloramphenicol from binding to the
ribosomes thus stopping the drug from inhibiting protein synthesis. The gene for CAT is also
located on a bacterial plasmid in the cytoplasm and produces a ~213 amino acid protein.

Figure 4: CAT inactivation of chloramphenicol (Rottig, A and Steinbuchel, A, (2013)

Acyltransferases in bacteria. Microbiology and molecular biology reviews 77 (2) p277-321).

The Kirby-Bauer antibiotic testing method (also known as disc diffusion

antibiotic sensitivity testing)

One method to analyse bacterial resistance to antibiotics is to grow cultures of the bacterium
of interest on agar plates which have localised zones where an antibiotic is present. In this
method, paper discs are soaked in a solution of antibiotic and then placed on the agar. The
antibiotic will diffuse out of the disc into a small area of the agar plate around the disc – this
ensures that there is a limited area in which the antibiotic is present in the agar, with the
potential to inhibit bacterial growth. The bacterial culture is then spread onto the plate and
incubated overnight. Following incubation, the growth of the bacteria on the plate is analysed
to determine whether the bacterial strain was able to grow in the presence of the antibiotic.

If a bacterial strain does NOT have a gene that confers resistance to an antibiotic, then it will
NOT be resistant to antibiotic present on the disc and it will NOT grow in the limited area of
the plate around the disc where the antibiotic is present. This results in a ring or area around
the disc containing no bacterial growth – the so-called zone of inhibition (ZOI). This is
illustrated in figure 5:

Figure 5: Agar plate with bacterium that is inhibited by the antibiotic A

In contrast, if the bacterial strain growing on the agar plate HAS a gene that confers
resistance to antibiotic A, then the bacteria IS able grow in the presence of the antibiotic.

Therefore, the bacterial strain will grow all over the plate and will not be affected by the
presence of the antibiotic. Refer to figure 6:

Figure 6: Agar plate with bacterium that is resistant to antibiotic A

Multiple discs (with different antibiotics) can be placed on a single agar plate. Consider the
following example: an agar plate is set up with two discs which have been soaked in
antibiotic A and antibiotic C and a bacteria strain is spread over the plate and allowed to grow
overnight. The bacterial culture is observed to grow around the disc soaked in antibiotic A
but it does not grow in an area around the disc soaked in antibiotic C.

This indicates that this bacterial strain is resistant to antibiotic A (this antibiotic does not
inhibit growth) whilst it is inhibited by antibiotic C as there is no bacterial growth around the
disc and a large zone of inhibition around the antibiotic C-soaked disc, refer to figure 7.

Figure 7: Agar plate with bacterium that is resistant to antibiotic A but is

inhibited by antibiotic C
 Laboratory 6: Antibiotic Resistance: A scenario*.
*This scenario is purely fictional – all patients, treatments & locations are given only for the
purposes of this laboratory & is not representative of the treatment given at a New Zealand
hospital.

You are a researcher in the clinical laboratory that analyses patient samples from the local
hospital, Infaustus Hospital. Three patients have been admitted with E. coli (bacterial)
infections and are not responding to the initial treatment. To ensure that an effective treatment
regime is given to the individual patient, the E. coli is first tested to determine whether it has
resistance to any antibiotics. This allows for an individualised treatment; the patient will be
prescribed an antibiotic that will be effective against the strain of bacteria that they have.

Three individual patient’s samples (Patient 1, Patient 2 and Patient 3) need to be tested and
compared to three E. Coli control strains (Strains X-Z; strains of E. coli for which the
resistance to antibiotics is known).

The E. coli control strains are labelled as follows:

• Strain X – this E. coli strain does not contain any plasmids encoding enzymes that
result in resistance to ampicillin or chloramphenical.

• Strain Y – this E. coli strain contains a plasmid expressing the bacterial gene for β–
lactamase that confers resistance to ampicillin.

• Strain Z - this E. coli strain contains a plasmid expressing the bacterial gene for CAT
that confers resistance to chloramphenicol.
Analysis of Patient Samples
In this lab you will analyse the E. coli samples from the patients using two different
techniques:

Part 1: analysis of plasmid DNA isolated from bacteria samples to determine

whether they contain plasmids expressing antibiotic resistance genes.

Your colleague has purified the plasmid DNA from each of the patients and the control E.
Coli strains and digested the plasmids using DNA restriction enzymes (restriction
endonucleases). The digestion of the DNA will release a “signature” DNA fragment
reflecting the presence of an antibiotic resistance gene (if the bacteria contains a resistance
gene).

You will analyse this plasmid DNA digest on an agarose gel. If the plasmid present in the
patient sample does contain a gene for an enzyme that confers antibiotic resistance, it will be
visible as a distinct band of DNA of known size (see the introduction to this laboratory).

To determine the type of antibiotic resistance, take note of the approximate size of the DNA
fragment and compare this to the expected size of the gene for β-lactamase or
chloramphenicol acyltransferase. You can do this by comparing the DNA fragment in the
patient sample with the DNA fragment from the control bacterial strains as well as comparing
the fragments to a DNA molecular weight standard (called a DNA ladder).
Part 2: comparison of the growth of bacteria in the presence of antibiotic to
confirm resistance of the bacteria to the indicated antibiotic.

Your colleague has also prepared a series of cultures of each of the patient samples or control
strains grown on agar plates containing antibiotic-soaked discs. There are 6 plates (one for
each of the patient samples and one of each of the bacterial controls). On each plate are 2
discs which have been soaked in either ampicillin (A) OR chloramphenicol (C). You will
analyse the plates and compare the growth of the bacterial sample in the presence of the
antibiotic discs.

Part 1: Analysis of plasmid DNA from

patient samples
The process for DNA plasmid sample preparation is summarised in the schematic figure 8
below.

Figure 8: Schematic of the steps taken to analyse plasmid DNA for patient samples; DNA size is given in base
pairs (bp) (figure by J. McIntosh using clipart from Literary R&R: MIA for patient and clipart for the flask).
How can we use the DNA ladder to estimate the DNA fragment size?

The DNA ladder is used to give an indication of the approximate size of the DNA fragments
of the samples on the agarose gel. In this laboratory, the DNA ladder has 3 bands, the size of
these bands are: 330 bp, 700 bp and 1670 bp (see figure 8 part 5). To determine the size of
the DNA fragments in your patient samples, carefully compare the location of the fragments
to the bands seen in the ladder. To guide you, there are two worked examples below, to
indicate how you would analyse the DNA fragment in Sample 2 (example 1) and Patient P
(example 2).

Example 1: This is strain Y which is known to have antibiotic resistance to ampicillin. The
DNA fragment is between 770 bp and 1670 bp long. This strain contains the gene for β-
lactamase, so we can predict that the DNA fragment on the gel would be approx 860 bp,
which corresponds to the known size of the β-lactamase gene. The large fragment (plasmid
backbone) observed in this sample (that is >1670 bp) is the remaining DNA plasmid, once the
DNA fragment (encoding β-lactamase) has been removed.

Example 2: The fragment size of patient P appears to be the same as that of Strain Z and can
be estimated to be between 330 bp and 700 bp. Strain Z is known to have resistance to
chloramphenicol and therefore must express the CAT gene. The DNA fragment observed in
Strain Z and patient P is approximately the expected size for the CAT gene; therefore, we can
conclude that the plasmid from patient P’s bacterial sample contains the CAT gene for
antibiotic resistance. The large fragment (plasmid backbone) observed in this sample (that is
>1670 bp) is the remaining DNA plasmid, once the DNA fragment (encoding CAT) has been
removed.

Experimental Methods:
Equipment:

1. Gel chamber including comb plastic bulb pipette

2. 500 ml Schott bottle of buffer labelled “TBE Buffer”

3. 50 ml Schott bottle of agarose (this is in the water bath) labelled “0.9% agarose”. 7 tubes of
DNA (labelled 1-7)

4. Micropipette and tips

5. 1 tube of loading dye (orange coloured dye in an Eppendorf tube)

6. Gloves - dispose of gloves in biohazard bag ONLY

Part 1.1 Pouring your agarose gel:

Put gloves on prior to starting.

1. Familiarise yourself with the parts of the gel chamber – figure 9 below indicates all the parts
that you should be familiar with.

Figure 9: Chamber for electrophoresis

2. Place the gel base in the chamber as illustrated in figure 10 image 2. Ensure that the gel base
is positioned with the seals (red ends) hard up against the green sides of the gel chamber. This
ensures that your agarose will not leak and you will form a viable gel.

3. Place the comb in the gel base at the end position as shown in figure 10 image 2.

4. Get the Schott bottle containing the dissolved agarose solution from the water bath
(CAREFUL: the water bath is HOT!). Gently swirl the bottle (before removing the lid) to
ensure a uniform solution. Don’t swirl too vigorously as you will create air bubbles in the
agar which will result in bubbles in your gel.

Pour ALL the agarose into the gel base.

5. Allow the gel to set for at least 15 minutes. During this time, the lab tutor will introduce the
lab to you.
6. Once the gel is set, rotate the gel base containing the gel so that the red ends face the white
ends of the gel chamber. The holes (called wells) should now be positioned above the black
stripe – as in Figure 10 image 3.

Figure 10: Preparation of an agarose gel

7. Pour the buffer in the labelled Schott bottle to just cover the gel, see figure 10 image 4. Please
ensure that the liquid does not go beyond the “max” line. The gel should be fully covered by
(or submersed in) the liquid.

8. Remove the comb by GENTLY pulling the comb directly upwards (see figure 10 image 4) –
the wells that are left in the gel are the holes where you will load your DNA samples.

9. Prior to loading your samples, please wash the wells of the DNA gel out with the buffer that
is in the gel chamber) using the plastic bulb pipette. Your demonstrator will show you how to
do this.
Part 1.2: Loading of the agarose gel:

1. You have been given the DNA samples (the digested DNA samples and a DNA
ladder) on your bench. Each sample contains 10 µl of DNA.

2. Using a P20 micropipette, add 5 µl of the loading dye (orange coloured solution in the
Eppendorf tube on your bench) to the digested DNA sample in the microfuge tube.
Flick the tube gently to mix the two solutions.

3. Tap the microfuge tube on the bench to collect the sample at the bottom of the tube.

4. Repeat steps 2 and 3 for each of your DNA samples.

5. Using a P20 micropipette, pipette up the first sample, the DNA ladder (15 ul). Insert
the tip of the pipette though the gel chamber buffer and load the sample into the first
well of the agarose gel. Your tutor will demonstrate the best way to load your DNA
samples.

Please share the loading of the samples with your lab partner to ensure you both get
experience with loading a DNA gel.

6. Using the method in step 5, load your samples including the ladder, in the order given
in the table below, into the agarose gel. Start with the DNA ladder in the first well on
the left of the gel (this is well 1). Use a new tip for each sample.

7. When all samples have been loaded, close the gel chamber by GENTLY applying the
lid. Do NOT move the chamber while doing this

8. Contact your demonstrator who will connect the power supply. Set the timer for 30
minutes and press start.

9. Whilst the gel is running – please start analysing the agarose plates you have been
given – See Part 2.
Part 1.3: Visualisation of the DNA on the gel and interpreting the results

1. Once the agarose gel electrophoresis has run for 30 minutes, please contact your
demonstrator who will turn off the power supply and ensure it is safe for you to
remove your gel.

2. Take your gel in the tray provided to the GelDoc to capture a visual image of your
sample. The demonstrator at the GelDoc will now the photo of your gel and print a
copy of your results.

3. Return to your bench with the picture of your gel and analyse the results by filling in
the table overpage. The first row has been filled in for you.

Well Number Sample Name Is there a DNA fragment released Approx Size of released DNA
(yes/no) fragment
1 DNA Ladder - -

2 Strain X No -

3 Strain Y

4 Strain Z

5 Patient 1

6 Patient 2

7 Patient 3

Table 1: A summary of the results seen in the agarose gel performed in Part 1 of the lab.
 Part 2: Analysis of Agar Plates

Wear gloves while handling the agar plates.

On your bench are 6 agar plates. Each plate has a different sample of E. coli (either the
controls or the patient samples) grown in the presence of antibiotics.

Note: there is one set of plates per two pairs (4 people) so please work with your fellow
students to analyse these results.

The plate are labelled according to the sample of E. coli that have been grown on them:

• Strain X – this E. coli strain does not contain any plasmids encoding enzymes that
result in antibiotic resistance.

• Strain Y – this E. coli strain contains a plasmid expressing the bacterial gene for β–
lactamase that confers resistance to ampicillin.

• Strain Z - this E. coli strain contains a plasmid expressing the bacterial gene for CAT
that confers resistance to chloramphenicol.

• Patient 1.

• Patient 2.

• Patient 3.

On each plate are two discs which have been soaked in either ampicillin or chloramphenicol
and are labelled:

A: ampicillin

C: chloramphenicol
 Work out the antibiotic resistance profile of the unknown bacteria samples from patients 1-3.
Fill in the table below with your analysis of the results. Note: the first two rows of the table
has been filled in for you.

Growth around Resistant to

Growth around Resistant to
Sample Chloramphenicol Chloramphenicol?
ampicillin disc? Ampicillin?
disc?

Sample X No No No No

Sample Y Yes Yes No No

Sample Z

Patient 1

Patient 2

Patient 3

Table 2: A summary of the results seen in the agar plates in Part 2 of the lab.

Part 3: Group activity

There will be a group activity to be completed during this laboratory:

1) Choose a culture and its traditional medicines that you would like to research into. Look into
this before you come to your lab.
2) Identify a compound of interest that may possess anti-microbial properties. You may choose
one listed on your resource sheet (on Canvas) or something entirely different.
 LAB 6 CLEAN UP INSTRUCTIONS

• Pay attention to your tutor for clean-up information.

• Please complete the assignment sheet and hand it in at the back of the lab.
APPENDICES
APPENDIX 1: USE OF GLOVES IN THE LABORATORY

1. Gloves protect you from hazards, and they protect your samples from potential
contamination from you.

2. When handling chemical and biological hazards in the laboratory, it is vital for your
safety (and the safety of others) that you to use disposable gloves.

3. Disposable latex gloves need to be worn when handling blood, blood products, or
other human or animal body fluids or tissues. Gloves must also be worn when using
chemicals/substances which are known to be mutagenic, carcinogenic, teratogenic,
toxic, or hazardous in any way. If you have a known latex allergy, nitrile gloves can
be provided.

4. Gloves are necessary when extracting DNA from samples as wearing them helps
prevent cross contamination with your DNA.

5. Since the outside of the glove is exposed to the hazardous substance, when you are
wearing gloves, do not touch anything other than lab equipment and designated
writing pens/pencils (including your own bare skin on face/arms, and your phone
etc!).

6. To avoid exposing others to the hazards that the gloves are protecting against, gloves
need to be removed PRIOR to leaving the laboratory.

7. Gloves should be removed in the correct manner (see below) and DISPOSED OF IN
THE DESIGNATED YELLOW BIOHAZADOUS BINS in the laboratory.

8. Immediately after removing your gloves, wash your hands with soap and water, and
then you may leave the laboratory.

Protocol for Removing Gloves:

1. Grip glove on the outside near the cuff and pull the glove down so that it comes off inside
out;

2. Hold the removed glove in the palm of your gloved hand;

3. Place two fingers inside the cuff of the glove on your gloved hand.

4. Peel off the glove so that it is removed from your hand inside out with the first glove
remaining inside it;

5. Discard gloves (one inside the other) in the yellow biohazard (mediwaste) bin/bag.
 Appendix 2: HOW TO USE AN AUTOMATIC
MICROPIPETTE

Push Button
1. To REDUCE volume turn the adjustment ring clockwise.
Adjustment ring
To INCREASE volume turn the adjustment ring anti-
Ejector Button
clockwise (F 1).

2. Select a coloured TIP according to the colour shown on

Volume Window
the top of the pipettor. Place the disposable tip on the
shaft of the pipettor. Press on firmly with a slight
twisting motion to ensure positive, airtight seal.

3. Depress the push-button to 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 and SMOOTHLY

while IMMERSED in the liquid to draw up the sample.

6. Wait 1 or 2 seconds, and then withdraw the tip from the

sample. If you have to, wipe any fluid from the outside of
the tip taking care not to touch the orifice of the tip.

7. To dispense the sample, place the tip end against the Tip
inside wall of the container and depress the push-button
SLOWLY to the first stop. Wait for a second 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 container. When the tip has been completely removed you can release the push-
button.

9. Remove the used tip by depressing the tip ejector button or remove manually.

10. NOTE: Never leave micropipette on the bench with liquid in the tip. Always store upright in
rack provided.
Pipettor Increments Tip Colour
10 - 100 µL 0.1 µL Yellow
40 - 200 µL 1 µL Yellow
200 - 1000 µL 1 µL Blue
1000 - 5000 µL 10 µL White
 APPENDIX 3: ELECTROPHORESIS OF DNA
Electrophoresis means, literally, to carry with electricity. DNA, as an organic acid, is
negatively charged. Hence when placed in an electric field, DNA molecules are attracted to
the positive pole and repelled from the negative pole.

We can separate different sized DNA fragments by electrophoresis. An agarose gel acts as a
molecular sieve through which smaller fragments can move more quickly than larger ones.
Thus, over a given time, smaller fragments migrate relatively further from the origin than do
the larger fragments.

Melted agarose (a polysaccharide derived from seaweed) is poured into a casting tray in
which a plastic comb has been inserted. As it cools the agarose solidifies to a gelatinous
substance consisting of a dense network of crosslinked molecules. The solidified gel slab is
immersed in a chamber filled with buffer solution containing ions needed to conduct
electricity. Removal of the comb leaves behind a series of wells into which the DNA samples
are loaded. Prior to loading the DNA is mixed with a loading dye consisting of sucrose and
one or more dyes. The movement of the dye allows us to monitor the migration of the unseen
DNA fragments.

Current is applied through electrodes at either end of the gel chamber. Following
electrophoresis the gel is removed and exposed to medium wavelength ultraviolet light. A
stain in the gel binds to the DNA band and fluoresces.

It is important to remember that a band of DNA seen in a gel does not represent a single
molecule of DNA, but rather the total fluorescence from millions of DNA fragments of the
same base-pair length.

Courtesy of Amanda Harper and BIOSCI101 Laboratory Guide.

 Appendix 4: THE SPECTROPHOTOMETER
PRINCIPLES

The spectrophotometer is an instrument that measures the fraction of incident light of a given
wave-length, transmitted by a solution. Readings are commonly recorded as Absorbance (or
optical density (OD)), equal to log10 (incident/transmitted light intensity), since this quantity
is often proportional to the concentration of the light-absorbing compound (Beer’s Law).

Both the colorimeter and the spectrometer measure Absorbance, but the latter has greater
advantages of accuracy, sensitivity, broad spectral range, and sharp selection of wavelength.

The spectrophotometer is used principally for three different types of determinations in

enzymology.

1. The concentration of a compound can be determined by measurement of Absorbance

at one wavelength, under conditions where no changes occur, provided that the extinction
coefficient of the compound is known.

2. The course of a reaction can be observed in an 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.

3. 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 that trace such plots automatically. This type of plot is called a spectrum.

OPERATION

Familiarise yourself with the spectrophotometer by following the instruction steps attached to
the instrument. If in doubt, consult a demonstrator.

SPECTROPHOTOMETER CELLS (CUVETTES)

During this Biochemistry course you will only encounter cells made from plastic. In later
courses you may use glass and quartz cells. PLASTIC cells normally need to be optically
matched each time they are used, because their surfaces are easily scratched, thereby altering
their original matching.

You can generally avoid this if you use a maximum of two cells each time. One cell used as
the blank or reference throughout all measurements; the other cell used for all other
measurements. If you want even greater accuracy you can use just one cell for the blank and
all the samples.
 Appendix 5: INTRODUCTION TO SPECTROSCOPY

PRINCIPLES OF PHOTOMETRY

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.

The absorption spectrum of riboflavin, in which one plots the amount of light absorbed at
given

wavelengths, illustrates this point.

Absorption spectrum of riboflavin (2.2 x 10-5 M in 0.1 M sodium phosphate, pH 7.06)

Riboflavin appears yellow to the eye. The ultraviolet absorptions having maxima at 260 and
370

nm are not visually recorded, but can be recorded with special instruments.

Measuring light absorption aids us in both the identification and quantification of substances.
For example, the absorption spectrum above is characterisation of riboflavin, and the amount
of absorption by riboflavin at a given wavelength is a function of the riboflavin
concentration.
Quantitative photometric measurement is based on two formalized laws.

The first, Lambert’s Law states that the proportion of incident light absorbed (A) by a
medium

is independent of light intensity, and depends on the pathlength of the sample.

The second, Beer’s Law states that the light absorbed (A) is directly proportional to the
number of molecules of absorbing substance through which the light passes. Thus, if the
absorbed substance is dissolved in transparent solvent, the absorption of the solution is
proportional to its molar concentration.

Lambert’s Law A l

Beer’s Law Ac

Where A = absorbance = Log I

l = light path in cm

ε = extinction coefficient

c = concentration in moles per litre

Io = intensity of the incident light

I = intensity of the transmitted (emergent) light

A combination of these two laws, often termed the Beer-Lambert Law gives the equation:

A = ε.c.l

At a concentration of 1 mol/L and light path of 1 cm, the coefficient ε is called the molar
extinction coefficient or with a concentration of 1 mmol/L the mmolar extinction coefficient.

(ε having the dimension cm-1 M-1 having the dimension cm-1 mM-1 respectively).
 Appendix 6: THE HANDLING OF ENZYMES

1. Heavy Metals

Many enzymes are inactivated by traces of heavy metals. Copper is a contaminant of the
laboratory tap water so this water should not be used for any biological experiment -
especially enzyme experiments. Similarly ice is manufactured from this tap water, so ice
should not be allowed to come into contact with any biochemical experiment - especially
enzyme experiments.

2. Temperature

Enzymes are generally inactivated by heat, so they should be kept on ice. For many
experiments it is recommended that a portion of the enzyme solution is pre-equilibrated to
room temperature, or to the reaction temperature just before use. AVOID excessive pre-
equilibration as this will denature (inactivate) the enzyme.

3 pH

Extremes of pH inactivate enzymes. This is one of the reasons why reactions are carried out
in

buffer, and why enzymes are stored in buffer.

4. Mixing/Foaming

Surface denaturation (foaming) results in the loss of enzyme activity. For most enzyme-
containing reactions, inversion over parafilm is the recommended mixing technique. Avoid
vigorously stirring enzyme solutions with stirring rods, avoid vigorous mixing with a vortex
mixer - unless you are mixing AFTER terminating an enzyme reaction.
Appendix 7: RECOMMENDED ASSAY TECHNIQUE

1. Preparation - Before starting each experiment carefully read the method again. Especially
check that you have all reagents and materials required, and that any equipment needed is
turned on - waterbaths are at the required temperature.

2. Pipetting - the accuracy of each experiment depends very much on your pipetting skills. It is
essential that you read through and follow the instructions given for correct use of volumetric
pipettors.

3. Timing - Assays must be incubated for exactly the time stated. Always start the reaction at
timed intervals (eg 30 or 60 seconds) and then stop the reaction at similar intervals so that all
tubes have exactly the same incubation time. Stopclocks will be provided.

4. Mixing - the accuracy of each experiment also depends upon correctly mixing reagents
together. When adding any reagent (and particularly after adding the substrate, or the reagent
used to stop any reaction) all tubes must be thoroughly mixed.

5. Temperature Control - Place tubes in the waterbath for a few minutes to warm up before
starting any reaction. Check the water level is sufficient to cover all tube contents, and check
the temperature of the waterbath.

Blanks and Controls - Samples may not give zero measurements where the reaction has occurred
without the enzyme being present. Controls (or Reagent Blanks) containing all reagents except the
enzyme should always be run concurrently. Either set the spectrophotometer to zero using this
control, then directly read the absorbance(s) of other tubes against this; or (RECOMMENDED
METHOD) set the spectrophotometer to zero against water or buffer (sometimes called a blank), and
subtract the control reading from all other tubes. Controls should also be used when the time or the
temperature of the experiment is altered as part of the experiment.
 Appendix 8: BIOCHEMICAL UNITS

You will be required to carry out some calculations using the unit millimolar (mM). This is a
concentration unit. You need to be familar with molarity (M), millimolarity (mM),
micromolarity (M), and nanomolarity (nM).

Definitions and relationships between these terms are given below.

MOLARITY (M) = the number of moles of solute per litre of solution (mol/L)

Molar concentrations are usually given in square brackets eg. [H+] = molarity of H+.

To calculate M, we must know the weight (in g) of dissolved solute and its molecular weight,
MW. MOLES = Weight(g)

MW

Mr, which you will be familiar with from chemistry, is relative molecular mass. MW is Mr
expressed in grams.

Dilute solutions are expressed in terms of millimolarity, micromolarity, nanomolarity and so

on. 1 mmol (millimol) = 10-3 mol

1 µmol (micromol) = 10-6 mol

1 nmol (nanomol) = 10-9 mol

1 pmol (picomol) = 10-12 mol

1 = 10-3 M = 1 mmol/L 1 µmol/mL

mM =

1 = 10-6 M = 1 µmol/L = 1 nmol/mL

µM

1 nM = 10-9 M = 1 nmol/L = 1 pmol/mL