Biology of cells: Week 7 Lent 2021

TOPIC : PCR Analysis of a Food-Borne Pathogen

Aim of the practical:

In this practical you will combine PCR analysis and restriction mapping to identify which
strain of Escherichia coli is responsible for an outbreak of food poisoning in a Cambridge
College.
The practical is also intended to stimulate you to think generally about the application of
molecular biological approaches to forensic science, diagnostics, food science and
pathogen typing, and also, to introduce you to the fascinating branch of microbiology
dealing with bacterial pathogenicity.

Preamble
There has been an incidence of food poisoning at Coli College, Cambridge. Three days after
a recent Founders meal, nearly all of the attending students and Fellows have been taken
seriously ill. Under-cooked chicken served at the meal is the suspected cause of the
outbreak. Fortunately, some samples of uncooked chicken meat remain in the Kitchen
refrigerator, which were removed by officers of the regional Health and Safety Executive
(HSE) for analysis. The affected students/Fellows present with symptoms including sudden
stomach cramps, abdominal pain and severe, watery diarrhea. By day 4 after the meal,
some of the patients are ejecting visibly bloody diarrhea, and are hospitalized.
The poultry was sourced from one of two local establishments; Balls battery farm or
Hudsons “organic” Home Farm. However, the receipts for the delivery in question cannot
be found, so on this occasion it is not clear who supplied the meat. The Balls establishment
is clean, well ventilated and rodent-free, but the birds are maintained in high-stress
conditions – a factor that is known to exacerbate flock infection rates. At Hudsons farm,
the birds are free-ranging and form part of a larger “working farm” complex that
specializes in raising rare breads. Hudsons has educational aspirations, and supplements
its income by encouraging paying members of the public to walk around and experience
at first hand the “ethically correct” rearing of animals and poultry for the food industry.
Members of the public are allowed to pet the animals on Hudsons farm – a known risk
factor in spreading Escherichia coli infections between farms.
Both Balls and Hudsons farms are contacted by the HSE, but these suppliers report that
none of their stock appears ill. This is not unusual since most infected chickens are known
to be passive carriers of the disease. Chicken stool samples from both farms are collected
by the HSE and sent to the Public Health Laboratories (PHL) for microbiological analysis.
The PHL determines that chicken stool samples from both farms contain a potentially
hemolytic variant of E. coli known as strain CB1. Subsequent analysis (enzyme profiling) of
bacteria isolated from the raw chicken meat in the College kitchen refrigerator confirmed
that these too were E. coli strain CB1. Diarrhea samples from patients presenting with food
poisoning also contain CB1.
Not all CB1 variants are necessarily hemolytic (i.e. disease-causing). Virulence in CB1 is
known to be largely mediated through expression of the vir operon. Expression of the vir
operon is repressed in many CB1 isolates through the action of the VirR protein.

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Biology of cells: Week 7 Lent 2021

The VirR protein is a DNA-binding protein that binds to and occludes the -10 site of the vir
operon σ70 promoter. Sequence analysis of VirR suggests that this protein also contains a
ligand-binding domain, and it is postulated that in the presence of a putative low-
molecular weight inducer, VirR dissociates from the DNA and transcription of the vir
operon proceeds. In this regard, the regulatory system is very much like the archetypal lac
operon that we met in the lectures. However, the chemical nature of the VirR inducer is
still subject to debate, although a number of laboratories have suggested that it is likely to
be a diffusible furan derivative. Neither E. coli nor humans produce this type of furan,
although a number of non-pathogenic bacteria are known to produce and secrete these
compounds in quantity.
CB1 serovar OX2 carries the wild-type gene (ca. 800 bp in length) for virR, and consequently
exists as a harmless gut commensal in many hosts including some people. However, CB1
serovar PaIN carries a small (25 bp) insertion that disrupts the virR reading frame and
thereby inactivates the VirR protein. This loss-of-function mutation in virR enables means
that transcription of the vir operon is no longer repressed, leading to constitutive
expression of the associated pathogenicity factors. The wild-type virR gene contains a
single HindIII restriction enzyme site. However, the insertion in serovar PaIN disrupts the
HindIII recognition site and instead introduces a cleavage site for the restriction enzyme
BamHI. There are no BamHI cleavage sites in the virR gene of serovar OX2.

In this practical, you will use PCR to amplify the virR gene from various environmental and
clinical isolates associated with the Coli College outbreak. These isolates include;
(i) Chicken excrement from Balls Farm (Sample “A”).
(ii) Chicken excrement from Hudsons Farm (Sample “B”).
(iii) A sample of the diarrhea derived from one of the ill students (Sample “C”).
(iv) A sample of the diarrhea derived from one of the ill Fellows (Sample ”D”). This
particular case of food poisoning may not be directly linked to the Coli College
outbreak since the Fellow was not present at the Founders meal. However, this
Fellow does regularly eat in College and could have picked up the bacterium that
way, although it should be stressed that the College kitchens are kept
scrupulously clean. This Fellow also regularly purchases organic meat from the
Hudsons farm outlet on Cambridge Market for personal consumption. Indeed, on
the day of the Founders feast, this individual had just returned from a lecture tour
of the Middle East and had eaten a hurriedly-cooked dinner of chicken at home –
a meal that she shared with her husband (who had remained in the UK during her
absence). The Fellow’s husband did not appear to suffer any adverse
consequences.
(v) A sample of the uncooked chicken meat kept in the College refrigerator (Sample
“E”).

Once the virR gene has been amplified from each isolate, you will use simple restriction
mapping to identify the source of the Coli College outbreak.

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Biology of cells: Week 7 Lent 2021

DEPARTMENT OF BIOCHEMISTRY : COSHH & RISK ASSESSMENT FORM Page 1 of 1

File ref: CSH_Cells_ Lent Date _Mar 2021 Group: NST1A BIOLOGY OF CELLS COURSE Location:ELEMENTARY LAB,
WK6 ZOOLOGY DEPT

Procedure: PCR Analysis of a Food-Borne Pathogen.

Signature: Name and status of assessor: Mark Leach – Technician in charge of Biochemistry practicals in Date: Mar 2021
NST1A Biology of cells course.

Signature: Senior demonstrator (Assessment Agreed): Dr Martin Welch Date: Mar 2021

Substances / Procedures Quantity Frequency Hazards Avoid contact with Waste Disposal Emergency Action
Lungs, mouth, eyes, skin
Handling microbiological 80ul Once Potential infection hazard. Wear gloves Autoclaving Standard
samples
Skin
Thermal cycling 10 Once Burn hazard – do not lift lid when cycler is hot N/a Standard

Ethidium Bromide 3ul Once Mutagen. Toxic. Wear nitrile gloves. Avoid using if Lungs,mouth, eyes, skin Collection for disposal Standard
pregnant.

Agarose gel electrophoresis 15 Once Electric shock hazard. Ensure lid is in place when Skin N/a Standard
current is applied and tank is not leaking.
Eyes, skin
Gel visualisation under UV 4 Once Radiation burns. Do not bypass safety features on N/a Standard
light Gel doc-it system

What measures are required to control risk? EMERGENCY ACTION

Engineering: Lids on gel tanks, Containment of UV light.
Personal Protective Equipment. Lab coat. Gloves
Management: Good Laboratory Practice. Advice from
demonstrators. Warning signs and labels. Smallest volumes of
harmful substances provided
Checks on control measures: Electrical safety testing. Gel
tank leak tests. Trays to contain spillage.
Is health surveillance or exposure monitoring required ?
No
SPILLAGE - Standard Procedures. Special procedures must be detailed above or on attached sheets.
Solvents - Extinguish naked flames. Volumes >1000ml - evacuate lab and call emergency services; volumes <1000ml, use booms in
spill packs to confine if necessary and absorb into sand or granules. Evaporate in fume hood or dispose via chemical waste company
Acids/Bases - Neutralize acids with excess sodium bicarbonate and bases with 2-6M HCl. Sweep or mop up carefully. Dissolve or
dilute in water and dispose via sink with flush. For large volumes, spill granules may be needed. Dispose via chemical waste company
Solid Reagents - Sweep up carefully. Avoid breathing dust and place in labelled container. Dispose via chemical waste company
EXPOSURE / CONTAMINATION - Standard Procedures. Special procedures must be detailed above or on attached sheets.
Mouth, Eyes, Skin Exposure - Flush area of contact with plenty of water, contact a First Aider; Lungs - Remove to fresh air,
contact a First Aider. If swallowed - Contact a First Aider, get details of substance ingested, and seek medical attention.
immediately. If casualty unconscious - Contact a First Aider immediately, call an ambulance.

Other Risks Ensure that other risks associated with the procedure have been adequately assessed and that you are familiar with these assessments and have received appropriate training.

Tick required assessments Radioactivity: [ ] Genetic Modification: [ ] Manual Handling: [ OK ] Electricity: [OK ] Equipment: [ OK ] Other (describe): [ ]

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Biology of cells: Week 7 Lent 2021

HEALTH AND SAFETY NOTICE

Wear gloves at all times when handling microbiological samples. Vinyl gloves are adequate. This
is not only to prevent you from contaminating yourself with any harmful microbes; it is also to
ensure that you do not contaminate the samples!!! PCR amplifies DNA templates, and is such a
sensitive technique that even tiny amounts of commensal bacteria from the skin can introduce
sufficient DNA to yield a spurious product. With some forensic PCR techniques, contamination
with as few as 5 cells is sufficient to yield a visible product!
Wear gloves at all times when handling Ethidium Bromide (EtBr). Nitrile gloves offer superior
protection against EtBr contamination. EtBr is a suspected mutagen and is toxic. The agarose gel
electrophoresis you will carry out incorporates EtBr which allows the DNA bands in the gel to be
visualised under UV light. Do not allow it or anything containing it (like the agarose gel or the used
TAE buffer) to come into contact your skin, or contaminate any surface. Don’t touch the Gel doc–
it door, keyboard or mouse with potentially EtBr contaminated gloves. Report ALL EtBr
contamination, no matter how minor, to the classroom technician, teaching assistants, or senior
demonstrator.
Wash your hands after leaving the laboratory.

PROTOCOL

Firstly, we need explain how the class is organised:

• The same colour ice buckets on the same bench designate a team of 5 – 10 students.
• Each team will process a set of 5 samples (A,B,C,D,E).
• Generally, 2 teams will share a thermal cycler, and each team will run a gel.

Each team should nominate a leader to check that all tubes from their team are in their
designated thermal cycler along with those from the adjacent team (where applicable) before the
cycler programme starts. The team leader should also co-ordinate gel loading for their team.

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Biology of cells: Week 7 Lent 2021

PART A

Each team will process 3 µL samples of A, B, C, D and E.

Each student pair placing has an ice bucket with one (or two) samples, and 80 µL of PCR master
mix, “M”, comprising;

• An equimolar mixture of dATP, dGTP, dCTP and dTTP, each at 200 µM final concentration.
• Reaction buffer (16 mM ammonium sulfate, 67 mM Tris-HCl (pH 8.8 at 25 °C), 0.01%
Tween-20).
• MgCl2 (2 mM)
• virR 5’ primer (1 µM)
• virR 3’ primer (1 µM)
• Taq polymerase enzyme (1 unit)

Step 1. You should have 3 PCR tubes per sample. Label 1 tube with the sample letter, another
with the sample letter plus “B”, and the third with sample letter plus “H” (For example, if you
have sample “A”, your three tubes should be labeled “A”, “AB” and “AH”).

Step 2. Pipette the entire contents (80 µL) of your PCR master mix “M” into your 3 µL sample,
and mix carefully by slowly pumping the contents up and down in the pipette tip several times.

Step 3. Carefully dispense 25 µL of your sample mix into each of your 3 PCR tubes. For efficient
thermal cycling, the liquid needs to sit nicely in the base of the tube without riding on an air
bubble, and should not be not splattered on the tube walls.

Step 4. Cap the tubes. A couple of light taps on the bench may be required to settle the mix in the
base of the tube.

Step 5. Take your labelled PCR sample tubes to your designated thermal cycler which is set to
hold at 4 °C for loading. Lift the lid and place the tubes in the heating block in a sensible
configuration.

Step 6. Once all 15 PCR tubes from your team are in the cycler and the lid of the machine has
been closed, the leader can press “SKIP” and (are you sure you want to skip this stage?) “OK”. Do
not attempt to alter the programme, or press STOP at any stage.
To enable the process to amplify virR, the IABOC programme executes 33 consecutive heating
cycles of 30 seconds at 94 °C, 30 seconds at 60 °C, and 1 minute at 72 °C. At the end of the
sequence, the IABOC programme will hold for 5 minutes at 72 °C and then cool to hold 4 °C.

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Biology of cells: Week 7 Lent 2021
THE THERMAL CYCLING TAKES AROUND 90 MINUTES. THIS IS A GOOD TIME TO GO FOR LUNCH
AND PONDER THESE QUESTIONS:

• What is an enzyme “unit”?

• In these experiments, the Taq DNA polymerase is present in the PCR master mix from the
start. Some researchers prefer to add the Taq polymerase only after the mixture has
reached 94ºC. Why might this be?

• Why are three different temperatures employed during each cycle of the PCR reaction?

• How do we choose what annealing temperature to use?

• Can we alter the annealing temperature of a primer in any way?

• What is the purpose of the long (5 minute) terminal extension step at 72 °C?

WHEN YOU COME BACK FROM LUNCH, YOU WILL FIND THREE MORE TUBES IN YOUR ICE
BUCKETS: RESTRICTION ENZYMES BamHI (B), HindIII (H), AND SAMPLE BUFFER (S).

Don’t worry if the “time remaining” on the thermal cycler seems high. It includes the 30 minute
enzyme incubation you are about to set up.

PART B

Step 7. Check the thermal cycler has paused the IABOC programme, and is holding the samples
at 4ºC. Take your tube(s) labelled with the single sample letter (i.e. A, B, C, D or E, as appropriate),
add 5 µL sample buffer “S”, and leave the tube(s) on ice.
DO NOT ADD “S” TO TUBES “XB” AND “XH” UNTIL STEP 13.

Step 8. Add 2 µL of BamHI (B) to tube “XB” and 2 µL of HindIII (H) to tube “XH”. Make sure that
you add the enzyme directly in to the PCR mix, not on to the side of the tube. Gently mix by
pipetting up and down several times and return the tubes to the thermal cycler.

Step 9. Once all 10 tubes per team are back in the thermal cycler, the leader can press “SKIP” and
(are you sure you want to skip this stage?) “OK” to execute the final part of the IABOC programme
- 30 minutes at 37 °C, and then hold at 4 °C. This incubation is optimal for the activity of the
restriction enzymes.

WHILE THE ENZYMES ARE DIGESTING YOUR SAMPLES, YOUR TEAM SHOULD PREPARE THE
AGAROSE GEL. THE GEL WILL ACCOMMODATE YOUR TEAMS 15 SAMPLES AND A “SIZING
LADDER” (L)

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Biology of cells: Week 7 Lent 2021
Step 10. Get a bottle of molten agarose for your team from the 55 °C incubator, and layer 200 µL
along the top & bottom edges of tray above the rubber gaskets. Two applications should suffice.
This small amount of agarose mix will set almost straight away, and should give you a good seal.
Next, place the 16 well comb in slots above the colour strip with the screws facing you.

Step 11. Ask a demonstrator to add 3 µL of 10 mg/mL Ethidium Bromide (EtBr) to the molten
agarose, replace the lid, swirl gently to mix and pour the gel mix into the tray. Remove any bubbles
from the gel surface by stroking them away with a pipette tip, and leave the gel undisturbed for
about 10-15 minutes to set.
(Please add about 50 ml of tap water to the empty agarose bottle, replace the lid, and swirl. This
prevents any remaining agarose setting in the bottle, making it a lot easier to clean).

Step 12. After the gel has set, lift the gel tray out, (take care not to touch the surface of the gel,
or let it slide off the tray), rotate the tray ¼ turn anti-clockwise, and replace the tray and gel in
the tank so that the comb and colour strip at left hand end of the tank. Pour in all 300 ml of the
1x TAE running buffer to the “max fill mark” on the side of the tank. Remove the 16 well comb
from the gel (care – EtBr contaminated!) and leave it on the perspex tray.

Step 13. Check the thermal cycler has finished the IABOC programme, and is holding the samples
at 4 °C. Remove your “XB” and “XH” tubes, and add 5 µL of sample buffer (“S”) to both tubes, and
leave on ice.

Step 14. A demonstrator will show you how to load samples in to the wells. Gels should be loaded
with 20 µL in alphabetical order: “A”, “AB”, “AH”, “B”, “BB”, “BH” etc, from left to right, in wells
1 – 6. Load well 7 with 10 µL DNA sizing markers “L”, then continue loading wells 8 – 16 with 25
µL of the remaining 9 samples in alphabetical order.

Step 15. When all the samples have been loaded, push the lid down on to the tank and check that
the leads are plugged in to the power supply. Set the power supply to 120 V, 1000 mA, and 45
mins, and press “RUN”. You should see fizzing in the buffer at the gel terminals and the dye front
start to migrate after a few minutes.

While the gel is running, answer the “General Questions and Numerical Problems” on the next
page. The demonstrators will help you if you get stuck.

Step 16. When the power pack alarm goes off to signal time’s up, turn the power supply off and
disconnect the leads from the power supply. Grip both sides of the lid and push down on the
white lugs with your thumbs to remove the lid.

Put a pair of nitrile gloves on. Lift the gel tray from the tank, and without allowing the gel to slide
off, carefully tilt for a few seconds to drain the excess (EtBr contaminated) buffer back into the
gel tank. When most of the buffer has drained off, put the tray and gel in the plastic box for safe
transport to the “Gel-Doc-it” systems, where you’ll be able to view your gel under UV light
through a safety window, have it photographed and get a print. Return the gel tray to the plastic
box, and leave it by the waste bucket for disposal.

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Biology of cells: Week 7 Lent 2021
Look carefully at the banding pattern on the photographs. Use a ruler to measure the distance
migrated by each of the markers and draw a graph of log10[marker size (in kbp)] vs distance
migrated. The DNA ladder markers are 10 K, 8 K, 6 K, 5 K, 4 K, 3 K, 2.5 K, 2 K, 1.5 K, 1 K, 0.8 K, 0.6
K, 0.4 K and 0.2 K base pairs. The 10 K and 1 K bands should have the highest intensity.
Also measure the distance migrated by each of the bands in samples A-E, AH-EH and AB-EB. What
size (in kbp) of DNA does each band in the samples correspond to? On the basis of your data,
answer the questions below.

1) Which serovar, OX2 or PaIN is associated with samples A-E?

2) Which serovar, PaIN or OX2 is associated with the poultry on (i) Balls farm and (ii) Hudsons
farm?
3) Which CB1 serovar is associated with the Coli College outbreak?
4) Which CB1 serovar caused illness in the Coli College Fellow (sample D)? Can you suggest
why she became ill and her husband did not?

General questions and numerical problems.

(i) Starting with 1 picogram (10-12 g) of template DNA, and assuming that each round of
PCR exponentially amplifies the DNA, how many cycles would be required in order to
obtain about 1 µg of product (i.e., enough to yield a visible band on an agarose gel)?

(ii) Taq DNA polymerase lacks 3’→5’ exonuclease activity. Consequently, and as might be
expected from first principles, Taq exhibits a high error rate (around 3 x 10-4 errors per
base pair incorporated). Assuming that we start amplifying from a single copy of the
800 bp virR gene, approximately how many rounds of amplification can occur until you
can be certain that at least one error has been introduced into the product? What are
the consequences if this error happens to have been introduced at a very early cycle
of the PCR? Should Taq be used for cloning purposes?

(iii) In a typical PCR reaction, the annealing temperature is chosen to be just a degree or
so below the theoretical melting temperature of the oligonucleotide primer with its
complimentary template (why?). However, once the annealing step is complete, the
temperature is rapidly elevated, often to several degrees above the theoretical
melting temperature of the oligonucleotide. Why does the primer not dissociate from
the template when the temperature is raised this way?

(iv) Taq synthesizes DNA at a rate of about 1000 bp per minute at 72 °C. How long would
it take for a molecule of Taq to theoretically replicate the entire E. coli genome (4.6 x
106 bp)? Two molecules of the endogenous PolI enzyme can bidirectionally replicate
the E. coli genome in 30 minutes. How many bp per minute can PolI synthesize? How
many turns of B DNA does the PolI replisome need to unwind each second?

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Biology of cells: Week 7 Lent 2021
(v) Your PCR reactions contained 1 unit (U) of Taq. 1 U of Taq polymerase is defined as
the amount of enzyme required to incorporate 10 nMol of dNTP into DNA in 30
minutes under optimal reaction conditions (72 °C). However, your PCR mixtures (25
µL volume) contained 200 µM dNTP. Assuming that these mixtures contained
unlimited template DNA, how long would it theoretically take for all of the dNTP
present to become incorporated into high molecular weight DNA?

(vi) The open reading frame of the wild-type virR gene is 800 bp long. The average
molecular weight of an amino acid is 110 Da. The VirR protein is known to be a
homodimer. What is the molecular weight of the VirR protein?

(vii) 1 µg of a particular DNA sample contains 1012 molecules. Assuming that Avogadros
Number is 6 x 1023 and that the average weight of a base pair is 650 Da, how many
base pairs does the DNA contain?

(viii) Assuming that the average E. coli cell can be approximated as a sphere with a diameter
of 1 µm, and that each cell contains an average of 1 genome, calculate the
concentration of a single gene in the cell.

(ix) Assuming that there is only a single binding site for VirR in the E. coli genome, and that
the Kd (dissociation constant) of VirR for its cognate DNA binding site upstream from
the vir operon is 3 nM, what fraction of the potential binding sites in the ensemble of
cells in a culture will be occupied if the total VirR [dimer] concentration in the cell is 3
nM. Assume that the concentration of DNA in the cell is the same as you calculated in
question (viii).

(x) Primers virUP and virDOWN anneal to regions 100bp upstream and downstream of
the virR ORF, respectively. The virR5’ and virR3’ primers used in today’s practical
anneal precisely to the ends of the virR ORF. A PCR reaction was carried out using the
virUP and virDOWN primers and a genomic DNA template. When the products were
resolved on an agarose gel, a large number of bands were seen, including three
closely-spaced bands of approximately the expected size (ca. 1 kb). The presence of
multiple bands is indicative of mis-priming. Assuming that you can excise the three ca.
1 kb size bands from the gel, and that the DNA from each can be extracted and
purified, outline a strategy that would enable you to determine which of the three
bands correspond to the virR amplicon ( “amplicon” is the technical term for an
amplified segment of DNA).

(xi) What strategies might you employ to try and increase the specificity of priming by
virUP and virDOWN (see Q(x))?

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Biology of cells: Week 7 Lent 2021

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