This document describes the production of Short Chain Variable Fragments (ScFv) that specifically bind to E. coli for use in detecting E. coli in water samples. Chickens were immunized with bacterial glycoconjugates to produce antibodies. RNA from the chickens was used to generate a ScFv gene library displayed on phages. ScFvs that bound strongly to E. coli were isolated through biopanning. The purified phage was used to produce soluble ScFv in E. coli for assessment of binding specificity to E. coli and Enterobacter cloacae via dot blot analysis. The ScFv showed strong binding to E. coli samples and negligible binding to E. cloacae, indicating potential for use

1. The Production of
Short Chain Variable Fragments
for the detection of
E. coli in Water
By
Denis Ryan
Submitted in part fulfilment for the award of the B. Sc. (Hons) in Applied Biology and
Biopharmaceutical Science, Galway-Mayo Institute of Technology
Galway-Mayo Institute of Technology
2016

2. Declaration
I declare that this project is completely my own work and that all sources of information
used have been acknowledged via complete references.
Signed: _______________________
Date: _______________________

3. ~ 1 ~
Acknowledgements
First and foremost I would like to thank Shane Deegan and Marta Utratna for taking me
under their wings for the past couple of months, and for their assistance with lab-work,
thesis writing and reviewing. The experience in Aquila has been fantastic and I’ll be
very sorry to leave.
I would also like to thank Trish O’ Connell for reviewing my thesis before submission
and for her support as my GMIT supervisor.
Finally, I would like to thank Kevin and Bernie Ryan and Raizel Burstein-Frame for
their advice regarding my thesis, and minor proof-reading.

4. ~ 2 ~
Table of Contents
List of Tables and Figures..........................................................................................................3
Abbreviations .............................................................................................................................4
Abstract ......................................................................................................................................6
Introduction................................................................................................................................7
Aims .........................................................................................................................................10
Materials...................................................................................................................................11
Methods....................................................................................................................................15
Transformation of E. coli with pComb3.1, and culturing on Ampicillin-LB plates ............16
Preparation of E. coli starter culture for the expression of soluble ScFv.............................18
Formation of bacterial pellets from transformed E. coli for lysis and ScFv purification.....19
Preparation of Ni-NTA affinity resin for ScFv purification.................................................20
Lysis of bacterial cells and ScFv purification ......................................................................21
Formulation of buffers for SDS-PAGE................................................................................23
Casting of SDS-PAGE gels and electrophoresis of ScFv sample ........................................24
Concentration of ScFv from elutions 1-6, and assessment of specificity for E. coli ...........27
Results ......................................................................................................................................30
Discussion ................................................................................................................................32
Conclusions ..............................................................................................................................33
Risk Assessment.......................................................................................................................34
References ................................................................................................................................36

5. ~ 3 ~
List of Tables and Figures
Figure 1: Polyacrylamide gel of ScFv elutions ...............................................................30
Figure 2:X-Ray film showing luminescence ..................................................................30
Figure 3: 11-245kDa protein ladder ...............................................................................31

6. ~ 4 ~
Abbreviations
APS – Ammonium Persulphate
cDNA – Complementary DNA
Da – Daltons
dH2O – Deionised water
ddH2O – Double Distilled water
DNA – Deoxyribonucleic Acid
DTT – Dithiothreitol
E. cloacae – Enterobacter cloacae
ECL – Enhanced Chemiluminescent substrate
E. coli – Escherichia coli
ELISA – Enzyme Linked Immunosorbent Assay
g – Grams / G- force (depending on context)
HCl – Hydrochloric acid
His – Histamine
HRP – Horse Radish Peroxidase
IPTG - Isopropyl β-D-1-thiogalactopyranoside
kDa – Kilodaltons
LB – Luria Bertani (broth or agar)
L – Litre
M – Molar
mM – millimolar
MgCl2 – Magnesium Chloride
mg – milligram

7. ~ 5 ~
ml – millilitre
MUG – 4-Methylumbelliferyl-β-D-glucuronide
NaCl – Sodium Chloride
NAPES – Next Generation Analytical Platforms for Environmental Sensing
Ni-NTA – Nickel-nitrilotriacetic acid
ONPG – ortho-Nitrophenyl-β-galactoside
PBS – Phosphate Buffer Solution
PCR – Polymerised Chain Reaction
RNA – Ribonucleic Acid
rpm – Revolutions per minute
ScFv – Short Chain Variable Fragment
SDS – Sodium dodecyl sulphate
SDS-PAGE – SDS Polyacrylamide Gel Electrophoresis
spp. – species
TEMED – Tetramethylethylenediamine
µl – microlitre
V – Volts
VH – Variable Heavy (chain)
VL – Variable Light (chain)

8. ~ 6 ~
Abstract
This project was undertaken in order to produce Short Chain Variable Fragments (ScFv)
that can specifically bind to E. coli. The ScFv is to be employed as a bio-receptor that
will be integrated into a water detection system for highly efficient detection of E. coli
in water samples.
ScFv production was achieved through immunising chickens against a non-specific
mixture of glycoconjugates using Freund’s adjuvant. After immunisation, the chickens
were euthanised and total RNA was isolated from the spleen and bone marrow. RNA
encoding the Variable Heavy and Light regions (VH and VL) of antibodies was
amplified using PCR, and converted into cDNA. The cDNA amplicons were conjugated
to form VH – VL sequences (ScFv genes) which were inserted into pComb3.1 vectors
for transformation into E. coli TOP10F. Successful transformation led to the generation
of chicken ScFv phage libraries. The expressed ScFv specific to E. coli was then
isolated using a process known as phage display. The ScFv-displaying phages were
assessed for binding to E. coli through biopanning. Phages which displayed strongest
binding were isolated and purified through repeated biopans. These initial stages of the
project were previously carried out by Aquila Bioscience.
The purified phage was then eluted and used to infect E. coli TOP10F for soluble ScFv
expression. ScFv specificity for E. coli was assessed via Dot Blot analysis. Three
bacterial samples (E. coli 01, E. coli 0157:H7 and Enterobacter cloacae) were taken and
blotted onto nitrocellulose paper. ScFv purified from E. coli TOP10F was blotted onto
each bacterial sample, followed by a wash. Anti-His tag antibodies were blotted onto
each bacterial sample, followed by a wash. HRP labelled anti-rabbit antibodies were
blotted onto each sample, followed by a wash, and exposed to ECL substrate. Binding
of ScFv to the bacterial samples was indicated by generation of light and observed using
X-Ray film.
It was observed that ScFv displayed strong binding to the two E. coli samples and
negligible binding to the E. cloacae sample. This result indicates that ScFvs may be
used in a bacterial water analysis kit for the detection of E. coli, with the possibility of
being extended to other bacteria.

9. ~ 7 ~
Introduction
Analysis and detection of microbial contamination is highly important in a number of
sectors, such as water analysis, food processing and healthcare industries. Successful
detection and quantification of objectionable organisms is critical to ensure consumer
safety and prevent the distribution of contaminated product. To this end, water analysis
is of particular importance as the use of water is widespread and diverse (bathing,
drinking, swimming, etc.). In situations where the water supply of a population is
contaminated with pathogenic organisms, the consequences for public health can be
profound, as was seen during the Cryptosporidium outbreak in Ireland, 2004. During the
outbreak, it was reported that approximately 432 individuals were infected with
Cryptosporidium, the majority of whom were children under the age of 5 years (Garvey
et. al., 2007).
Traditionally, bacterial water analysis has relied on various culturing techniques which
are able to detect and quantify the populations of key indicator organisms (e.g.
Escherichia coli, Clostridium perfringens, Enterobacter spp.) (Madigan et. al., 2012).
Examples of culturing techniques include the use of:
 MacConkey Agar
 The Colilert Technique
 Membrane Filtration
While these and other culturing techniques have proved to be useful indicators of water
purity, there are some disadvantages associated with their use. The main disadvantage is
the time required for results to be generated. For all of the above techniques, bacteria
must either be cultured (MacConkey Agar and Membrane Filtration) or enzymatically
assessed (Colilert Technique). Both processes require an incubation period of
approximately 24 hours, assuming no problems arise during incubation (e.g., power
failure in incubator, operator error during process, incorrectly used medium, poor
aseptic technique, etc.).
Accurate detection of bacteria may also be a concern for these techniques. The Colilert
technique can provide inaccurate results due to its being an enzyme-based assay. If
other bacteria are present which are capable of metabolising its defined substrates,

10. ~ 8 ~
ONPG or MUG, false positives and inflated bacterial populations may be reported
(Köster et. al., 2003). A similar concern is found in the use of selective media.
MacConkey Agar contains agents, such as bile salts, which actively inhibit the growth
of background organisms in water samples. However, due to a variety of biocidal agents
commonly found in water (chlorine, copper and zinc) selected indicator organisms may
be stressed, and therefore difficult to culture on the medium. This would lead to false
negatives or a lesser degree of contamination being reported (Köster et. al., 2003).
Aquila Bioscience is working with a large consortium of experts from across Europe as
part of an FP7 EU grant. The project they are undertaking, NAPES, intends to create
low cost, deployable, autonomous environmental sensor platforms using highly specific
detection methods for determination of bacterial contaminants in water supplies
(NAPES, 2016). ScFv (an antibody fragment) was chosen as the analytical molecule
for use in NAPES. Theoretically, antibodies and antibody fragments could be used to
identify bacteria in water samples as bacteria possess a wide range of proteins on their
cell surfaces. However, this wide range of proteins also makes identifying bacteria using
antibodies quite difficult. Whole cells are regarded as “complex antigens” (Kemeny et.
al., 1988) and antibodies applied to whole cells are capable of cross reacting with other
strains instead of binding to one specific strain. Therefore, the ability to produce ScFvs
which are capable of binding to a specific species of bacteria is critical to their
successful use in NAPES.
A ScFv is a fusion protein of the VH and VL regions of an antibody, the gene for which
was converted from RNA to cDNA using PCR overlap extension (Andris-Widhopf et.
al., 2000). The ScFv will be integrated into the NAPES detection platform and, in
combination with an extremely sensitive detection device, will be able to detect minute
quantities of E. coli in a matter of minutes. (Deegan, 2016)
The RNA for this ScFv was previously obtained by Aquila Bioscience from chickens
immunised against a mixture of glycoconjugates. This mixture non-specifically raised
the chickens’ immune systems, resulting in a variety of antibodies being produced.
After successful immunisation, the chickens were sacrificed and total RNA was
obtained from the spleens and femur bone marrow (Andris-Widhopf et. al., 2000).

11. ~ 9 ~
As many diverse ScFv genes were produced from the total RNA, Aquila Bioscience
purified the genes for ScFvs which specifically possessed binding to E. coli using phage
display. A phage library was produced by ligating ScFv genes into pComb3.1 vectors
which were used to transform individual E. coli TOP10F colonies. Viral production was
induced by co-infection of the colonies with Helper phage, and the resulting phage
particles were purified and concentrated (Cunningham et. al., 2013). This concentrated
solution of ScFv-displaying phages was purified through biopanning, where a 96-well
titre plate was coated with E. coli, and aliquots of the phage solution were added to each
well. After an incubation period, the wells were washed to remove unbound phage, and
the bound phage was eluted and retained. This solution was then repeatedly introduced
to the plate, washed and eluted, resulting in a solution of ScFv-displaying phage with
strong affinity for E. coli (Andris-Widhopf et. al., 2000).
Using the viral DNA from this purified solution, this project aims to transform a sample
of E. coli TOP10F cells and induce expression of soluble ScFv. Once expressed, this
ScFv will be assessed using a Dot Blot assay to determine the degree of binding against
three bacterial samples: E. coli 01, E. coli 157:H7 and E. cloacae.

12. ~ 10 ~
Aims
1. To transform E. coli TOP10F with ScFv DNA
2. To isolate and purify ScFv from transformed E. coli TOP10F via Nickel Affinity
Chromatography
3. To produce an antibody fragment (ScFv) capable of selecting E. coli over other
cell types, and confirm this using a Dot Blot

13. ~ 11 ~
Materials
Luria Bertani Agarose Powder Rotary Incubator
Manufacturer: Sigma Aldrich Manufacturer: Grant-Bio
Lot Number: BCBP4967V Make: Orbital Shaker-Incubator
Batch Number: L3022-1KG
2-20µl Pipette Ampicillin Sodium Salt
Manufacturer: Eppendorf Manufacturer: Sigma Aldrich
Make: Research Plus Lot Number: BCBP 8492V
Model Number: G24284D Batch Number: A9518-5G
20-200µl Pipette Electronic Balance
Manufacturer: Eppendorf Manufacturer: Ohaus
Make: Research Plus Make: Scout Pro
Model Number: G25373D Model Number: B319313677
1-1000µl Pipette Magnesium Chloride
Manufacturer: Eppendorf Manufacturer: Sigma Aldrich
Make: Research Plus Lot Number: SLBH739V
Model Number: G25915D Batch Number: M8266-IKG

14. ~ 12 ~
Pipette Filler Incubator
Manufacturer: VWR Manufacturer: Thermo Scientific
Make: Smoothie Make: Heratherm
Model Number: 612-454
Autoclave -80° C Freezer
Manufacturer: Rodwell Manufacturer: Thermo Scientific
Model Number: ABASSADOR Make: Forma -86C ULT Freezer
Serial Number: 1931 Model Number: 906
Sterile Loops Heating Block
Manufacturer: Sarstedt Manufacturer: Corning LSE
Lot Number: 5081211 Make: Digital Dry Bath Heater
Reference Number: 86.1562.010 Model Number: 6787-SB
Expiry Date: 3 / 2018 Serial Number: 1127421
15 ml Vial Sterile Spreaders
Manufacturer: Sarstedt Manufacturer: Sarstedt
Lot Number: 4043001 Lot Number: 5085M
Reference Number: 62.554.502 Reference Number: 86.1569.005
Expiry Date: 7 / 2017 Expiry Date: 4 / 2018

15. ~ 13 ~
Low Temperature Freezer Vial 10 ml Pipette
Manufacturer: VWR Manufacturer: Sarstedt
Lot Number: 708904055 Lot Number: 4169E
Reference Number: 479-0801 Reference Number: 86.1254.001
Expiry Date: 03 / 2015 Expiry: 6 / 2017
Vortex Centrifuge 1
Manufacturer: Biocote Manufacturer: Hettich
Make: Vortex Mixer Make: Rotofix 32A
Catalogue Number: SA8 Serial Number: 0025914-03
Serial Number: R800007463 Reference Number: 1206
Virkon Detergent Centrifuge 2
Manufacturer: VWR Manufacturer: Thermo Scientific
Lot Number: 1411BAOC56 Make: Heraeus Pico 17
Manufacture Date: 1/11/14 Model: 40929153
Expiry Date: 1/11/17
Labroller Ni-NTA Affinity Resin
Manufacturer: Labnet International Inc. Manufacturer: QIAGEN
Serial Number: A406178 Lot Number: 151018842
Model Number: H5500-230V-EU Material Number: 1018244

16. ~ 14 ~
Hotplate Power Pack
Manufacturer: Biocote Manufacturer: Omni PAC
Make: Stuart Heat-Stir Serial Number: 151019158
Catalogue Number: UC152 Model Number: CS-300V
Serial Number: R600003124
Vivaspin 500 columns Nitrocellulose Membrane
Manufacturer: Sigma Aldrich Manufacturer: GE Healthcare
Catalogue Number: Z614025-25EA Catalogue Number: 10600014
Lot Number: 2599539 Lot Number: G9972438
Anti-His Antibodies Electrophoretic Rig
Manufacturing: Sigma Aldrich Manufacturer: BioRad
Catalogue Number: SAB1306085-40TST Make: Mini Protean Tetra System
Lot Number: SH090811BJ Catalogue Number: 1658005
Glass Plates Short Plates
Manufacturer: BioRad Manufacturer: BioRad
Catalogue Number: 1653311 Catalogue Number: 165330

17. ~ 15 ~
Methods
Preparation of Sterile Luria Bertani Broth
1. Three stoppered flasks (400ml, 100ml and 100ml) were collected and brought to
an electronic balance.
2. A plastic weigh boat was placed onto the balance and the balance was zeroed.
3. Using a plastic spoon, 2g of the LB powder was accurately weighed.
4. The 2g of LB powder was taken and transferred to the 400ml flask.
5. Taking a 100ml graduated cylinder, 100ml of distilled H2O (dH2O) was
accurately measured.
6. The 100ml dH2O was transferred to the 400ml flask.
7. The flask was stoppered and shaken to ensure complete solubilisation of the LB
powder.
8. After the LB powder had dissolved, the above procedure was repeated twice
more for the 100ml flasks, using 1g of LB powder and 50ml of dH2O.
9. The stoppers of the three flasks were loosened and brought to the autoclave for
sterilisation.
10. The flasks were placed into the autoclave and a temperature probe was inserted
into a 400ml flask of H2O.
11. The autoclave was set to run for 15 minutes at 121°C, and left overnight.
12. After the sterilisation run had completed, the three flasks were removed from the
autoclave and stored at room temperature for future use.

18. ~ 16 ~
Transformation of E. coli with pComb3.1, and culturing on Ampicillin-LB plates
1. Three vials (containing E. coli TOP10F cells, pComb3.1 ligated with ScFv, and
Ampicillin [50mg/ml] respectively) were taken from the -80°C freezer and
defrosted on ice.
2. A Bunsen burner was lit and the flame turned to blue to create an aseptic
environment.
3. Once the vials had fully defrosted a 2-20µl pipette was used to transfer a 2µl
aliquot of plasmid directly to the vial of E. coli cells. The vial was gently
inverted to ensure homogeneity.
4. The E. coli cells were transferred to an ice bath and left to incubate for 30
minutes.
5. After the 30 minutes had elapsed, the cells were removed from the ice bath and
transferred to a heating block set to 42°C for 60 seconds, after which they were
transferred back to the ice bath for 2 minutes.
6. After 2 minutes, the cells were removed from the ice bath and 450µl of LB broth
was added to the vial using a 1-1000µl pipette.
7. The vial was gently inverted to ensure homogeneity and then transferred to a
rotary incubator. The vial was inserted horizontally, taped in place using
autoclave tape and left to incubate for 60 minutes (160rpm, 37°C).
8. While the cells were incubating, an LB plate was taken. A 25µl aliquot of
Ampicillin was transferred to the LB plate and spread across the surface using a
sterile spreader. The plate was left standing on the bench to dry.
9. After the 60 minutes has elapsed, the cells were removed from the rotary
incubator and 2µl of Ampicillin was pipetted directly into the vial. This was
inverted gently.
10. From the vial, 50µl of solution was removed and used to inoculate the
Ampicillin-LB plate using a sterile spreader.
11. The plate was labelled with the date and cell type, transferred to an incubator set
to 37°C and left overnight to culture.
12. The next day, the plate was removed from the incubator and inspected for
colony formation / contamination.
13. A Bunsen burner was lit and the flame turned to blue to create an aseptic
environment.

19. ~ 17 ~
14. Using a sterile 10ml pipette and pipette filler, 5ml of sterile LB broth was
transferred to a stoppered 15ml vial.
15. Using a 2-20µl pipette, 10µl of Ampicillin was transferred to the vial, which was
then stoppered and inverted.
16. Using a sterile inoculation loop, a single colony was taken from the centre of the
plate and was used to inoculate the 15ml vial of LB broth.
17. The vial was labelled with the date and cell type, and transferred to the rotary
incubator for 6 hours (160rpm, 37°C, horizontal).
18. After the 6 hours had elapsed, the vial was removed from the rotary incubator
and visually inspected for turbidity.
19. Using a 1-1000µl pipette, 500µl of the liquid culture and 500µl of 50% glycerol
were transferred to a 2ml Freezer vial.
20. The vial was inverted gently to ensure homogeneity, labelled with the date and
cell type and transferred to the -80°C freezer.

20. ~ 18 ~
Preparation of E. coli starter culture for the expression of soluble ScFv
1. An aseptic environment was created by lighting a Bunsen burner and turning the
flame to blue.
2. The previously made glycerol stock of transformed E. coli TOP10F cells was
removed from the -80°C freezer and left to thaw on ice.
3. While the stock was thawing, 5ml of sterile LB broth was pipetted into a
stoppered 15 ml vial using a sterile 10ml pipette and pipette filler.
4. Taking a 2-200µl pipette, 10µl of an Ampicillin stock (50mg/ml) was taken and
added to the 15ml vial. The mixture was inverted 20 times.
5. Once the glycerol stock had thawed, a small aliquot was taken up via capillary
action, using a pipette tip. This was transferred to the 15ml vial, which was
gently inverted 20 times.
6. The vial was taken and placed inside a rotary incubator horizontally. The vial
was held in place using autoclave tape, and left to incubate until turbid (160rpm,
37°C).
7. Once the broth had become turbid, the vial was removed from the incubator.
8. Taking a 2-200µl pipette, 50µl of the starter culture was taken and transferred
into 50ml of pre-prepared, sterile LB broth.
9. Using a sterile 10ml pipette and pipette filler, 2.5ml of 1M MgCl2 was pipetted
directly to the sterile LB broth.
10. 100µl of IPTG was added to the broth, and the solution was gently swirled.
11. The lid of the flask containing the broth was loosened and held in place using
autoclave tape. The flask was transferred to the rotary incubator and allowed to
culture overnight (160rpm, 37°C).
12. The next day, the flask was removed from the rotary incubator and visually
inspected for growth.

21. ~ 19 ~
Formation of bacterial pellets from transformed E. coli for lysis and ScFv
purification
1. The previously prepared E. coli culture was taken from the rotary incubator.
2. Using a sterile 10ml pipette and pipette filler, the 50ml solution was divided into
four 12.5ml portions. Each of these portions was pipetted into 15ml stoppered
vials.
3. Each vial was weighed using an electronic balance to ensure each contained
approximately the same volume. If this was not the case, small portions of
solution were transferred from one vial to another via pipette until all vials
contained the same volume.
4. The stoppers on the vials were tightened and brought to a centrifuge.
5. The vials were inserted into the centrifuge in a cross pattern.
6. The lid of the centrifuge was closed and the centrifuge was set to run at 4000 g
for 30 minutes.
7. After the 30 minutes had elapsed, the vials were removed.
8. The supernatant was poured from each vial into a stoppered flask, and the pellets
were retained.
9. The vials containing pellets were stoppered and placed into a -20°C freezer for
storage.
10. The stoppered flask was taken and Virkon powdered detergent was added to the
pooled supernatant.
11. The lid of the flask was tightened and shaken briefly to ensure good mixing, and
the flask was disposed of.

22. ~ 20 ~
Preparation of Ni-NTA affinity resin for ScFv purification
1. A 150µl aliquot of Ni-NTA beads was taken and transferred to a 1.5ml
Eppendorf.
2. The meniscus was observed and marked on the Eppendorf using a black marker,
and the tube was centrifuged at 2rpm for 2 minutes.
3. After 2 minutes had elapsed, the Eppendorf was removed from the centrifuge
and the supernatant (methanol) was removed and disposed of.
4. The Ni bead pellet was re-suspended using 1ml of PBS (1X), and the solution
was centrifuged again at 2rpm for 2 minutes.
5. The PBS supernatant was removed from the Eppendorf and disposed of. The
beads were re-suspended using lysis buffer (PBS and 20mM Imidazole, 350mM
NaCl, 1% Triton X) up to the original meniscus.
6. The above procedure was repeated, providing two samples of washed Ni-NTA
beads.

23. ~ 21 ~
Lysis of bacterial cells and ScFv purification
1. The vials containing pellets were removed from the -20°C freezer and thawed on
ice.
2. Each pellet was re-suspended in 10ml of lysis buffer (PBS and 20mM Imidazole,
350mM NaCl, 1% Triton X)
3. The four solutions were pooled into one 50ml stoppered vial and placed into a
250ml beaker of ice.
4. The beaker containing the stoppered vial was transferred to a sonicator and the
cells were sonicated for 10 cycles (30 seconds) followed by 10 cycles of rest (30
seconds).
5. After sonication, the solution was centrifuged at 28000g for 30 minutes. The
supernatant was retained in a 15ml stoppered vial labelled “ScFv lysate 1” and
the resulting pellet disposed of.
6. One sample of the prewashed Ni-NTA affinity resin was taken and transferred to
the 15 ml vial.
7. The vial was taped onto a labroller and left to rotate for 2 hours.
8. After 2 hours had elapsed, the vial was removed from the labroller and
centrifuged at 1000g for 4 minutes.
9. The supernatant was poured off into another 15ml vial which was retained and
labelled “ScFv lysate 2”.
10. The Ni bead pellet was suspended in 1ml of 1X PBS and transferred to a 1.5ml
Eppendorf.
11. The Ni beads were centrifuged at 2rpm for 2 minutes, after which the PBS was
removed and disposed of.
12. The Ni beads were re-suspended in 1ml lysis buffer and centrifuged at 2rpm for
2 minutes, after which the lysis buffer was removed and discarded.
13. Taking a 20-200µl pipette, 200µl of elution buffer (200µl PBS and 200mM
Imidazole) was used to resuspend the Ni beads.
14. The beads were centrifuged at 2rpm for 2 minutes.
15. While the beads were centrifuging, three Eppendorfs were taken and each
labelled Elution 1, 2 and 3 respectively.
16. After 2 minutes, the beads were removed from the centrifuge and the
supernatant was transferred to “Elution 1”.

24. ~ 22 ~
17. 200µl of elution buffer was added to the Ni beads twice more, which both
supernatants being transferred to “Elution 2” and “Elution3” respectively.
18. 1ml of lysis buffer was added to the Ni beads, which were left aside for storage.
19. The above procedure was carried out again using “ScFv lysate 2” and each
elution was transferred to “Elution 4, 5 and 6”.
20. The six elutions and both bead samples were transferred to the -20°C freezer for
storage.

25. ~ 23 ~
Formulation of buffers for SDS-PAGE
1. A plastic weigh-boat was placed onto an electronic balance, and the balance was
zeroed.
2. Using a plastic spoon, 30.25g of Trizma base was accurately weighed out. This
was transferred to a 200ml beaker and solubilised in 180ml of ddH2O using a
magnetic stirrer.
3. Using a pH meter and 5M HCl, the pH of the Tris solution was corrected to be
pH 8.8.
4. The Tris-HCl solution was transferred to a 500ml graduated cylinder, and its
volume assessed.
5. Using ddH2O, the volume was brought up to 250ml to form a 1M solution of
Tris-HCl, and the entire volume was transferred to a 500ml stoppered flask,
which was labelled.
6. Using 45.40g of Trizma, the above procedure was repeated to provide a 1.5M
solution of Tris-HCl, pH 6.8.
7. Using the electronic balance, 2.5g of SDS, 36g of glycine and 7.5g of Trizma
base were weighed out and added to a 500ml beaker. These were solubilised in
500ml ddH2O, transferred to a 500ml stoppered flask labelled “5X Running
Buffer”,
8. A further 4g of SDS was weighed out and solubilised in 20ml of ddH2O to
create a 20% SDS solution.
9. A 15ml stoppered vial was taken and labelled “SDS Loading Buffer”.
10. 2ml of 20% SDS, 1.2ml of 1M Tris-HCl, 0.154g of DTT and approximately
1ml of glycerol were transferred to the 15ml vial. A dash of bromophenol blue
powder was added to the solution, which was inverted briefly.
11. Another 15ml stoppered vial was taken and, using a 100-1000µl pipette, 1ml of
ddH2O was pipetted into the vial.
12. Using the electronic balance, 1g of APS was weighed out. This was transferred
to the 15ml vial and inverted to ensure a homogenous solution. The vial was
labelled “10% APS”.
13. The buffers were taken and stored at room temperature for future use.

26. ~ 24 ~
Casting of SDS-PAGE gels and electrophoresis of ScFv sample
1. The six ScFv elutions and previously prepared buffer solutions were gathered.
2. Two gel casts were prepared by taking two glass plates and two short plates. The
short plate was pressed against the glass plate until their sides and bottoms were
flush. This was then kept in place using a clamp.
3. Taking the 20% SDS solution, 5ml were pipetted into a 15ml vial and diluted
using 5ml of ddH2O. The vial was labelled “10% SDS”.
4. Another 15 ml stoppered vial was taken and labelled “12% Resolving Gel”.
5. Aliquots of the buffer solutions were transferred into the Resolving Gel vial as
follows:
 3.4 ml of ddH2O
 4ml of 30% acrylamide
 2.6ml of 1.5M Tris-HCl pH 8.8
 100µl of 10% SDS
 4µl of TEMED
6. To this solution, 100µl of 10% APS was pipetted and the vial briefly inverted.
7. Using a 5ml pipette and pipette filler, two gels were prepared by pipetting 5ml
of the resolving gel solution into the gel casts.
8. Once the resolving gel had been pipetted into the two casts, the gels were
overlaid with 400µl of isopropanol and left to set for approximately 1 hour.
9. After the gels had set, the isopropanol was removed from the cast.
10. A 15ml vial was taken and labelled “5% Stacking Gel”.
11. Aliquots of the buffer solutions were transferred into Stacking Gel vial as
follows:
 3.4 ml of ddH2O
 850µl of 30% acrylamide
 650µl of 1.0M Tris-HCl pH 6.8
 50µl of 10% SDS
 5µl of TEMED

27. ~ 25 ~
12. To this solution, 100µl of 10% APS was pipetted and the vial briefly inverted.
13. Using a 100-1000µl pipette, the stacking gel solution was used to overlay the
resolving gel until the gel cast was full. This was repeated for the second gel cast.
14. Well combs were inserted into both gel casts and inspected for air bubbles.
15. The two gels were left to set for approximately 2 hours.
16. After the stacking gels had set, the best gel was removed from its clamp and
placed into an electrophoretic rig.
17. 200ml of the 5X running buffer was taken and diluted with 800ml of ddH2O to
produce 1L of 1X running buffer.
18. The gel was submerged in 1X running buffer, and the tank of the electrophoretic
rig half filled.
19. Taking the six elutions, 15µl of each sample was taken and pipetted into
separate 1.5ml Eppendorfs.
20. To each Eppendorf, 15µl of SDS loading buffer was added.
21. The Eppendorfs were heated to 95°C for 5 minutes using a heating block.
22. After 5 minutes had elapsed, the samples were removed from the heating block.
23. The well comb of the gel was removed and, using a 2-20µl pipette, 10µl of an
11–245 kDa protein ladder and each of the six samples were pipetted into the gel
wells from left to right.
24. The rig was closed and set to run at 100V for approximately 2 hours.
25. After the 2 hours had passed, the power supply was turned off and the gel
removed from the rig.
26. The short plate was separated from the glass plate and the stacking gel was
removed from the resolving gel.
27. The resolving gel was placed into a container of ddH2O to remove any tracking
dye from the gel surface. The ddH2O was removed and replaced with Coomassie
Brilliant Blue.
28. The container was closed and placed inside a rotary incubator overnight to stain
the protein in the gel (50rpm, 32.6°C).
29. The next day, the container was removed from the rotary incubator and the
Coomassie stain was recovered.
30. The gel was swirled gently with ddH2O three times to remove excess stain.
31. Once washed, the gel was soaked in approximately 20ml of acetic acid, and the
container was closed.

28. ~ 26 ~
32. The container was transferred to the rotary incubator (50rpm, 32.6°C) and left to
destain for 1 hour and 30 minutes.
33. After 1 hour and 30 minutes, the container was drained of acetic acid and briefly
rinsed with ddH2O.
34. Another 20ml of acetic acid was introduced to the gel, and the above procedure
repeated.
35. Once the gel had been adequately destained, the banding pattern of the gel was
visualised using a white light trans-illuminator and a photograph was taken.

29. ~ 27 ~
Concentration of ScFv from elutions 1-6, and assessment of specificity for E. coli
1. The six ScFv elutions were removed from the -20°C freezer and allowed to thaw.
2. A sample of nitrocellulose membrane was taken and cut to size (8cm x 3cm)
using a scissors.
3. Three bacterial samples (E. coli 01, E. coli 0157:H7 and Enterobacter cloacae)
suspended in LB broth were centrifuged at 1000 g for 2 minutes
4. After the samples had centrifuged, the LB broth was poured off and disposed of.
5. Using a sterile 5 ml pipette and filler, three 5ml aliquots of PBS were used to
suspend each bacterial pellet.
6. Using a 2-20µl pipette, 5µl of each suspended bacterial sample was taken and
spotted onto the nitrocellulose membrane.
7. The locations of the spots were marked using a marker and the membrane was
allowed to dry at room temperature.
8. Once the nitrocellulose membrane was sufficiently dry, the membrane was
transferred to a 50ml stoppered vial and left aside.
9. While the membrane was drying, ScFv elutions 1-3 and 4-6 were pooled into
two separate Eppendorf tubes.
10. The pooled solutions were transferred to two Vivaspin 500 columns and were
centrifuged at 15000 g for 8 minutes to concentrate the ScFv.
11. Once the Vivaspin columns had been centrifuged, both concentrated ScFv
solutions were pooled into one Eppendorf tube and left aside.
12. A 100ml sample of 10X PBS was taken and diluted with 900ml of ddH2O to
form a 1L 1X PBS solution.
13. 500ml of the 1X PBS was transferred to a 500ml stoppered flask and a 250µl
aliquot of 100% Tween was added to form a 500ml solution of 1X PBS – 0.05%
Tween Buffer.
14. Using an electronic balance, 5g of Marvel skimmed milk powder was accurately
weighed out and transferred to a 100ml beaker.
15. The milk powder was solubilised using 100ml of PBS – Tween Buffer on a
magnetic stirrer to form a 100ml solution of Blocking Buffer.
16. The vial containing the membrane was taken and the surface of the membrane
was flooded with 15ml of the blocking buffer.

30. ~ 28 ~
17. The vial was inserted into the rotary incubator horizontally for 3 minutes (50rpm,
25.5°C) to incubate.
18. While the membrane was incubating, two 15ml vials were taken and labelled
“ScFv in Milk” and “Rabbit Anti-His in Milk” respectively.
19. Using a sterile 10ml pipette and filler, two 10ml aliquots of the blocking buffer
were pipetted into both 15ml vials.
20. 30µl of the concentrated ScFv solution was added to “ScFv in Milk” and 10µl of
Rabbit Anti-His-tag antibody were added to “Rabbit Anti-His in Milk”. Both
vials were inverted to ensure homogeneity.
21. The membrane was removed from the rotary incubator and drained of the
blocking buffer.
22. The “ScFv in Milk” solution was used to flood the membrane, which was then
incubated for 1 hour (50rpm, 25.5°C).
23. After the hour had elapsed, the membrane was removed from the rotary
incubator and drained of the “ScFv in Milk” solution which was retained.
24. The membrane was flooded with 15ml of the PBS – Tween buffer and incubated
for 3 minutes, three times in order to wash the excess ScFv from the surface of
the membrane.
25. Once the surface of the membrane was adequately washed, it was flooded with
the “Rabbit Anti-His in Milk” solution and left to incubate in the rotary
incubator for 1 hour (50 rpm 25.5°C)
26. When the hour had elapsed, the membrane was removed from the incubator and
drained of the “Rabbit Anti-His in Milk” solution which was retained.
27. The membrane was washed again with 15ml PBS – Tween buffer three times.
28. Once adequately washed, the membrane was flooded using a premade solution
of HRP tagged anti-rabbit antibody in 10ml of milk and incubated for 1 hour
(50rpm, 25.5°C)
29. After the hour had elapsed, the membrane was washed as previously described,
and brought to a dark room.
30. The membrane was removed from its container.
31. The membrane was flooded with ECL substrate and left to incubate for 3 to 5
minutes.
32. Once the HRP had reacted with the ECL substrate, a sheet of X-Ray film was
placed over the membrane.

31. ~ 29 ~
33. After 5 minutes, the X-Ray film was removed from the membrane and
developed.

32. ~ 30 ~
Results
Figure 1: Polyacrylamide gel of ScFv elutions arranged from left to right: 11-245kDa protein
ladder, Elution 1, Elution 2, Elution 3, Elution 4, Elution 5, Elution 6 and Loading Buffer
Figure 2:X-Ray film showing luminescence detected from three bacterial samples spotted onto
nitrocellulose membrane: E. coli 01, E. cloacae and E. coli 0157:H7.
ScFv at approx. 27kDa

33. ~ 31 ~
Figure 3: 11-245kDa protein ladder with sizes for different bands (NEB, 2016)

34. ~ 32 ~
Discussion
The results obtained from the SDS-PAGE (Figure 2) indicate that it is possible to
genetically modify a sample of E. coli to produce soluble ScFv which can be recovered
for future use. When the gel was stained, it was observed that the majority of bands
were found in Lane 1 (Elution 1), each weighing approximately 58, 27, 25 and 11 kDa
respectively. The most relevant band was that found at the 27kDa position. As ScFvs
weigh approximately 27kDa (Shen et. al., 2005), it is reasonable to assume this band
represents the ScFv produced by E. coli and purified using the Ni-NTA resin. The other
bands were likely to be contaminating proteins derived from the host cell which were
not adequately removed during purification. As this purification served as proof of
concept, total ScFv purity was not expected nor desired (hence the decision to use Ni-
NTA beads in the bacterial solution over a true Ni-NTA column).
The results of the Dot Blot (Figure 1) indicate that it is possible to produce ScFvs which
are capable of distinguishing between different species of bacteria. After the X-Ray film
had developed, it was observed that two black dots had formed over the E. coli 01 and E.
coli 157:H7 samples, and no dot had formed over the E. cloacae sample. The formation
of black dots on the film was a positive result which indicated successful binding of
ScFv to the bacterial sample. As black dots only appeared over the E. coli strains, and
not the E. cloacae sample, it is reasonable to assume that the ScFv produced is specific
to E. coli and will not recognise different (but closely related) species of bacteria.

35. ~ 33 ~
Conclusions
The aim of this project was to investigate if ScFvs specific to E. coli could be produced
for use in NAPES to analyse water samples. Based on the results previously described,
there is a strong indication that this is possible.
Preliminary experiments indicate that ScFvs may be raised to recognise a specific
species of bacteria, but have trouble in distinguishing between different strains (i.e.,
ScFv could distinguish between Escherichia coli and Enterobacter cloacae, but not
Escherichia coli 01 and 157:H7). The extent of this specificity should be investigated
further, using a wider range of sample bacteria commonly encountered during routine
water analysis. Additionally, it may be worth investigating if this protein can be applied
to common bacteria found in other sectors which require microbial analysis (e.g.,
healthcare, food and pharmaceutical industries).

36. ~ 34 ~
Risk Assessment
Biological Chemical Physical Fire
Inhalation of powder irritationL L L L Refer to container for health
risks and associated hazards.
Wear lab coat and goggles at all
times.
Wear face mask when
preparing media.
Wear gloves when preparing
media.
Work in fume cupboard when
preparing media
Student, lecturer,
Risk Assessment
Hazard Action Required Responsible Persons
L Student, lecturer,Infection.
Cuts.
Burns.
Damage to eyes,skin
and respiratory
system.
L L L Wear lab coat and goggles at all
times.
Wash hands with germicidal
soap upon entry and egress of
the lab.
Sterilise workstation before
and after work.
Cuts.
Infection
L L L
Student, lecturer,
Student, lecturer,
Only use microbial cultures
provided by lab technicians.
Only use Group 1 and Group 2
organisms.
Employ correct aseptic
technique.
Only store microbial cultures in
designated
containers/cupboards.
Autoclave all biological waste
before disposal.
Report all spillages to
supervisor.
L L L L
Wear goggles.
Check all glassware for cracks.
In case of broken glass,
carefully remove the glass from
the area and dispose of in the
designated glassware bin. If
the glassware has been or is
suspected of biological
contamination, place in the
sharps container to be
incinerated.
L
Infection. L L L L
Infection. Student, lecturer,
Only use microbial cultures
provided by lab technicians.
Dispose of in biological hazard
bag for autoclaving.
Report all spillages to
supervisor

37. ~ 35 ~
Wear lab coat and goggles at all
times.
Wear gloves when carrying out
stain.
Wear face mask when carrying
out stain.
Wash hands/eyes immediately
if contact occurs and seek
medical attention as
appropriate.
L Student, lecturer,Chemical irritation.
Damage to eyes,
skin, respiratory
system and clothing.
L L L
Chemical irritation.
Damage to eyes,
skin, respiratory
system and clothing.
L M M L Wear lab coat and goggles at all
times.
Wear gloves when handling
concentrated acid.
Wear face mask when handling
concentrated acid.
Wash hands/eyes immediately
if contact occurs and seek
medical attention as
appropriate.
Student, lecturer,

38. ~ 36 ~
References
1. Andris-Widhopf, J., Rader, C., Steinberger, P., Fuller, R. and Barbas, C.F.
(2000). “Methods for the generation of chicken monoclonal antibody fragments
by phage display”. JIM. 242, pp. 159 - 170
2. Cunningham, S., Starr, E., Shaw, I., Glavin, J., Kane, M. and Joshi, L. (2013).
“Development of a convenient competitive ELISA for the detection of free and
protein-bound non-human Gal-a-(1,3)-Gal epitope based on novel highly
specific chicken ScFvs”. Anal. Chem. 85, pp 949 - 955
3. Deegan, S. (2016). Personal Communication, Aquila Bioscience, Ireland
4. Garvey, P. and McKeown, P. (2007). “Epidemiology of Cryptosporidiosis in
Ireland, 2004”. HPSC. pp.2-6
5. Kemeny, D.M. and Challacobe, S.J. (1988) “Application of ELISA to
Microbiology”. In ELISA and Other Solid Phase Immunoassays: Theoretical
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and Sons, Chichester, pp. 325 – 326
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P., Guillot, E., Mabilat, C., Newport, L., Niemi, M., Payment, P., Prescott, A.,
Renaud, P. and Rust, A. (2003). “Analytical Methods for Microbiological Water
Quality Testing”. In Assessing Microbial Safety of Drinking Water: Improving
Approaches and Methods (Fewtrell, L., ed.), IWA Publishing, London, pp. 237 –
253
7. Madigan, M., Martinko, J., Stahl, D., and Clark, D., (2012) “Wastewater
Treatment, Water Purification, and Waterborne Microbial Diseases”. In Brock
Biology of Microorganisms, 13 (Espinoza, D., ed.) Pearson, San Fransisco, pp
1033 – 1035
8. NAPES (2016) [Online] Available at: http://www.napes.eu/ [Accessed 12/5/16]
9. New England Biolabs (2016) “Color Prestained Protein Standard, Broad Range
(11–245 kDa)” [Online] Available at: https://www.neb.com/products/p7712-
color-prestained-protein-standard-broad-range-11-245-kda [Accessed 10/5/16]
10. Shen, Z., Stryker, G.A., Mernaugh, R.L., Yu, L., Heping, Y. and Xiangqun, Z.
(2005) “Single-Chain Fragment Variable Antibody Piezoimmunosensors”. Anal.
Chem. 77,(3), p. 797