Clostridium chauvoei Toxin A analysis
Title page
Clostridium chauvoei Toxin A analysis
Author
Lea Elena Haaning Normand
Education
Academy Profession (AP) graduate in Chemical and Biotechnical Science
Class:
5laba0911
Number of signs:
45.726 signs (without spaces)
Supervisor:
Professor Joachim Frey,
Student counselor Astrid Koggersbøl Skadborg
Abstract
This project deals with Clostridium chauvoei Toxin A analysis, and the wish to produce an
inactive toxin. A recombinant plasmid has been created, by cleaving the Toxin A gene and
clone a Kanamycin resistant gene into the Toxin A gene. The process has included plasmid
extraction, digestion, cloning, protein expression and protein purification. All steps have
been successful, and have resulted in a recombinant plasmid containing the inactive Toxin
A gene. The theory has been proven by adding purified protein from the recombinant
plasmid on a blood agar plate, along with the protein of the full toxin, and check if there is
a hemolytic effect.
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Clostridium chauvoei Toxin A analysis
Table of contents
Regulation ................................................................................................................................................... 4
Introduction................................................................................................................................................. 5
Materials and Methods ................................................................................................................................ 6
Solutions and agars ............................................................................................................................. 6
Plasmid Vector .................................................................................................................................... 6
Praxis .............................................................................................................................................. 6
Insert .................................................................................................................................................. 7
Praxis .............................................................................................................................................. 7
Extraction of plasmid .......................................................................................................................... 7
Homemade method -Without column................................................................................................. 7
Chemicals and theory behind. ......................................................................................................... 7
PeqGOLD MiniPrep Extraction kit (Peqlab, web) .................................................................................. 8
Praxis .............................................................................................................................................. 9
Digestion............................................................................................................................................. 9
Praxis .............................................................................................................................................10
Ligation ..............................................................................................................................................11
Praxis .............................................................................................................................................12
Electroporation ..................................................................................................................................12
Praxis .............................................................................................................................................13
Heat shock transformation .................................................................................................................14
Praxis .............................................................................................................................................14
Protein expression and SDS gel ..........................................................................................................15
Praxis .............................................................................................................................................16
Protein purification ............................................................................................................................17
Praxis .............................................................................................................................................18
Hemolytic activity (toxicity text) .........................................................................................................19
Results and discussion ............................................................................................................................... 20
Extraction and digestion of the vector ................................................................................................20
Extraction and digestion of the insert......................................................................................................... 22
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Clostridium chauvoei Toxin A analysis
Ligation of cctA and Km; Electroporation and digestion.............................................................................. 23
Extraction of plasmid; transformation into expressing E. coli strain ............................................................ 25
Expression of the protein and SDS gel ........................................................................................................ 27
Protein purification .................................................................................................................................... 28
Toxicity test ............................................................................................................................................... 31
Conclusion ................................................................................................................................................. 32
Literature/references................................................................................................................................. 34
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Clostridium chauvoei Toxin A analysis
Regulation
For a better understanding reading this project, this section covers the structure of this report.
This final examination project covered approximately 7 week; 5 weeks of laboratory work, and 2 weeks
of data processing.
For every section in this report, the theory of the method will be described followed by a description of
the experiment in praxis. This will show the reader how the work is performed step by step.
Moreover, the result and discussion will be in the same section, since all results have been validated
step by step before continuing.
References will throughout the project be referred using APA reference system (AAU, studiehåndbog,
huminf, web).
A few words will be shortened throughout this report.
___________________________
Lea Elena Haaning Normand
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Clostridium chauvoei Toxin A analysis
Introduction
Presentation of Clostridium chauvoei. (Hereafter C. chauvoei).
C. chauvoei is an anaerobic, motile gram positive bacterium, capable of form spore and gas producing.
C. chauvoei is the pathogenic agent of “blackleg”, a severe global disease of cattle, sheep and other
domestic animals. Infections of ruminants with C. chauvoei cause myonecrosis (necrosis of muscular
tissues), gas gangrene and serious toxemia (blood poisoning) with high mortality rate. Recently a toxin
has been detected and identified as, belonging to the leucocidin superfamily of bacterial toxins: the CctA
toxin. It is secreted by all strains of C. chauvoei tested as far, originating from the whole globe and
isolated during the last 60 years (Frey et al. 2012, web). This toxin is also suggested to be the main
virulence attribute of C. chauvoei.
In spite of the efficacy of current blackleg vaccines, there is no knowledge on which antigens in current
formulations are protective. As a result commercial blackleg vaccines comprise chemically toxoid
supernatant and inactivated bacteria. The potency of vaccine batches currently needs to be monitored
by a challenge model in guinea pigs (European Pharmacopoeia 7.7 p. 5333). In this test the vaccine
under assessment is administered to guinea pigs, which are later challenged with a virulent strain of
C. chauvoei, together with a group of unvaccinated control guinea pigs. For the test to be valid all of the
control animals must die within three days and for the vaccine to pass the test at least 90% of the
vaccinated guinea pigs must survive for at least five days post challenge.
The main goal of the project is to finally develop an in vitro test for vaccine batch release to replace the
animal test that is currently legally compulsory.
Therefore a recombinant CctA protein that is no more toxic, respectively no more hemolytic has to be
constructed.
This project represents the first step in this approach.
Problem formulation:
Can we produce an inactive toxin gene of cctA by a deletion of a part of the cctA coding region
using a pre-existing plasmid containing the cctA gene in Escherichia coli? (By in vitro
mutagenesis)
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Clostridium chauvoei Toxin A analysis
Materials and Methods
Solutions and agars
First is added a short list of solutions and agars that have been used repeatedly throughout this project.
o
o
o
o
o
o
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LB-broth. Made with Difco™ LB-Broth powder, containing: Tryptone, Yeast extract and sodium
chloride.
LB agar plates. Made with Difco™ LB-agar powder, containing: Tryptone, Yeast extract, Sodium
chloride and agar.
LB agar plates containing Amp + Km in a concentration of 50 µg/ml. The agar plates containing
antibiotic are made with adding antibiotic in the agar, and then casting the plates.
Sterile H2O.
Sterile 10% glycerol.
50 mg/ml Kanamycin
100 mg/ml Ampicillin
See Appendix 1.1, 1.2, 1.3 for volume and calculations.
Plasmid Vector
This section will describe what is understood by a plasmid vector and mention some special features a
plasmid vector contains. A plasmid is a piece of double stranded circular DNA, used to carry a foreign
piece of DNA into a host cell, where it can be replicated. Therefor it is also call a vector. Plasmid vector
are used in molecular cloning. The plasmid vector contains 3 very important features; an origin of
replication, an antibiotic resistant gene, which are used as a selective marker and a cloning site, where
the foreign piece of DNA is inserted. Important to know about the plasmid vector is, which antibiotic
resistant gene it contains and what copy number plasmid it is, when extraction method is chosen.
The vector has to be purified and cleaved open, before it is ready to use. Afterwards the plasmid is ready
to obtain a foreign piece of DNA, an insert.
Praxis
The plasmid vector chosen for this experiment, is a recombinant plasmid, containing the Toxin A gene
(CctA) called (pJFFCctANusA1), taking from E. coli strain JF 4415 (Frey et al. 1012, web). CctA stands
for Clostridium chauvoei toxin A. This plasmid is a High copy number plasmid and has an Amp resistant
gene. Note for a later mentioning: 6x histidine tags have been genetically engineered into this plasmid.
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Insert
To be able to create a clone or a recombinant plasmid, an insert is needed. An insert is the DNA piece of
interest. It is the DNA sequence wanted to be cloned into a vector.
Praxis
In this experiment plasmid pHP45Ω-Km is chosen. However, in this case only a part of pHP45Ω-Km, a
Kanamycin resistant gene (Km), is wanted as the insert. This insert needs to function by stopping the
expression of the CctA gene. Moreover, a double selection of antibiotic is created. In further mentioning
this insert will be referred to as Km.
Extraction of plasmid
The needed plasmids are inside an E. coli host; therefor they must be extracted and thereby isolated
before they can be used for further experiments. Whether you use one method or choose another, it
will be based on the same general principals. However, few differences appear. The next section will
describe a homemade protocol using phenol/chloroform extraction and an extraction method using a
kit.
Homemade method -Without column
The first method to be described is the homemade method. This method is based on using
phenol/chloroform for extraction the DNA.
Chemicals and theory behind.
See Appendix 2.1 for preparation of chemicals and calculations.
To extract a plasmid from a host, colonies grown in nutrient broth is needed. After growth the cells
are harvest by centrifugation and supernatant is removed. The cells are now ready for lysis. First is
added:
o
Tris-HCl as a pH stabilizer and EDTA that inactivates nucleases (DNase and RNase) which can
be harmful to the plasmid DNA by degradation.
o
Lysis buffer (Alkali) containing NaOH and SDS, with a high pH value. The SDS is a detergent
and denatures protein. SDS is often used to lyse Gram negative bacteria, like E. coli as it
degrades the lipid membrane of the cell. NaOH is a base that denatures the genomic DNA and
the Plasmid DNA. However, plasmid DNA does not denature completely.
o
Neutralization buffer is a mix of acetic acid and potassium acetate with a pH at 5.5, to make
the plasmid and genomic DNA renature. The lower pH value is the reason for the DNA to
renature, because the pH value will return to neutral after the alkaline treatment of NaOH and
SDS. The genomic DNA is not able to renature correctly, and will instead precipitate as a white
clump. After a centrifugation, the genomic DNA will lay as a pellet (Qiagen, web). The plasmid
DNA however, is tied together as rings, renatures, and will stay in the supernatant. The
supernatant is removed to a new tube for further treatment.
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Clostridium chauvoei Toxin A analysis
o
Phenol/chloroform/isoamyl alcohol (PCIA). PCIA is added to the supernatant and function by
removing all the proteins, by splitting into an organic phase and an aqueous phase. Phenol will
denature and precipitate proteins, chloroform will furthermore denature and precipitate
proteins, but at the same time provide a greater mass volume, assure the organic phase will
be at the lower of the tube. Isoamyl alcohol will prevent foam and deactivate RNase activity
(Sigma Aldrich 1, web).
The pH is of high importance using PCIA extraction. A pH value of 7 or higher will keep the
DNA negatively charged and in the aqueous phase. If the pH drops below 7, there will no
longer be a negatively charge in the DNA and it will go into the organic phase (Sigma Aldrich 2,
web).
After centrifugation the upper aqueous phase is transferred to another tube for further
treatment.
o
Chloroform/isoamyl alcohol is added, to remove further proteins and deactivate DNase. After
centrifugation the aqueous phase is transferred to another tube.
o
Isopropanol is added to the sample to make the DNA precipitate.
It is kept cold (-20 °C) for at least 20 minutes. Keeping the sample with isopropanol cold, will
make more DNA precipitate.
However, it will also produce more salt, so washing 1-3 times with 70 % EtOH is necessary to
remove the salt, before re-suspending DNA. (Prof. Joachim Frey, Head of Institute, University
of Bern).
o
RNase A: 50 µg/ml in TE-buffer. RNase A in TE-buffer is highly stable, also towards alkaline
compounds and detergent, and will remove RNA from the sample.
The DNA is re-suspended in TE-buffer or H2O.
The full protocol can be found in Appendix 2.2.
Caution must be taken when using PCIA
Phenol is local anesthetic and corrosive. It injures skin and in particular eyes. Chloroform is an
anesthetic, which affects the central nervous system. Isoamyl alcohol can cause respiratory irritations
and eye irritations.
PeqGOLD MiniPrep Extraction kit (Peqlab, web)
Using extraction kit is easier and less time-consuming than the homemade method. The main
principles are the same for both methods. However, kit extraction uses a column to bind the plasmid,
premade solution and no phenol, chloroform, isoamyl alcohol or isopropanol. In this matter the kit is
also safer to use. The solutions for the kit will not be discussed any further, because they are based on
the same principals as the solutions mentioned for the homemade method. The full protocol for
extraction of DNA using PeqGOLD MiniPrep extraction kit can be found in Appendix 2.3
The reason for using two extraction methods during this experiment is to optimize the homemade
method, so it will provide as good results as the kit, and thereby excluding using the kit in the future.
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Praxis
To begin this experiment the chosen plasmid has to be extracted from the bacterium host, in order for
the plasmid to be used for cloning. In this case a glycerol stock of the chosen bacterium containing
plasmid pJFFCctANusA1 is collected, and a small mass of bacterium is streaked out on LB agar plates.
The plates which contains Ampicillin (hereafter Amp) in a concentration of 50 µg/ml, is incubated
overnight at 37°C, in order to obtain colonies. Parallel, the bacterium JF 287 containing the plasmid
pHP45Ω-Km is grown up on LB plates containing 50 µg/ml Kanamycin (hereafter Km).
When colonies appear, a colony swab is taken, using a sterile plastic inoculation needle, and
inoculated in a sterile tube containing 3 ml LB broth + Amp for bacterium containing pJFFCctANusA1
and 3 ml LB-broth + Km for bacterium containing pHP45Ω-Km, in a final concentration of 50 µg/ml.
The tubes are incubated overnight, shaking, at 37°C.
Plasmid pJFFCctANusA1 is extracted using both the homemade method and the extraction kit. Both
methods are chosen in order to compare the efficiency of extraction the Plasmid DNA and DNA
amount that is the end-result of the two methods.
However, in this experiment the plasmid pJFFCctANusA1 extracted by PeqGOLD MiniPrep extraction
kit is chosen for the further experiments, because the homemade method provide a smaller amount
of DNA and still need to be optimized.
PCIA is combined with high danger, therefor; steps containing the use of PCIA and
Chloroform/isoamyl alcohol are done using Laminar flow, wearing nitrile gloves and eye protection.
Plasmid pHP45Ω-Km is extracted using only PeqGOLD MiniPrep extraction kit, to obtain the best
result.
In this project the DNA have been re-suspended in both TE-buffer with RNase A in a concentration of
50 µg/ml and H2O alone.
After re-suspension, 100 µl of solution containing the plasmid is kept at -20°C. The extracted plasmid
is now ready for further experiments.
Digestion
This section will describe the basic theory of digestion. Digestion of the vector and the insert is
necessary, in order prepare them for cloning. Digestion with restriction enzymes means that enzymes
will recognize and cleave a specific base sequence in double stranded DNA.
The enzymes used for cleaving are restriction enzymes that are naturally found inside different bacteria,
where they function by cleaving foreign DNA to protect the cell. Up until now, more than 3000 enzymes
have been detected from all around the world. Of these 3000 enzymes, 200 different sequences are
listed, which means that most restriction enzymes are duplicates (Kielberg et al, 2003, page 77).
There exist different types of restriction enzymes (New England Biolabs 1, web), but since knowledge
about the different enzymes is not relevant for this experiment, they will not be discussed any further.
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Depending on the restriction enzymes, there are two different ways to cleave DNA strings. One will
make overhangs of single stranded DNA. This is called “sticky ends overhang”. These overhangs can be
cleaved to give a 5’ overhang or a 3’ overhang (see picture 1 below). Cleavage with overhang is the
most common way to digest plasmids. The other way of cleavage is called “blunt end” and will cleave
the DNA right through (picture 2).
Picture 1. 5’ overhang created with HindIII
Picture 2. Blunt ends
Created by SmaI
Most enzymes used for cloning recognize sequences of 4, 5 or 6 bases. The enzymes work by breaking
the phosphodiester bonds between 2 nucleotides. After cleaving, a free 5’- phosphate group on one
nucleotide, and a free 3’- OH group on the other nucleotide is created. Cleaving will now provide a
linear piece of DNA, ready for ligation.
Praxis
The next step for producing a recombinant plasmid is to digest the vector with the appropriate
restriction enzymes, and thereby make the vector ready for cloning. For plasmid pJFFCctANusA1 (the
vector) the wish is to cleave the DNA sequence in one specific place, where the toxin A is located. In the
middle of toxin A gene is a HindIII restriction site.
Using HindIII restriction enzyme to cleave means that, the cctA gene will be cleaved, split apart and
should no longer be active. Cleaving will then create new 5’ overhang ends. See Appendix 3.1 for HindIII
restriction site in the cctA gene. The plasmid is firstly double digested in order to obtain a clearer
fragment and check if the vector was okay. (Prof. Joachim Frey, Head of Institute, University of Bern).
The Km gene originates, as mentioned, from another plasmid pHP45Ω-Km. The Km gene has a HindIII
restriction site in both ends, which means: This enzyme is the one that has to be used in order to get the
whole Km gene (see picture 3 below).
Picture 3. Plasmid pHP45Ω-km with the Km gene
This plasmid is however, has also an Amp resistant gene, which is not needed.
To use only the Km resistant gene, the Amp gene has to be removed. In order to do this, a restriction
enzyme that separates these two is needed.
pHP45Ω-Km is cleaved one time with HindIII alone, in the hope of separation and thereby being able to
extract the Km gene directly from the gel.
However, using HindIII alone makes the separation of the two genes so small, that a gel extraction is not
possible. Therefore pHP45Ω-Km was also cleaved with PvuI and HindIII. The enzyme PvuI is chosen after
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Clostridium chauvoei Toxin A analysis
inspection of the plasmid, showing all restriction sites for the Km and the Amp gene.
The inspection of the plasmid and restriction site is done in collaboration with Prof. Joachim Frey.
PvuI shows to cleave in the Amp gene, dividing it into 2 fragments, and leaves the Km gene untouched.
Cutting the Km gene with HindIII will give a fragment on approximately 2000 base pair. The Amp
fragment is 2320 base pair in total.
The digestion of both plasmids is carried out as followed:
Table 1. Digestion volume
The different compounds are mixed in an Eppendorf tube. For pHP45-Km a double digestion has taken
place. All restriction enzymes used are from Roche, and the choice of cutting buffer is chosen by their
recommendations. The amount of cutting buffer and restriction enzyme are standard in this institute
(Institute of Veterinary Bacteriology, Bern University). If more DNA is needed, less H 2O is added. For
temperature and time of digestion, see Appendix 3.2.
After digestion, an analytic agarose gel electrophoresis is performed, to check if the digestion has been
successful, and to obtain approximately measure on the sizes of the DNA fragments.
Gel




Electrophoresis was performed with a 0,8% agarose gel in 0,5x TBE buffer.
RedSafe is used as dye for the DNA.
λ-Hind is used as a marker.
Loaded on the gel is the total amount of digestion.
See Appendix 3.3 for agarose weight and volume gel.
After confirmation of successful digestion, both plasmids are digested again, using the double amount of
plasmid volume (See table 1 for prior volume) and are now ready for ligation.
Ligation
When the chosen vector and insert has been cleaved with restriction enzymes, a ligation can be made,
using DNA ligase. Ligation is the cloning step, where a new recombinant plasmid is created. This ligase
enzyme works by synthesize the phosphodiester bond between the 3’-OH group on one nucleotide and
the 5’-phosphate group on another nucleotide (New England Biolabs 2, web). If the overhangs are
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cleaved with the same restriction enzyme or restriction enzymes that give matching overhangs, the base
pairs will match with the plasmid and the insert and they will be “glued” together.
In the living organism DNA ligase is the essential enzyme that connects the small fragments after the
DNA polymerase in the replication.
Praxis
After digestion of plasmids pJFFCctANusA1 and pHP45-Km are cleaved open with matching 5’ HindIII
overhangs and they are ready to be ligated. In this experiment, T4 DNA ligase has been used.
The T4 DNA ligase is an enzyme from bacteriophage T4.
The ligation is made like this:
The ligation buffer must be added in the amount of 10 x the total volume. The ligation is incubated in
the fridge overnight.
When ligation is finished, there should in theory have been made a new clone. This is recombinant
plasmid.
Electroporation
After ligation a clone should have been created. However, in order to investigate if the cloning has been
a success and to isolate the new plasmid, the mix of ligated plasmid and insert must be transformed and
replicated. This will mean that the newly made recombinant plasmid has to be transformed into a host
cell, usually a fast growing bacteria like E. coli. The plasmid, which contains an origin of replication, will
be replicated by the bacterium as the bacterium’s own DNA.
After transformation it is essential to place the cells on antibiotic agar plates which match the resistant
gene inside the plasmid, in order to select clones. Bacterium who does not contain the plasmid will not
be able to grow on antibiotic plates, while the bacteria who have obtained the recombinant plasmid
during transformation will contain both antibiotic resistant genes and grow.
Electroporation is a common and very efficient method for transformation, especially if the bacteria’s
needs to obtain larger fragments of DNA. By electroporation the cells will be exposed to a brief, but
efficient electro pulse. The electro pulse will open pores on the cell membrane and the DNA will pass
through into the cells (Primrose, S.B. and Twyman, R.M, 2006, p 25). The competent cells used for this
method of transformation, needs to be prepared in a specific way without salt. If the level of salt is too
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Clostridium chauvoei Toxin A analysis
high, it can cause a discharge of electricity, meaning lack of transformation. Directly after the electro
pulse, a nutrient broth is added, to make the cells recover after electroporation.
The electro competent cells are prepared as described in Appendix 4.1
Praxis
After ligation of pJFFCctANusA1 + Km the two should now be linked together, creating a new
recombinant plasmid. The new plasmid now needs to be isolated and therefor transformed into an E coli
DH5α bacterium. At the same time this will also prove if the ligation has been successful, and by that
obtain the plasmid.
Based on previous experience using electroporation as a transformation method in this institute
(Institute of Veterinary Bacteriology, Bern University), the parameters for transforming E. coli by
electroporation, is chosen as showed in table 2.
Table 2. Electroporation parameters.
1 ml LB-Broth is added to the electroporation cuvette directly after electroporation and incubated 1.5
hours at 37°C, shaking.
After incubation, 50 µl of the ligation mix is added on 15 LB agar plates, containing Amp + Km in a
concentration of 50 µg/ml.
Since the new plasmid should contain both antibiotic genes, it is crucial to add the bacterium on agar
plates containing both Km and Amp as selective antibiotics.
50 µl of the control is as well added on LB Amp + Km agar plates (same antibiotic concentration).
Furthermore plates containing Amp alone is used as control.
All plates are incubated two days at 37°C.
After growth, in total, 9 colonies are picked and grown in liquid media overnight, shaking at 37°C (5
colonies from 1 Amp + Km plate and 4 colonies from 1 Amp plate). 6 of these are furthermore extracted,
using the PeqGOLD MiniPrep extraction kit.
A 100 µl solution containing the plasmid is saved at -20°C.
The extracted recombinant plasmid will be called cctA::km_copy (1, 2, 3, 4, 5, and 9) (DH5α)
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After extraction of the plasmid, a new digestion is performed, using restriction enzyme HindIII (same
protocol Appendix 3.2). After digestion an agarose gel electrophoresis is made, to investigate the size of
the plasmid, and there by confirm a successful cloning.
Electroporation is chosen, because it is a rather large fragment to transform into the E. coli bacterium.
Gel




Electrophoresis was performed with a 0,8% agarose gel in 0,5x TBE buffer.
RedSafe is used as dye for the DNA.
λ-Hind is used as a marker.
Loaded on the gel is the total amount of digestion. Note this gel is only for analytical purpose.
See Appendix 3.2 for agarose weight and volume TBE.
After investigation of the recombinant plasmid cctA::km_copy (1, 2, 3, 4, 5, 9) (DH5α) on the agarose
gel, the chosen of these can be transformed into an express vector.
Heat shock transformation
This method of transformation is also often used. The principle with this method is to destabilize the
membrane of the bacteria with CaCl2 and a heat shock. This facilitates uptake of DNA and particular
plasmids.
The transformation is carried out by placing the tubes, containing competent cells and plasmid on ice for
30 minutes. One theory about the success of the method says, mixing the cells with cold calcium
chloride will create holes in the membrane of the cell (BiotechArticles, web). It is proven that plasmid
DNA and E. coli works well together at low temperature in calcium chloride solution (S.B. Primrose and
R.M. Twyman, 2006, p 24).
After incubation on ice, the cells are exposed to a heat shock. This will make the plasmid enter the cells.
Finally the tubes are placed back on ice. A nutrient broth is added to make the cells recover the
transformed bacteria.
For Heat shock transformation, cells dissolved in calcium chloride are needed.
Praxis
In this experiment the recombinant plasmid needs to be transformed into a special E. coli strain
(BL21(DE3)), in order to express the protein corresponding to the cctA gene. In this case two clones,
cctA::km_copy5 and cctA::km_copy9 (DH5α) are chosen for further transformation.
Firstly, competent cell of the express vector, dissolved in calcium chloride is needed. In this project the
E. coli strain BL21(DE3) is chosen. See protocol for preparation of competent cells for this experiment in
Appendix 5.1.
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2 x 100 µl of competent BL21(DE3) cells are kept on ice, and 2 µl of plasmid (cctA::km_copy5 and
cctA::Km_copy9(DH5α)) is added in each of their tube. See Appendix 5.2 for the protocol of Heat shock
transformation done in this experiment.
1 ml LB broth is added to both tubes, and they are incubated 1 hour at 37°C. After incubation, 50 µl
liquid from each tube is added on 2 agar plates, containing a mix of both Amp and Km of 50 µg/ml. The
plates are incubated overnight at 37°C.
After the transformation and incubation overnight, the only 2 colonies appeared from
cctA::Km_copy5(DH5α) are picked from the plate and randomly 4 colonies are picked from the plate
containing cctA::Km_copy9(DH5α).
All are grown up in 3 ml LB broth containing Amp and Km of 50 µg/ml.
Plasmid is extracted using PeqGOLD MiniPrep extraction kit (method mentioned earlier). The extracted
plasmid is digested with HindIII and electrophoresed to check the size of the plasmid and confirm a
successful transformation with E. coli BL21(DE3).
Gel




Electrophoresis was performed with a 0,8% agarose gel in 0,5x TBE buffer.
RedSafe is used as dye for the DNA.
λ-Hind is used as a marker.
Loaded on the gel is the total amount of digestion. Note this gel is only for analytical purpose
See Appendix 3.2 for agarose weight and volume TBE.
The new recombinant E. coli will be called cctA::km_copy5 (BL21) and cctA::km_copy9 (BL21). Next is to
figure out, if the protein of this plasmid can be expressed.
Protein expression and SDS gel
After transformation into an express vector, it is possible to express a protein one specific gene is coding
for, from the plasmid. After expression of this protein an SDS protein gel electrophoresis can be
performed to separate the proteins and analyze them.
Protein expression is commonly used in the world of biotechnology, and means to produce a specific
protein, for example in the purpose of use in the medical industry (Britannica, web). To express a
protein, translation in the bacteria containing the plasmid, must be induced. For that, the chemical
isopropyl β-D-thiogalactosid (hereafter IPTG) is commonly used and works as an inducer for
transcription of cloned genes on express vectors.
Transcription is the beginning of producing a new protein. Inside the cell the process of gene expression
is progressing as following: transcription from DNA to mRNA, and translation from mRNA to the protein
the following gene is coding for. Transcription will copy the DNA string into a single stranded RNA string,
but will still be identical to the template. Now the translation can begin, by translating the base pair into
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amino acids. When all the amino acids have been translated (in triplets), the protein is produced
(Kielberg et. al, 2003, p. 34).
After induction and expression of the protein, the samples can be prepared for SDS analysis.
SDS gel electrophoresis is a method to separate proteins and possibly identify proteins. The molecular
mass of the proteins is the factor for separation of the proteins. Proteins are built in chains by the 20
different amino acids.
In order to separate the proteins by SDS gel electrophoresis, the proteins must be denatured in order to
obtain the primary structure of the protein, which is linear. To denature the protein, samples are mixed
with a loading buffer containing Β-mercaptethanol which breaks the disulfide bonds in the proteins.
Bromphenol blue which is a dye that visualizes the samples, so they can be followed through the gel.
Furthermore it contains glycerol that will provide a greater density and thereby make the sample stay in
the bottom of the well (ucsf, University of California, San Francisco, web). The buffer also contains SDS
that is a detergent which dissolves the membrane of the protein, strengthen them and provide the
proteins with a negative charge (Department of Biology, Davidson College, web). After mixing, the
samples are heated to 95°C for 15 minutes to complete denaturing (Experimental Biosciences, web).
To separate the proteins, a SDS protein gel is needed. The SDS protein gel is build up in a 3- dimensional
network, making it more difficult for bigger proteins to pass through, and smaller proteins to pass
quicker, because of their smaller molecular mass.
The SDS-PAGE electrophoresis has the same principle as an agarose gel electrophoresis. The samples
move from the cathode towards the anode or from negative to the positive pol, through the 3dimensionel network.
On the SDS gel; samples are loaded along with a protein marker with known sizes of protein as a
standard.
After running, the gel must be stained. Often a compound called Coomassie Blue is used for staining.
After a certain staining time, the gel is de-stained, before it is possible to see the bands of the proteins
and possibly the marker. After de-staining, band should appear clear, and can now be measured for size.
Praxis
Even when there have been growth of new colonies on Km and Amp plates after transformation, the
ability of being able to express the protein of cctA::km_copy 5 and cctA::km_copy 9 (BL21) have still not
been confirmed. Therefor an expression of these two clones is carried out. Moreover, an SDS protein gel
is needed to prove expression of the protein, and thereby also a successful transformation into the
express vector. This means that this step is only carried out for analytical purpose.
For further mentioning:


cctA::km_copy5 (BL21) will be referred to as JF 5509.
cctA::km_copy9 (BL21) will be referred to as JF 5510.
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For the protein expression, a few colonies of strain JF 5509 and JF 5510 containing the new plasmids are
inoculated in each 3 ml LB-broth containing Km and Amp in a concentration of 50 µg/ml. The bacteria
are grown at 37°C until they appear dense. This takes approximately 2-3 hours. At this time, the bacteria
are suspected to be in the logarithmic (or exponential phase) and are making a lot of cell divisions. Then
IPTG is added in a final concentration of 1mM and continues to grow for 2 hours. See Appendix 6.1 for
stock solution and calculation of IPTG. At the same time, tubes without IPTG are grown as controls.
While samples are incubated, the SDS gels are prepared. Chamber is build up, to contain 4 x 10% gels.
See Appendix 6.3 for chemicals and amount in the gel.
The compounds in SDS gels are highly toxic and the SDS gels are prepared under Laminar flow, see
Appendix 6.4 for safety on SDS gels.
When the samples are ready, they are centrifuged in order to obtain a pellet. Supernatant is removed
and pellet is re-suspended in 1ml PBS solution. 300 µl sample and 300 µl SDS loading buffer is mixed in a
separate tube. See Appendix 6.2 for full protocol.
In the SDS chamber, running buffer containing glycine, SDS, Tris and H2O is added. Afterwards the
samples JF 5509 and JF 5510 and negative control are, in this project, loaded on the gel in the amount of
30 µl. A protein marker is loaded in the amount of 10 µl. The negative controls are the non-induced
samples, prepared in the same way. A 10 % gel is used for the analysis and run for approximately 2
hours in total.
The gel is stained in Coomassie Blue for approximately 40 minutes. Coomassie Blue is a mix of
Coomassie R250 which is a chemical dye, Methanol, acetic acid and H2O. Afterwards the gel is destained in a mix of Methanol, acetic acid and H2O until bands appear.
The gel rest in H2O to regain its square shape.
When an expression of the protein have been confirmed by this analytic SDS protein gel, a large amount
of LB-broth containing bacterium and plasmid can now be prepared for the purpose of protein
purification.
Protein purification
Protein purification can be done in different ways. In this project the purification will be done by affinity
chromatography, using nickel nitrilotriacetic acid columns (hereafter Ni-NTA columns). The affinity is
towards the 6x histidine tags, which have been genetically engineered into original recombinant plasmid
(pJFFCctANusA1, mentioned in the section of the plasmid vector).
Firstly the Ni-NTA column and the samples has to be prepared for denature condition. For the samples it
means that the proteins need to be in a buffer that will separate their 3- dimensional structure
(secondary, Tertiary and Quaternary structure) and make them soluble. This happens when the sample
is dissolved in GuHCl (guanium chloride). The proteins need to be soluble for purification in Ni-NTA
column (Prof. Joachim Frey, Head of Institute, University of Bern). The Ni-NTA column is, at the same
time prepared for handling the denatured protein. To make the samples ready to be dissolved in GuHCl,
they are sonicated, which is a mechanic way of cell lysing. Sonication will disrupt the cells and release
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the protein (They University of Sydney, web). After a centrifugation step, the supernatant containing
dissolved protein is kept to load onto the Ni-NTA column.
Before applying the supernatant to the column, a sample called Crude Extract is collected from the
supernatant. The Crude Extract, which contains all the protein from the supernatant, will be used as a
control on an SDS protein gel after purification through the column. The Crude Extract is used to check
if protein has been present in the sample before loading it onto the column.
The protein is eluted from the column by dropping pH values of the buffer.
After elution from the column, the protein needs to be dialyzed, which will separate small molecules
and salt from the protein. The protein is transferred into a special dialyze-membrane, which allows the
smaller molecules to go out to the buffer, but keep the bigger molecules, like proteins, inside the
membrane. After dialyze the liquid from the membranes will be removed and saved for analysis (NCBI,
web).
Collected from the Ni-NTA column is Flow Through. Flow through is the first sample to pass through the
column, before elution. This is used as a control, to see how well the Ni-NTA column is holding back the
proteins.
Praxis
When the protein expression and thereby also a successful transformation into the express vector
BL21(DE3), has been confirmed by an analytic SDS-gel electrophoresis, purification of the protein from
the plasmids can be started. Purifying the protein is necessary before a test for hemolytic activity can be
performed. Therefore, cultures of 50 LB-broth with strain JF 5509 and JF 5510 containing the new
recombinant plasmids are grown until OD600 has reached ≈ 0.5.
Table 3. Culture growth
When OD600 has reached ≈ 0.5 (mid exponential growth phase), 50 µl IPTG is added in a final
concentration of 1 mM. Growth is continued for 2 hours. After induction, the samples and the Ni-NTA
columns are prepared by following Qiagen protocol for denaturing condition, see Appendix 7.2, 7.3.
Sample JF 5509 and JF 5510 are loaded on each their Ni-NTA column.
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The protein was eluted with PNG (See Appendix 7.6 for PNG content) by the pH values of 7.0, 6.0, 5.5,
5.0 and 4.5. Total 10 ml from each pH value was collected by gravity flow. After elution, all fraction
number 2 is transferred into a dialyze-membrane for dialyze. After dialyze, 10 µl of each fraction are
analyzed on a 10 % SDS-gel, to figure out in which pH value the protein has been eluted.
When the correct pH value is found, the protein is analyzed again on a 10% SDS-gel along with the full
protein of toxin A from C. chauvoei (NusA::CctA) for comparison between the size of the full protein and
the newly made recombinant protein. The protein from NusA is added as a negative control in order to
compare size.
After purification, the recombinant protein will be referred to as NusA::CctAΔ. Δ stands for “deletion”.
The toxin protein should now be shortened from the new recombinant plasmid, and the purified protein
will prove the deletion in the next step.
Hemolytic activity (toxicity text)
The final stage for this project is to test if the CctA toxin gene has been successfully deleted and
inactivated from the plasmid. To perform such a test, the protein containing the full toxin as a positive
control is needed and NusA protein without the toxin as a negative control is needed.
This test will been done on a blood agar plate, where hemolytic activity will be expected for NusA::CctA,
and no hemolytic activity for NusA::CctAΔ (the recombinant protein)
25 µl of CctA toxin (purified protein)(NusA::CctA),
25 µl the purified recombinant protein (NusA::CctAΔ) and
25 µl of NusA protein (negative control) is spotted on a TSA agar plate with 5 % sheep blood, and
incubated 6 hours. Afterward the plate was studied under light, in order to check for hemolytic activity.
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Results and discussion
Is this section I will process and discuss my results which is obtained from the prior experiments.
Extraction and digestion of the vector
This first section will discuss the results for the plasmid vector. However, the insert will also be
mentioned.
Picture 4. Gel electrophoresis of vector and insert
Table 4. Location of samples
The plasmid vector is here extracted from the bacterium. Furthermore it has been digested with
carefully chosen restriction enzymes. For this gel electrophoresis (see picture 4), plasmid
pJFFCctANusA1 is the most important, since it needs to act as the cloning vector. These samples can be
found in slots no 4-9 (see table4).
Sample in slot 4, 5, 6 is extracted using PeqGOLD MiniPrep extraction kit, digested with different
enzymes. The kit is mentioned in methods.
Sample in slot no 7, 8, 9 are extracted using the homemade method with phenol/chloroform and no
column, and digested with different enzymes (see table 4). pJFFCctANusA1 has been digested with
different enzymes in order to check differences in cleavage efficiency. However, the double digested of
pJFFCctANusA1 (HindIII and XhoI) cleaves two places, leaving a fragment on 7336 base pair and a
fragment on 577 base pair. The small fragment on 577 base pair is not possible to see on the gel. This
could mean the amount of DNA in this small fragment is too low to make the fragment visible on this
gel.
For plasmid pHP45Ω-Km, which cleaved with HindIII alone, two bands are visible. They are very close to
each other (see slot 2 and 3 on picture4) and this means that HindIII cannot be used alone to cleave
pHP45-Km in order to extract the Km gene directly from the gel.
The theoretically size for Km gene alone is approximately 2000 base pair and the Amp gene is 2320 base
pair. Based on calculations made from measuring the picture from the gel electrophoresis), the sizes of
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the two fragments will be considered respectively to be pHP45-Km and Amp gene (see table 5 for base
pair size).
Graph 1. Standard curve for calculation of vector and insert
Table 5. Calculated base pair
For sample pJFFCctANusA1, extracted by kit (Slot 4, 5, 6 on the gel), the calculated fragment size is not
so far from the theoretically size of the original plasmid (pJFFCctANusA1), which is 7913 base pair, (see
table 5). Considering measurement uncertainty, by measuring migration on a picture of a gel, calculated
7600 base pair and thereby a difference of 313 base pair will be considered as sufficient. Moreover,
migration of all samples is the same length, which indicates the plasmid is the same.
The base pairs for both plasmids are calculated based on the correlation coefficient from a standard
curve. The curve is made from measuring the migration in mm of the marker and takes the logarithm of
migration in mm. See Appendix 8.1 for sizes of λ-HindIII marker. This provides a correlation coefficient.
Afterwards migration of the samples are measured in mm, and used for calculation of base pair. Shown
is a R2 value on 0,978, which is not considered high. This value should be around 1. However, in
microbiology and giving the uncertainty by measuring in this way, the calculated sizes would imply that
it is respectively the plasmids pJFFCctANusA1 and pHP45-Km. The migration is measured with a ruler on
a picture of the gel.
See Appendix 8.3 for mid calculations of base pairs.
As seen on the gel photo (picture 4), the plasmid extracted with the homemade method leaves a trace
of RNA. It is also seen that the amount of DNA is a little less, than the plasmid extracted with the kit.
This means the homemade method still needs to be optimized in order to provide the same good results
as the extraction kit. For this reason, further plasmid extraction is carried out using PeqGOLD MiniPrep
extraction kit only.
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Extraction and digestion of the insert
This section will discuss the new digestion of the insert pHP45-Km. The plasmid has been double
digested using HindIII and PvuI restriction enzymes. After digestion an agarose gel electrophoresis has
been performed.
Table 6. Location of samples
Picture 5. Gel electrophoresis of insert
Looking at the gel above, it is possible to see 3 bands which are well digested. Samples in slot 2 and 3
are the same (see table 6). The reason for this is that the bacteria culture was divided in two for the
extraction. Looking at the bands (picture 5) it is consistence with the amount of fragments this digestion
with HindIII and PvuI should give. The theoretical size of the Km fragments is 2000 base pair and 2320
for the Amp gene.
Graph 2.Standard curve for calculation of insert
Table 7. Calculated base pair of the insert
The calculated size for this gel is not completely the same as the theoretically calculated. Sample
pHP45_1a is supposed to be the Km resistant gene. pHP45_2a and pHP45_3a is the Amp gene, cleaved
with PvuI (see table 7 for calculated sizes). Despite that the calculated Km gene is approximately 300
base pair bigger than theoretical size, it is believed to be the correct gene. This assumption is partly
based on the inaccuracy that is linked together with measuring and calculated base pair from a gel
photo, and that the amount of fragments is exactly as wanted.
Moreover, the determined size of the Amp gene by gel electrophoresis is as well 305 base pair more
than the theoretical size. Since it is 300 base pair in difference for both genes, it can be the result of
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Clostridium chauvoei Toxin A analysis
measure failure. The failure can be connected to the starting point of measure migration has not been
completely accurate for all bands. Also the gel is slightly tilted, which would make the inaccuracy even
bigger.
The calculated sizes of the fragments for pHP45Ω-Km are based on the correlation coefficient from a
standard curve. Linearity is seen with a R2 value is 0,98, which is rather good. The migration is measured
with a ruler on a picture of the gel. Base pair are calculated the same way as mentioned in the section
for the vector.
Based on discussion with Prof. Joachim Frey about calculated size and measurement uncertainty, this
plasmid, digested with HindIII and PvuI restriction enzymes is used as the insert for further cloning. See
Appendix 8.4 for mid calculations.
Ligation of cctA and Km; Electroporation and digestion
The ligation of the plasmids and following electroporation is first evaluated by growth of colonies on
selective LB agar plates.
The two pictures below are the results of the plasmid after electroporation. The plates contain both Km
and Amp. It also shows that two samples did not contain the insert (see plate on the right,
cctA::Km_copy 6 and 7 (DH5α). The plate on the left shows cctA::Km_ copy 1-3 (DH5α). Pictures of the
remaining cctA::Km_copy4, 5 and 6 (DH5α) are not shown in this project, because of a contamination of
the plate before a picture was taken.
Picture 6. cctA::Km_copy 1-9 (DH5α).
Based on the bacteria growth on the LB agar plates and lack of growth, the experiment was continued
with the extraction for the mentioned 6 copies (1, 2, 3, 4, 5 and 9).
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Table 8. Location of samples
Picture 7. Gel electrophoresis of
electroporated samples.
Table 5 shows which samples corresponding to which slots on the gel (picture 7). Table 8 shows the
location of the plasmid. The 2 bands from the gel show the plasmid and the insert, after digestion with
HindIII. Amount of bands are consisting with the number of fragments after digestion with HindIII. Also
to be seen on the gel is, that all sample have migrated the same length on the gel.
Table 9. Calculated base pair
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The difference between the theoretical size and the measured size (table 9) is not critical and is
generally seen on agarose gels, and I trust this plasmid to be the newly made recombinant plasmid
cctA::km_copy (1, 2, 3, 4, 5, 9) (DH5α). I make that assumption based on numbers of fragments, the
same length of migration for all samples and that fact that the bacteria containing this plasmid, has been
grown up using two types of antibiotic as selective markers. Moreover, the bands calculated individually
with a difference of 242 base pair for the vector JF 4415 and 121 base pair for pHP45-Km is acceptable,
again considering the uncertainty of the measuring method. In total, a difference of 121 base pair when
the two fragments are calculated together is considered accurate.
Graph 3. Standard curve for calculation of DH5α clone.
The calculated size of the fragments for cctA::Km_copy1, 2, 3, 4, 5, 9 (DH5α) is based on the correlation
coefficient from a standard curve. The R2 value is 0,9677, which is not high, but based on uncertainty
about the measuring method, the value is considered sufficient. Calculations of base pairs for this gel, is
measured on another gel picture. See picture in Appendix 8.5.
Extraction of plasmid; transformation into expressing E. coli strain
After transformation into an expression E. coli strain BL21(DE3), a digestion of the extracted plasmid is
performed. All digestion is added on an agarose gel for analytic purpose. The results of transforming
into an express vector will be discussed in the following.
Picture 8. Gel electrophoresis BL21.
Table 10. Location of samples
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Clostridium chauvoei Toxin A analysis
Transformation was done with cctA::Km_copy5 and
cctA::Km_copy9.
After transformation, and following incubation on plates
overnight, 2 colonies from cctA::Km_copy5 and 4 colonies
from cctA:Km_copy9 were picked to extract and digest
(see picture 9; agar containing colonies after transformation).
Extraction from express vector and following digestion with
HindIII shows very nice bands (see picture 8). In table 10 the
samples and corresponding slots are listed.
Picture 9. Transformation plate
Table 11. Calculated base pair, BL21.
The calculated sizes of the recombinant plasmid cctA::Km_copy_5 and cctA::Km_copy9 (BL21) are
considered to be sufficient. The difference is calculated to 234 base pair, which is considered as an
acceptable difference above the theoretically size. See table 11.
As mentioned in the section of extraction and digestion of the vector and section of extraction and
digestion of the insert, the calculated difference for the vector is still around 300 base pair.
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Graph 4. Standard curve for calculation of
BL21.
The calculated size of the fragments for cctA::Km_copy1, 2, 3, 4, 5, 9 (BL21) is based on the correlation
coefficient from a standard curve. The R2 value is 0,9854, which is considered to be sufficient. In the field
of microbiology a R2 value of 0,98, will be calculated as sufficient.
However, to be sure the plasmid is 100 % correct, sequencing or a PCR with specific primers for
respectively the cctA gene and the Km resistant gene must be performed.
Afterwards an analysis on agarose gel can be performed.
Expression of the protein and SDS gel
The following will discuss the result of protein expression by performing an analytic SDS protein gel.
Table 12. Location of samples
Picture 10. Analytic SDS gel
This SDS gel is made for an analytical purpose see picture 10. The goal was to see if there has been an
expression of the protein in each 2 clones; JF 5509 and JF 5510. There has not been made any certain
calculations. The form of the gel is not optimal for measuring, so the validation of expressed protein is
based on visibility.
For JF 5509 there is a small difference between the induced and non-induced, (slot 1, 2 and 3).
For JF 5510, the difference between induced and non-induced is rather large (slot 4 and 5).
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Clostridium chauvoei Toxin A analysis
Both JF 5509 and JF 5510 are chosen for further experiments, despite the small difference of protein
expression in JF 5509.
Protein purification
As the protein from previous expression and analysis on SDS protein gel has shown to be in the correct
measure range, protein purification has been performed. The following will show the results for the
protein of JF 5509 and JF 5510.
First is analysis of the different fractures eluted by dropping pH values.
Picture 11. SDS cctA::Km_copy 9
Table 13. Location of samples.
Picture 12. SDS cctA::Km_copy 5
Table 14. Location of samples
The SDS gels seen in picture 11 and 12 is made, to investigate in which pH value the protein has been
eluted, during the protein purification with NI-NTA column. Location of samples of the two gels is listed
in table 13 and 14.
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Clostridium chauvoei Toxin A analysis
Seen on these pictures, the protein has been eluted with pH value 5.5, and pH 5, for both JF 5509 and JF
5510.
The Flow Through samples is to be found in slot number 7. Since it is more or less empty, it indicates,
that the Ni-NTA columns hold back the protein sample rather good.
The Crude Extract, seen in slot number 8 shows all the proteins before loading the sample onto the
columns. This also indicates a good efficiency of the Ni-NTA column.
There are two bands visible for JF 5509. The size is not calculated, but is estimated to be the correct
protein for an inactive Toxin A gene.
JF 5509 and JF 5510 are both used for continued comparison of the full Toxin A.
Afterwards fragment eluted by pH 5.5 from JF 5509 and JF 5510 are analyzed on a SDS protein gel, along
with positive control; the full Toxin A from C. chauvoei and a negative control; NusA without the Toxin A.
See picture 13 below.
Picture 13. SDS gel. All samples
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Clostridium chauvoei Toxin A analysis
After running a SDS protein gel, size in kDa is calculated.
Graph 5. Standard curve for calculated kDa
Table 15. Calculated kDa
The calculated sizes in kDa, along with location of the samples, shown in table 15, shows for all samples
10 kDa more than the theoretically size, which is calculated on molecular mass. Since 10 kDa is
consistent for all samples, it could indicate measure uncertainty, by measuring migration in mm on the
gel pictures see Appendix 8.2 for protein marker size. Moreover, the SDS gels are bended, which would
make the measuring even more inaccurate. However the differences measured are within the range of
determination that is possible on a SDS gel
The size for NusA::CctAΔ, the new inactive toxin, is smaller than the full toxin. The reason is that the
insert is placed in the middle of the CctA toxin gene, which will allow the protein to be expressed up to a
certain point. After that, the insert will result in a frame shift, providing a stop codon for translation.
Therefore the recombinant protein is smaller than the protein from NusA::CctA full Toxin A.
The calculated sizes for my experiment are calculated based on the correlation coefficient from a
standard curve. See Appendix 8.7.
Based on calculations for the two visible bands for JF 5509(slot 4) it is believed that JF 5509 still contains
a rest from the full toxin as well.
The size of the protein is calculated based on the correlation coefficient from a standard curve which
shows linearity. The R2 value is 0,9819, which is considered good.
Based on the calculations and the visibility of two bands from JF 5509, this is not chosen for further
experiments.
The purified protein from JF 5510 is continued for the final test.
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Clostridium chauvoei Toxin A analysis
Toxicity test
The final test for this experiment is a toxicity test made on a blood agar plate. This test can be made
when the purified protein has been obtained.
Picture 14. Hemolytic activity
The test will show hemolytic activity of NusA::CctA, which is the full protein of Toxin A, NusA::CctAΔ,
which is the newly made recombinant protein JF 5510) and NusA, the protein without the toxin as
negative control.
Seen on picture 14 showing the blood agar plate, is a clear hemolytic zone on the spot from NusA::CctAfull toxin. For NusA::CctAΔ no hemolytic activity is visible. For the negative control, there will be no
hemolytic activity as well, which means that the CctA toxin is deleted and inactivated in the new
recombinant plasmid.
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Clostridium chauvoei Toxin A analysis
Conclusion
This project represented the beginning of a large study concerning a toxin found in Clostridium chauvoei.
This toxin, Toxin A, is the main virulence factor of Clostridium chauvoei that causes severe worldwide
sickness called “blackleg” in cattle, sheep and other domestic animals. The sickness can lead animals to
the death.
However, a vaccine is available against “blackleg”, but because the protective antigen is still unknown,
all batches are tested on guinea pigs, before their commercial availability.
In order to reduce the use of laboratory animals, researchers are trying to develop an in vitro test.
This project has been the beginning of this large study. It successfully produced a non-toxic derivative
protein out of the toxin CctA.
Once, at the end of this experiment, all the results have been processed, it would be possible to give an
answer to the problem formulation:
Can we produce an inactive toxin gene of cctA by a deletion of a part of the cctA coding region using a
pre-existing plasmid containing the cctA gene in Escherichia coli? (By in vitro mutagenesis)
In order to prove the creation of an inactive toxin gene it was necessary to carry out several steps.
By using a pre-existing recombinant plasmid containing the cctA gene, it has been possible to inset a
kanamycin resistant gene in the region of the toxin gene. This is proven by growth on double selective
antibiotic plate, and afterwards digestion and analyze on agarose gel electrophoresis.
Afterwards the protein in the newly made recombinant plasmid was expressed, in order to compare it
with the size of the full protein from the toxin gene.
By analyzing on SDS protein gel, it is shown that the recombinant protein is smaller than the toxin
protein, which proves that it does not have the full toxin gen expressed.
In the extension of measuring protein size, the recombinant clone was proven to contain the inactive
toxin gene, by performing a test for hemolytic activity on a blood agar plate. On the plate was added:
-
NusA::CctA, the full toxin;
NusA, protein without the toxin and functioned as a negative control;
NusA::CctAΔ, the new recombinant protein.
NusA::CctA, the full toxin, showed hemolytic activity. The NusA::CctAΔ showed no activity, proving that
the toxin is inactive.
By these steps it can now be concluded that it is possible to produce an inactive toxin gene of cctA using
a pre-existing plasmid containing the cctA gene in Escherichia coli where a part of the coding region of
the cctA has been deleted
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Clostridium chauvoei Toxin A analysis
This gene construct will now be used for making a deletion mutant of cctA in Clostridium chauvoei for
development and proof of principle in a novel animal-free vaccine batch release test.
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Clostridium chauvoei Toxin A analysis
Literature/references

AAU, studiehåndbog, huminf:
http://www.studiehaandbog.huminf.aau.dk/kildehenvisninger-citaterlitteraturliste/kildehenvisninger/

BiotechArticles , web:
http://m.biotecharticles.com/Biotech-Research-Article/Principles-and-Techniques-BehindBacterial-Transformation-815.html

(Britannica, web):
http://www.britannica.com/EBchecked/topic/1357082/pharmaceuticalindustry/260317/Synthetic-human-proteins

Department of Biology, Davidson College:
http://www.bio.davidson.edu/genomics/method/SDSPAGE/SDSPAGE.html

Experimental Biosciences, web:
http://www.ruf.rice.edu/~bioslabs/studies/sds-page/denature.html

Frey. Et. Al, web 2012:
http://www.ncbi.nlm.nih.gov/pubmed?term=Frey%2C%20J.%2C%20Johansson%2C%20A.%2C%
20B%C3%BCrki%2C%20S.%2C%20Vilei%2C%20E.M.%2C%20Redhead.%20K.%5BAuthor%5D

Keilberg, Vivi ; Nørby, Søren; Rasmussen, Leif; DNA og RNA-en håndbog, 1st edition, Gads forlag,
2003.

New England Biolabs 1, web:
https://www.neb.com/~/media/NebUs/Files/Brochures/RE.pdf

New England Biolabs 2, web:
https://www.neb.com/products/m0202-t4-dna-ligase

Peqlab, web:
http://www.peqlab.co.uk/wcms/uk/products/index.php?do=getArticleDetails&which=12-694202

Primrose, S.B. and Twyman, R.M. – Principles of Gene Manipulation and Genomics, Seventh
edition, Blackwell Publishing, 2006.
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Clostridium chauvoei Toxin A analysis

Prof. Joachim Frey, Head of Institute, University of Bern

Qiagen, web:
http://www.qiagen.com/products/catalog/sample-technologies/dna-sampletechnologies/plasmid-dna/qiagen-plasmid-mini-kit#resources

Sigma Aldrich 1, web:
http://www.sigmaaldrich.com/etc/medialib/docs/Sigma/Product_Information_Sheet/1/p3803p
is.Par.0001.File.tmp/p3803pis.pdf

Sigma Aldrich 2, web:
http://www.sigmaaldrich.com/content/dam/sigmaaldrich/docs/Sigma/Datasheet/9/77619dat.pdf

They University of Sydney:
http://sydney.edu.au/science/molecular_bioscience/ohs/documents/sop/sop_sonication.pdf

Ucsf, University of California, San Francisco, web:
http://mullinslab.ucsf.edu/Protocols%20HTML/SDS_PAGE_protocol.htm
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Lea Elena Haaning Normand