Download Cloning and expression of proteins from Mycobacterium smegmatis

Cloning and expression of proteins from
Mycobacterium smegmatis
Erik Mattsson
Degree project in biology, 2007
Examensarbete i biologi, 30 hp, 2007
Biology Education Centre and Department of Cell and Molecular Biology, Uppsala University
Supervisor: Dr. Mikael Nilsson
Content
1
Summary
2
2
Abbreviation list
3
3
Introduction
3
3.1
3.2
3.3
3.4
3.5
3
5
5
5
6
4
Mycobacterium tuberculosis
Rational approach to pathogen inhibitor discovery
Mycobacterium proteins that are possible drug targets
Protein purification using affinity chromatography
Aims of the project
Results
4.1
4.2
4.3
4.4
4.5
Identification of Mycobacterium smegmatis homologues
Amplification of M. smegmatis genes and addition of His-tag
Construction and cloning of expression plasmids
Analysis and manipulation of expression plasmids
Protein production
7
7
9
12
13
16
5
Discussion
17
6
Materials and methods
17
6.1
6.2
6.3
6.4
6.5
6.6
6.7
6.8
17
18
19
19
19
22
23
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Bacteria and plasmids
Cloning
Growth media
Agarose gels and extracting DNA from them
Polymerase chain reactions
Plasmid preparation
Sequencing
Bioinformatics
7
Acknowledgements
24
8
References
25
1
Summary
Tuberculosis is a mayor health threat that causes death to 1.6 million people each year. In an
attempt to battle the disease I have focused on the extra ordinary thick and tough cell wall.
However, experience from the work at the Department of Cell and Molecular Biology tells
that working with genes and proteins from Mycobacterium tuberculosis (M. tuberculosis)
could be a troublesome task. To avoid this, an alternative approach is to work with the gene
homologues from Mycobacterium smegmatis (M. smegmatis).
In this report three open reading frames (ORFs) in M. smegmatis were identified as
homologous to the M. tuberculosis genes; Rv3782, Rv3790 and Rv3791. These ORFs were
identified in M. smegmatis as Msmeg6329, Msmeg6344 and Msmeg6347. In M. tuberculosis
these proteins are believed to be important for the cell membrane construction and therefore
vital for the bacterial survival.
New multi drug resistant (MDR) strains are developing in former Soviet Union countries.
The antibiotic treatment is not completed and this creates a breeding ground for MDR-TB to
develop. These proteins offer a chance to develop new drugs against tuberculosis (TB). The
development of new drugs is essential in the struggle to stop the ravage of tuberculosis.
The cloning of Msmeg6347 resulted in a plasmid with a correct insert. The production of
protein however was not successful. The progress of Msmeg6344 went to the stage of colony
polymerase chain reaction (PCR) where the result showed no success in ligating the gene into
the vector. The cloning of Msmeg6329 gene failed at an early stage due to faulty primer
designing and therefore was terminated.
2
2
Abbreviation list
Amp
bp
DMSO
EtBr
GC
GOI
IMAC
LB
LMW
Min
MDR-TB
NIAID
OD600
ORF
PCR
RAPID
RT
SDS-PAGE
TAE (buffer)
TB
Tet
Tm
WHO
Ampicillin
base pair
Dimethyl sulfoxide
Ethidium bromide
Guanin, Cytosin
Gene(s) of interest
Immobilized metal ion affinity chromatography
Luria Broth
Low molecular weight marker
Minutes
Multidrug resistant tuberculosis
National Institute of Allergy and Infectious Diseases
Optical density at 600 nm
Open reading frame
Polymerase Chain Reaction
Rational Approaches to Pathogen Inhibitor Discovery
Room Temperature
Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis
Tris Acetate EDTA
Tuberculosis
Tetracycline
Temperature of melting
World Health Organization
3
3
Introduction
3.1
Mycobacterium tuberculosis
The bacterium Mycobacterium tuberculosis is a pathogenic organism that is responsible for
causing the disease tuberculosis (TB). The disease is one of the most deadly on the planet and
is spread to one third of the world population [19]. The respiratory system is most commonly
affected but also vascular, lymphatic, nerve and bone tissue as well as joints might be
affected. Common symptoms are sustained cough, sometimes with blood, chest pain, fever
and chill [13].
The disease is continuously spread by speaking, spitting and sneezing [22]. According to the
World Health Organization (WHO) M. tuberculosis infects a new individual every second
[22]
. According to the United States National Institute of Allergy and Infectious Diseases
(NIAID) some two billion people are believed to be infected with M. tuberculosis [14]. An
infected host with a fully functional immune system can carry latent TB for a very long time.
Not only the developing countries are heavily affected by TB, but also countries of the
former Soviet Union have a growing problem [16].
In 2005 the disease killed approximately 1.6 million people [22]. An outbreak may occur when
the immune system for some reason is weakened, for example by old age, malnutrition or
other infections like HIV. Tuberculosis is the leading cause of death of HIV positive
patients [22]. As a single infectious organism, M. tuberculosis, kills more adult people world
wide than AIDS, malaria and tropical diseases combined [15].
The lethal combination of TB and HIV creates a huge problem for developing countries,
especially in Africa, where AIDS is widely spread. However, an enlarging threat from multidrug resistant strains of TB (MDR-TB) increases the problem significantly. Treatment of a
MDR-TB infection is 1000 times more expensive than ordinary treatments, and less
effective [21]. The treatment also has strong side effects, and even when treated, only 50% of
the patients survive. Abuse of antibiotics and interrupted treatment are creating a breeding
ground for MDR-TB laying a devastating future ahead. Countries from the former Soviet
Union like the Baltic states Estonia, Latvia and Lithuania are regarded as hotspots for larger
outbreaks of MDR-TB [22].
Mycobacterium tuberculosis is a rod-shaped, gram-positive, facultative aerobic bacterium
that has some exceptional chemical properties due to its cell wall composition. Also the
genome has a significantly high guanine and cytosine (GC) content, about 65.6% compared to
E. coli that has about 50.8% [8]. It has a very long generation time of 15-20 hours [20] which
makes it unsuitable to work with in vitro. The ability to produce a layer of mycolic acids,
arabinogalactan and peptidoglycan creates a very strong cell wall. The polysaccharide-rich
envelope resembles a capsule, giving M. tuberculosis a very strong defence against
antibiotics and the host’s immune system. It even gives the ability to stay dormant and
survive within the macrophages phagosomes [4].
Mycolic acids are complex large fatty acids with 60 to 90 carbon atoms [2]. A large number of
functional groups are in general associated with mycolic acids, such as cyclopropane rings,
epoxy esters, keto and methoxy groups. The low permeability provided by the mycolic acids
contribute to the resistance to therapeutic agents [2]. Mycolic acids also result in the acid
fastness or acid resistance of mycobacteria. Biological materials with this property form acid4
stable complexes with certain arylmethane dyes. Mineral or methanol acids do not decolorize
them [4].
3.2
Rational approach to pathogen inhibitor discovery
The Rational Approach to Pathogen Inhibitor Discovery (RAPID) is an interdisciplinary
approach to design drugs against pathogens used at the Departments of Computational
Chemistry, Medical Chemistry and Structural Biology at the Uppsala University. Many
different fields are involved such as computer science, organic chemistry and biotechnology.
The intent is to shorten the time it takes to develop new drugs and thereby save money, limit
suffering and mortality. By solving the three dimensional structure of essential proteins, the
time to design inhibitors can be shortened. A computer is used to evaluate the chemical
structure of potential inhibitors before testing them in vitro.
The classical drug designing strategy starts off with a number of chemicals that are tested
against certain targets. As a substance is discovered to be effective it enters a stage where it is
chemically altered and re-screened again to find a more effective composition. The chemical
alteration and synthesis is expensive and time consuming. This stage is iterated until an
effective chemical is found. The next stage is animal testing. This approach is more of a trial
and error approach, which is very costly and time consuming. In the end a lot of substances
are cancelled due to toxic side effects. This makes pharmaceutical research a very expensive
business.
3.3
Mycobacterium proteins that are possible drug targets
Proteins produced by the open reading frames (ORFs) Rv3782, Rv3790 and Rv3791 in the M.
tuberculosis are believed to be suitable drug targets because of their cell wall related function
[11] [14]
. There is evidence that the Rv3782 encoded protein is a glycosyltransferase that is
involved in the first stage of galactan synthesis. Galactan is an important part of M.
tuberculosis cell wall structure [11]. The proteins from the genes Rv3790 and Rv3791 are
involved in epimerization and forming of the D-arabinofuranose, an also important part of the
exceptional cell wall structure [10]. The inhibition of these enzymes may result in a much
weakened bacterium that the immune system can eliminate. However, experience from work
at the Department of Cell and Molecular Biology shows that working with M. tuberculosis,
both genetic material and purification of protein have been troublesome. An alternative might
therefore be to work with the closely related homologue proteins of M. smegmatis. The M.
smegmatis genes are referred to as the genes of interest (GOI).
3.4
Protein purification using affinity chromatography
Immobilized metal ion affinity chromatography (IMAC) is an affinity protein purification
system that can be based on a polyhistidine sequence added to the recombinant protein. Six
histidine residues known as a His-tag are added by the primer or are part of the vector. It can
be added at the N-terminal or C-terminal end of the protein. The histidine has the property to
bind to nickel or cobalt that has been immobilized on a gel matrix [12]. The recombinant Histagged protein can be added directly to the affinity gel from a lysate. High purity can be
5
gained without big losses of recombinant protein due to the strong bond between the His-tag
and the affinity matrix.
3.5
Aims of the project
The aim of this project was to identify M. smegmatis homologues of the M. tuberculosis
genes Rv3782, Rv3790 and Rv3791. Identified ORFs were then to be isolated by PCR and
cloned using E. coli and finally heterologous expressed and purified by affinity
chromatography.
Purified protein homologues will be used for further analysis with x-ray crystallography.
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4
Results
4.1
Identification of Mycobacterium smegmatis homologues
Since the goal was to use the M. smegmatis protein homologues as a model system for the M.
tuberculosis, the homologues ORF’s of M. smegmatis had to be identified.
To identify the M. smegmatis homologues to the M. tuberculosis genes Rv3782, Rv3790 and
Rv3791, a comparative analysis using the GenMycDB [3] server was performed. The
comparative analysis resulted in several hit candidates. The E-value was used to find the
candidate with highest sequence identity. The E-value represents the statistical likelihood that
the matches between the two sequences are due to chance, the lower E-value, the higher
sequence similarity. The sequences with the lowest E-value were chosen. According to the Evalue, the comparative analysis showed that homologues to M. tuberculosis genes Rv3782,
Rv3790 and Rv3791 were identified as Msmeg6329, Msmeg6344 and Msmeg6347
respectively. Both the DNA and the protein sequences were retrieved for the identified M.
smegmatis homologues.
In order to verify the sequence identity of the retrieved gene sequences from the comparative
analysis a paired alignment of the translated ORFs were undertaken using ClustalW [6] [7] [ 9].
From the visual inspection of the alignments one could see that the Rv3790 and Rv3791 had
a close to perfect match with Msmeg6344 and Msmeg6347 considering both amino acid
sequence and length of the translated protein. It was found that the homologous proteins only
differed in length with one amino acid. The alignment did not contain any inserts or gaps,
see Figure 2 and 3. However, the Msmeg6329 (Rv3782) had a lower degree of sequence
identity to its homologue. Inside the inserted sequence a stretch of five serine residues was
also visible in the alignment. The residues were located in the middle and are shown in
Figure 1.
The alignments are important to establish the similarity between the M. tuberculosis genes
and the M. smegmatis genes and the proteins produced by these genes. By aligning the
protein sequences from M. tuberculosis and its equivalent in M. smegmatis it could be seen
that the retrieved sequences indeed were homologous, and should be suitable as model
proteins for the M. tuberculosis.
Other important information for further manipulation of the genes such as sizes, weights and
GC% content was obtained by utilizing web-based sequence analysis tools [18] and is
summarized in Table 1 [8]. The gathered information on the Msmeg-gene sequences is
important while designing oligonucleotide primers for PCR, and analysis of generated
amplicons. Protein identity shows how many amino acids that are similar between the
proteins.
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Figure 1: Sequence alignment of Rv3782 and Msmeg6329 using ClustalW. In the consensus line the stars “*”
represents an amino acid identical to that in the compared sequences, while a “:” represents amino
acids with similar properties and the “.” represents a less similar amino acid. The E-value, which
represents the statistical likelihood that the matches between the two sequences are due to chance, is
0. The similarity between amino acids is graded and given a score where a high value represents high
extent of conservation. In this alignment the score was 1818 which is considered as a high score.
Figure 2: Sequence alignment of Rv3790 and Msmeg6344 using ClustalW. The E-value was 0 and the score
2832. See legend to Figure 1 for further explanations.
8
Figure 3: Sequence alignment of Rv3791 and Msmeg6347 using ClustalW. The E-value was 0 and the score is
1371. See legend to Figure 1 for further explanations.
Table 1: Data retrieved from bioinformatics analysisa
Gene b
Length
GC c
Sequence
identity d
Protein length e
Protein MW f
(base pares)
(%)
(amino acids)
(g/mol)
Msmeg6329 (Rv3782)
909
67
77 % (232)
302 (304)
34040.9
MSmeg6344 (Rv3790)
1383
56
84 % (383)
460 (461)
50575.7
MSmeg6347 (Rv3791)
762
68
85 % (215)
253 (254)
27038.2
a
Bioinformatics data for M. smegmatis open reading frames (ORF), and its M. tuberculosis homologues.
b
M. smegmatis ORF identified as homologous to M. tuberculosis gene. M.tb. ORF is presented in parenthesis.
c
Percentage of GC-content in ORF.
d
Percentage of amino acid sequence identity calculated by ClustalW analysis of the two identified homologous
ORFs. Number in parenthesis represents the number of identical amino acids in the pared alignment analysis.
e
Number of amino acids in the ORF. Number in parenthesis represents the protein length for M.tb. homologue.
f
Calculated molecular weight based on amino acid sequence in ORF.
4.2
Amplification of M. smegmatis genes and addition of His-tag
Polymerase chain reaction (PCR) was used to amplify Msmeg6329, Msmeg6344 and
Msmeg6347 genes from genomic M. smegatis DNA also including an N-terminal His-tag.
The His-tag was added in a separate PCR to lower the risk of unspecific primer binding due
to the DNA-sequence coding for the 6 histidins. A mutation at the end of the genes was
introduced by designing the primers to change a stop codon from TGA to TAA to increase its
strength. Figures 4-6 show the primary amplifications and Figures 7-9 show the His-tag PCR
results. Bands that are shown in the Figures, but not commented on, are to be regarded as
artefacts or false products. The Figures also have a 100 kb ladder where 0.5, 1.0 or 1.5 kbp
band is marked to simplify size estimation of the PCR products. Bands marked “cut out” in
the Figures were cut out and the DNA purified.
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Figure 4: PCR amplification of Msmeg6329. The
product was analyzed on a 1.25% agarose
gel. The expected size of the amplicon
gene is 909 kb. Lane 1: 100 bp ladder 3
µl; lane 2: PCR product 7 µl. The band
marked “cut out” in the figure was cut
out and DNA purified.
Figure 5: PCR amplification of MSmeg6344. An
agarose gel of 1.25% was used to visualize
the PCR product. The expected size of the
amplicon gene is 1383 kb. Lane 1: 100 bp
ladder 3.5 µl; lane 2 10 µl PCR product
Msmeg6344 using 1:10 diluted template;
lane 3: PCR product 10 µl Msmeg6344
using 1:10 diluted template and 1 µl
DMSO; lane 4: PCR product 10 µl using
undiluted template. The bands marked
“cut out” in the figure were cut out and the
DNA purified.
Figure 6: PCR amplification of Msmeg6347. The
figure shows the result after the PCR
reaction using MS6347-F and MS6347-R
primers and genomic DNA as template.
The product of expected size, 762 bp, was
isolated. Lane 1: 100 bp ladder 3 µl; lane 2:
PCR product 10 µl. The band marked “cut
out” in the figure was cut out and DNA
purified.
10
Figure 7: PCR adding His-tag to Msmeg6329.
Agarose gel of 1.25%. The figure shows the
result of the PCR reaction of MS6329-FHIS
and the MS6329-R primers using previous
amplified Msmeg6329 gene as template. The
primers added an N-terminal His-tag. The
product was estimated to 930 bp.
Lane 1: 100 bp ladder 3 µl; lane 2: PCR
product A 10 µl. The band marked “cut out”
in the figure was cut out and DNA purified..
Figure 9: PCR adding His-tag to Msmeg6347.
The previous PCR product of
Msmeg6347 gene was used as a
template and amplified with MS6347FHIS and MS6347-R primers. The
primers added a N-terminal His-tag. The
product was loaded on 1.25% agarose
gel. The size was estimated to 786 bp.
Lane 1: 100 bp ladder 3 µl; lane 2: PCR
product 10 µl. The band marked “cut
out” in the figure was cut out and the
DNA purified.
11
Figure 8: PCR adding His-tag to Msmeg6344.
The density of the agarose gel was 1%.
PCR adding N-terminal His-tag to the
Msmeg6344 gene by using previous
PCR product and the MS6344-FHIS
and MS6344-R primers. The expected
size of the fragment was 1407 bp. Lane
1: 100 bp ladder 3.5 µl; lane 2: PCR
product 12 µl. The band marked “cut
out” in the figure was cut out and DNA
purified.
4.3
Construction and cloning of expression plasmids
All the amplified and purified fragments with His-tag added, named His-Msmeg6329, HisMsmeg6344 and His-Msmeg6347, were elongated with an A at the 3’ end separately. The
fragments were then ligated into the pEXP5/CT vector and transferred into E. coli top 10-F
cells by heat shock. Transformed cells were spread onto LB-agar plates containing 100 µg
ampicillin / ml.
E. coli Top 10-F is a bacterium that is modified in the genes recA1 and endA1, which results
in more stable plasmids, and it is therefore suitable for cloning plasmids to high
concentrations that can easily be harvested.
Bacterial colonies of E. coli Top 10-F cells, transformed with the pEXP5/CT vector carrying
either Msmeg6329, Msmeg6344 or Msmeg6347 inserts were used as template in the colony
PCRs. The usage of a vector primer and an insert primer will show if the insert has the
correct orientation and therefore works as a screening process.
Colony PCR reactions were used to verify each ORF respective insert and orientation.
Figures 10 and 12 have brackets that mark PCR products that were regarded as genuine
products and therefore coming from clones carrying vectors with a correct oriented insert in
respect to the promotor. These clones were grown and used for plasmid harvesting. Figure 11
has no brackets which mean no correctly ligated plasmid was found.
Figure 10: Colony PCR of His-Msmeg6329 inserted in the pEXP5/CT
vector. The reaction used MS6329-F and T7 Term universal
primers. The PCR product was analyzed on a 1% agarose gel.
Lane 1: ladder 3 µl; lane 2-7: 10 µl of PCR product using
clones transformed with Msmeg6329 inserted in the vector.
The brackets mark the colonies that were isolated and grown.
12
Figure 11: Colony PCR to establish correct His-Msmeg6344 gene insert
in the pEXP5/CT vector. The product formed is believed to be
primer dimer artefacts from the Msmeg6344-FHIS and T7
Term universal primers. The PCR was analyzed on a 1%
agarose gel. The expected size of the amplicon was around
1450 bp. Lane 1: 100 bp ladder 3.5 µl; lane 2-7: PCR product
10 µl.
Figure 12: Colony PCR using a cloned vector with His-Msmeg6347 insert
as template. The reaction used MS6347-F and T7 Term
universal primers. The PCR product was analyzed on a 1%
agarose gel. Lane 1: ladder 3 µl; lane 2-7: 10 µl of PCR
product. The brackets mark the colonies that were isolated and
grown.
4.4
Analysis and manipulation of expression plasmids
Colonies of E. coli Top 10-F cells that were positive were grown and the plasmids harvested.
Figure 13A and B shows that the plasmid preparation was successful. The plasmid inserts
were sequenced, and results aligned with the original sequence. The results showed that HisMsmeg6329 and the His-Msmeg6344 were not identical with the sequences retrieved from
the database. The result of the His-Msmeg6347 showed that clone 5 and 6 only had small
errors at the end of the sequence.
13
A
B
Figure 13: Agarose gel electrophoresis of harvested plasmids. A. Vector pEXP5/CT with His-Msmeg6329 inserted.
Lane 1: 100 bp ladder 3 µl; lane 2 and 3: plasmid 6 µl; lane 4: plasmid 6 µl with no insert. The agarose
concentration was 1% (w/v). B. Plasmids of pEXP5/CT with His-Msmeg6347 inserted that were sent for
sequencing. Lane 1: Ladder 2 µl; lane 2-5: harvested plasmids 6 µl from clone 2-5. The agarose
concentration was 1% (w/v).
Clone 4 and 5 carried plasmids with His-Msmeg6347 insert. The plasmids had errors at the
end of the insert that was covered by the MS6347-R primer. A PCR reaction was used to
correct the errors in the sequences. The PCR product was then elongated with an A at the 3’
end, a procedure that enabled ligation by changing a blunt ended fragment to a sticky ended.
The pEXP5/CT-Topo linear vector requires fragments with sticky ends for ligation. The
result from the PCR after the product has been 3’ A elongated is shown in Figure 14.
Figure 14: PCR product after Taq 3’ A elongation of
His-Msmeg6347 procedure. A 1% agarose gel
was used. The PCR was an attempt to repair
the damage in the sequences by using the
MS6347-FHIS and the MS6347-R primers. In
lane 2 the clone 4 plasmids was used as
template and in lane 3 clone 5 plasmids was
used. Lane 1: 100 bp ladder 3.5 µl; lane 2: 6
µl Taq polymerase treated PCR product; lane
3: 6 µl Taq polymerase treated PCR product.
14
After the His-Msmeg6347 insert had been corrected it was ligated with pEXP5/CT and
transformed into TOP 10-F cells. These colonies were screened using a colony PCR. The
result is shown in Figure 15. The clone in lane 3 was cultivated and plasmids were harvested
and sent for sequencing.
Figure 15: Screening of colonies performed. The colony
PCR of transformed Top 10-F cells was using the
MS6347-FHIS and T7 term universal primers to
establish the inserts orientation. A positive result
was seen in lane 3. The gel has 1% density and
was run at 100 V for 20 min. Lane 1: 100 bp
ladder 3 µl; lane 2-6: PCR product 10µl mixed
with 2 µl dye.
The result of the plasmid harvest is shown in Figure 16. The plasmids that were harvested
were the new sequence corrected His-Msmeg6347 plasmids. These harvested plasmids were
sent for sequencing to evaluate the sequence-restoring reaction. The new sequence was
aligned with the original sequence and it showed that the insert now was correct and had the
added N-terminal His-tag and mutated stop codon.
Figure 16: Evaluation of plasmid preparation kit
of sequence-corrected HisMsmeg6347 containing plasmids. The
1% agarose gel. Lane 1: 100 bp
Ladder 3 µl; lane 2: plasmids 3µl.
15
4.5
Protein production
In order to produce protein, the plasmids encoding the correct His-Msmegs6347 gene were
used to transform E. coli BL21-AI cells. It resulted in colonies on an agar plate. However,
they did not grow when transferred into a liquid medium.
16
5
Discussion
The gene coding for the Msmeg6347 protein was correctly inserted in a pEXP5/CT vector.
For unknown reasons the transformed BL21-AI did not grow after being transferred from an
agar LB plate to liquid LB. The promotor for the inserted gene might have been leaking. If a
toxic product was formed, that could explain the lack of growth. This was never investigated
due to lack of time.
There are other organisms or strains of E. coli that may be more suitable for production of
protein then BL21-AI. The E.coli strain Rosetta-gami™ that can be purchased from Novagen
could be an example. If the proteins were toxic to bacteria, yeast could be used for protein
production.
The Msmeg6344 gene was amplified but in the ligation step it did not insert correctly into the
vector. Without a correct plasmid, no BL21-AI cells can be transformed and therefore the
lack of protein production is obvious. The insert in the plasmid was never sequenced so it is
impossible to tell if it was the correct gene that was amplified. This is unlikely due to the fact
that it was later discovered that the primers contained errors.
Another primer flaw was the Msmeg6329 primers that annealed at two sites within the gene.
This became obvious when a purified product was used as template in a PCR reaction and the
gel afterwards showed two bands. According to the size marker the top band corresponded to
the Msmeg6329 gene but after sequencing the plasmid one could see that this was not the
case. The appearance of two bands can be due to badly designed primers or an artefact due to
the high GC content of the genome. The most reasonable explanation is probably a
combination of both.
Working with a high GC content gives not only PCR problems but also sequencing
difficulties [17]. The result from the sequencing might show errors or deletions but this might
not be true because high GC content in an area within a gene can cause artefacts. One way
around this problem would be to use methods such as amino acid sequencing or liquid
chromatography coupled with a mass spectrometer to establish that the correct product is
produced.
17
6
Materials and methods
6.1
Bacteria and plasmids
The chemically competent E.coli Top 10-F bacteria were purchased from Invitrogen™. The
E.coli BL21-AI bacteria were purchased from Invitrogen™.
The plasmid used was pEXP5-CT/TOPO linear vector. It was purchased from VWR
International AB. An overview of the vector design is shown in Figure 18.
Mycobacterium smegmatis DNA was supplied by Dr. Mikael Nilsson at the Department of
Cell and Molecular Biology, Uppsala University.
Figure 18: The pEXP5-CT/TOPO linear vector.
6.2
Cloning
To add a 3’ A overhang, 17 µl PCR amplicon were treated with a mix of 0.5 µl dNTP of 2.5
mM stock solution, 2 µl Thermopol buffer and 0.2 µl Taq polymerase. All chemicals were
bought from New England BioLabs. as a kit. Incubation lasted for 10 min at 72ºC.
To create a ligation mix 1µl of 3' A elongated PCR product and 0.5µl pEXP5/CT –Topo
linear vector was mixed. Also 0.5 µl salt solution and 1µl water was added to the mix. The
mix was incubated for 20 min in RT and then put back on ice. All chemicals were supplied
by Invitrogen™.
The Invitrogen™ chemically competent E. coli Top10-F cells were thawed on ice. The
ligation mix and the competent cells were mixed and incubated on ice for 5 min and then heat
shocked at 42ºC for 40 sec and then put back on ice for 2 min. The cells were then spread on
LB-Amp plates containing 100 µg/ml ampicillin that had been preheated to 37ºC, and
incubated at 37ºC over night.
Competent E.coli BL21-AI cells were prepared according to the Chung et al. (1988) [5]. An
amount of 50 µl BL21-AI cells were grown in 2 ml LB at 37°C, aired by shaking and 5 µg/ml
tetracycline (Tet) was used as selective agent. When the optical density, measured at 600 nm
18
(OD600), was in the range 0.4 to 0.7 the cells were harvested. The cells were centrifuged at
3000 g for 15 minutes at 4ºC. The supernatant was removed and the pellet resuspended in 2
ml TSE buffer. The composition of TSE buffer was 85 ml LB, 4.5 ml dimethyl sulfoxide, 10
g polyethylene glycol 4000, 10 mM MgCl2, 10 mM MgSO4, pH 6.1. The cells were
incubated on ice for 20 minutes.
6.3
Growth media
Luria broth (LB) contained in one litre of liquid medium, 10 g tryptone, 5 g yeast extract and
10 g NaCl. LB agar plates also contained 12 g of agar per litre mixture. Ampicillin was added
when needed to a final concentration of either 50 or 100 µg/ml.
6.4
Purification of DNA using agarose gel electrophoresis
Agarose gels were prepared by dissolving agarose of BDH quality (VWR International AB)
in 40 ml of 1 x TAE buffer. The concentration of agarose gel was varied between 1-1.25%
(w/v). The gel was then melted by heating to boiling temperature using a microwave oven.
After cooling the melted gel to approximately 50 ºC, 2.4 µl ethidium bromide (EtBr 5mg/ml)
was added and the gel was cast into an electrophoresis cassette. Agarose gel electrophoresis
was performed at 100 V for 20-30 min in 1 x TAE buffer. The TAE buffer contained 40 mM
Tris, 20 mM acetic acid and 0.1 mM EDTA.
The 6x loading dye contained 25 mg Brome phenol blue (0.25%), 25 mg xylene xyanol
(0.25%), 4g sucrose (40%) and the volume was adjusted to 10 ml with H2O.
The 100 kb ladder size marker was purchased from New England Biolabs.
When isolation of bands was performed a QIAprep® Mini-M kit was utilized according to
the manufactures manual. In some cases the cut out gel piece was frozen (-20°C) before
purification.
6.5
Polymerase chain reactions
The compositions of the PCR reactions are presented in Tables 2 to 7. In Table 2 the primary
amplification of the genes is presented using the M. smegmatis genomic DNA as template. In
Table 3 the reaction of adding His-tags is described. Table 4 shows the colony PCR reactions.
In these reactions the template was added in form of bacteria by scraping with a pipette tip at
the chosen colonies. Table 5 describe the sequencing restoring PCR. In Table 6 the primers
are shown and in Table 7 the PCR programs are shown.
The dNTP concentration was 2.5 mM (New England Biolabs) and pfu buffer supplied by the
manufacturer was used accordingly provided manual (New England Biolabs). The pfu
polymerase was purchased from New England Biolabs. The concentration of the primer
solutions was 20 mM. All reactions were evaluated on agarose gels containing 0.3 µg
EtBr/ml of gel.
19
Table 2: Gene amplification from genomic DNA.
Msmeg6329
Msmeg6344
Genomic Msmeg DNA template
20 ng (1µl)
20 ng (1µl)
dH2O
16 µl
16 µl
Pfu buffer (10x)
2.5 µl
2.5 µl
dNTP (2.5 mM)
2 µl
2 µl
Primer 1 (20 mM)
1.25 µl MS6329-F
1.5 µl MS6344-F
Primer 2 (20 mM)
1.25 µl MS6329-R
1.5 µl MS6344-R3
1 µl
1 µl
Pfu enzyme (10 U/µl)
PCR program a
PCR-P1
PCR-P3
Total volume
25 µl
25 µl
a
The label PCR program is referring to PCR programs that are found in Table 7.
Msmeg6347
20 ng (1µl)
16 µl
2.5 µl
2 µl
1.25 µl MS6347-F
1.25 µl MS6347-R
1 µl
PCR-P1
25 µl
Table 3: Addition of His-tag to amplified PCR product.
Msmeg6329
Msmeg6344
PCR amplicon from
Approx. 0.1 ng (1 µl)
Approx. 0.1 ng (2 µl)
previous reaction used as
template a
dH2O
16 µl
15 µl
Pfu buffer (10x)
2.5 µl
2.5 µl
dNTP (2.5 mM)
2 µl
2 µl
Primer 1 (20 mM)
1.25 µl MS6329-HIS
1.5 µl MS6344-FHIS
Primer 2 (20 mM)
1.25 µl MS6329-R
1.5 µl MS6344-R3
1 µl
0.5 µl
Pfu enzyme (10 U/µl)
PCR program b
PCR-P2
PCR-P2
Total volume
25 µl
25 µl
a
In the Msmeg6329 reaction the template was diluted 1:100.
b
The label PCR program is referring to PCR programs that are found in Table 7.
Msmeg6347
Approx. 0.1 ng (1 µl)
16 µl
2.5 µl
2 µl
1.25 MS6347-FHIS
1.25 µl MS6347-R
1 µl
PCR-P4
25 µl
Table 4: Colony PCR
Msmeg6329
Msmeg6344
a
Msmeg6347
Bacteria
Bacteria
Bacteria
Template
dH2O
16 µl
16 µl
16 µl
Pfu buffer (10x)
2.5 µl
2.5 µl
2.5 µl
dNTP (2.5 mM)
2 µl
2 µl
2 µl
Primer 1 (20 mM)
1.25 µl MS6329-HIS
1.5 µl MS6344-FHIS
1.25 MS6347-FHIS
Primer 2 (20 mM)
1.25 µl reverse T7 Term
1.25 µl reverse T7 Term
1.25 µl reverse T7 Term
universal
universal
universal
1 µl
0.5 µl
1 µl
Pfu enzyme (10 U/µl)
PCR program b
PCR-P2
PCR-P2
PCR-P4
Total volume
24 µl
23 µl
24 µl
a
The bacteria template was added by touching an E. coli Top 10-F colony using a sterile tip, then transferring
the sample to the PCR reaction by pipetting up and down to mix the samples.
b
The label PCR program is referring to PCR programs that are found in Table 7.
20
Table 5: Sequencing correction PCR
Msmeg6347 (clone 4)
Msmeg6347 (clone 5)
Plasmid template
25 ng (0.5 µl)
25 ng (0.5 µl)
dH2O
16 µl
16 µl
Pfu buffer (10x)
2.5 µl
2.5 µl
dNTP (2.5 mM)
2 µl
2 µl
Primer 1 (20 mM)
1.5 µl MS6347-FHIS
1.5 µl MS6347-FHIS
Primer 2 (20 mM)
1.5 µl MS6347-R
1.5 µl MS6347-R
0.5 µl
0.5 µl
Pfu enzyme (10 U/µl)
PCR program a
PCR-P3
PCR-P3
Total volume
25 µl
25 µl
a
The label PCR program is referring to PCR programs that are found in Table 7.
Table 6: Primer sequences (5´to 3´) used for PCR or sequencing reactions
Name
Sequencea
Tmb
MS6329-F
ATGACGCACACTGAGGTCG
58
MS6329-R
TTATCGCTGGAACCTTTCGGC
58
MS6329-FHIS
ATGCATCACCATCACCATCACGGTACGCACACTGAGGTCGTC
58
MS6344-F
ATGTCAACGACCGAGTTTCC
60
MS6344-R
TTAGAGCAGTTGCAGGCGCCT
60
MS6344-FHIS
ATGCATCACCATCACCATCACGGTTCAACGACCGAGTTTCCGAC
60
MS6347-F
ATGTTCGACGCCGTAGGTAAC
62
MS6347-R
TTAGATGGGGAGCTTGCGG
62
MS6347-FHIS
ATGCATCACCATCACCATCACGGTTTCGACGCCGTAGGTAACC
62
MS6344-F2
ATGTCAACGACCGAGTTTCCGAC
70
MS6344-R2
CTAGAGCAGTTGCAGGCGCCTG
76
MS6344-R3
GCCTGTCAGAGCAGAGC
56
T7 forward
TAATACGACTCACTATAGGG
56
T7 Term universal
TATGCTAGTTATTGCTCAG
52
a
The underlined regions denote the annealing part of the sequence of which the temperature of melting (Tm)
was calculated.
b
Tm is a the temperature in degrees Celsius (°C) at which the primer was calculated to melt.
21
Table 7: PCR Programs.
Name
Number of cycles
Temperature
Time
PCR-P1
1
2
94°C
94°C
60°C
72°C
94°C
60→55°C a
72°C
72°C
4°C
2 min
2 min
1 min
2 min
2 min
1 min
2 min
5 min
∞
95°C
95°C
65°C
72°C
95°C
65→56°C a
72°C
72°C
4°C
2min
2min
1min
2min
1min
30sek
2min
5min
∞
95°C
95°C
63°C
72°C
95°C
63→50°C a
72°C
72°C
4°C
2min
1min
2min
1min
1min
30sek
2min
5min
∞
95°C
95°C
50°C
72°C
72°C
4°C
2min
2min
1min
2min
5min
∞
25
1
PCR-P2
1
2
25
1
PCR-P3
1
2
30
1
PCR-P4
1
29
1
a
The program is a touchdown program with a descending temperature starting from the higher value to end at
the lower one.
6.6
Plasmid preparation
Overnight cell cultures of E.coli TOP 10-F in 2 ml LB supplemented with 50 or 100 µg
Amp/ml were used for plasmid preparation. Plasmids were purified using a QIAprep®
Miniprep kit according to the manufacturer’s instructions. The DNA was eluted in 50 µl
buffer supplied by the manufacture. Plasmid preparations were evaluated using agarose gel
electrophoresis.
22
6.7
Sequencing
Purified plasmids were sent to sequencing to the Uppsala Genome Center, Uppsala
University. The samples sent consisted of 3 µl plasmid, 1.5 µl 1 mM T7 forward primer
(Table 6) or 1.5 µl 1 mM µl T7-term universal primers (Table 6) and 1.5 µl H2O. The
Uppsala Genome Center provided the sequencing results of the plasmids. Combinations of
forward and reverse results to complete sequences were made of all inserts. These full
sequences were aligned with the original sequence and compared.
6.8 Bioinformatics
The gene sequences were retrieved from http://www.dbbm.fiocruz.br/GenoMycDB using
Mycobacterium_tuberculosis_H37Rv as query and Mycobacterium_smagmatis_MC2 as hit.
The retrieved sequences were then aligned using the ClustalW tool at
http://www.ebi.ac.uk/clustalw/. The tools were used in their default settings. For retrieving
the molecular weight the ExPASy tool protparam was used at
http://www.expasy.ch/tools/protparam.html. Other information was found in TIGR CMD
database. The database is located at http://cmr.tigr.org/tigrscripts/CMR/shared/DnaMoleculeInformation.cgi
23
7
Acknowledgements
I would like to thank Dr. Mikael “Nisse” Nilsson for supervising me in my research work and
for giving me this opportunity. I also want to thank the rest of the Department of Cell and
Molecular Biology for being so nice and friendly. I wish you all the best!
24
8
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26