1.1
METHODS
1.1.1
1.1.1.1
MEDIA PREPARATION
PREPARATION OF 1X TBE BUFFER
1 litre of 1x TBE buffer was used for the period of running experiments during this project. Using a
digital weighing balance, 10.8 grams of Tris (base), 0.9 grams of EDTA and 5.5 grams Boric acid were
introduced into a 1000ml borosilicate glass reagent bottle and diluted to 800ml with RO water. The
mixture was homogenized on a magnetic plate while its pH was adjusted to ~8.2 using 5M HCL. The
total volume of the mixture was rounded to 1000ml using RO water and stored at room temperature
for the duration of the project.
1.1.1.2
PREPARATION OF 1% AND 2% AGAROSE GEL FOR GEL ELECTROPHORESIS
1% gel and 2% agarose gel were used in running the Gel electrophoresis samples. For preparing the
1% gel, 0.5 grams of agarose powder was weighed and introduced into a 50ml borosilicate glass
reagent bottle. Using a measuring cylinder, 50ml of prepared 1xTBE buffer (see section 3.2.1.1) was
used to dilute the solvent before subjecting the mixture to heat in a microwave for 1 minute. After
heating, 2μl of Gel red reagent was added to the mixture and set to cool before pouring it into an 8
well comb of Flowgen Nioscience Gel Electrophoresis pack. 2% agarose gel followed the same protocol
of preparation except that 1 gram of agarose powder was diluted in 50ml of 1X TBE buffer.
Prior to loading, 6X Gel Loading Dye was added to the samples and mixed (brief vortexing and
centrifugation). Also, 5μl of DNA marker (up to 5000bp) was loaded in the first well as standard for
the procedure. The gel settings were prepared to run at 100v, 200A for 45 minutes.
1.1.1.3
PREPARATION OF LB BROTH AND LB BROTH + AMPICILLIN
Two conical flasks of 250ml of LB broth were prepared for the transformation and cell culture stages
of the project. Using a digital weighing balance, 25grams of Tryptone, 1.25 grams of Yeast Extract, 1.25
grams of Nacl and 0.25 grams of Glucose were measured and introduced separately into two 250ml
flasks. Both mixtures were diluted with 250ml of RO water before autoclaving (done by Lab
technicians). After autoclaving, the mixtures were set to cool and 100μl of ampicillin was inoculated
into one set of the flask mixture (LB broth + Ampicillin) under sterile conditions. The prepared
mediums were stored at 4°C for the period of the laboratory sessions.
1.1.1.4
PREPARATION OF LB AGAR PLATES
250ml of LB agar was prepared for cell plating of the transformation samples. In a 500ml borosilicate
glass reagent bottle, 25grams of Tryptone, 1.25grams of Yeast Extract, 1.25grams of Nacl, 0.25 grams
of Glucose and 2.5 grams of Nutrient Agar (LB Agar) were weighed and added individually. The mixture
was diluted to 250ml of RO water and set for autoclaving. Under sterile conditions, 150μl of Ampicillin
was inoculated into the agar mixture and mixed by swirling before evenly distributing among 12 petridishes. The plates were left to solidify before storing them in the 4°C incubator.
1.1.2
CUTTING OF GEL BANDS
Complying with the cautionary operational procedures of using the U.V room, gel bands were excised
with a razor blade under the illumination aid of Ultraviolet light and placed in 10ml polypropylene
centrifuge tube. The mass of excised gel were measured using a digital weighing balance provided by
Coventry University.
1.2
PRIMER DESIGN
The four primer sets used in this project were designed with the simulation aid of Primer3 software.
The resulting sequences were sent to our collaborators at The Pirbright Institute for respective
oligonucleotide generation. The primers were designed specifically to be in used in the amplification
of the GP4 template, pcDNA-GP4 backbone, hypervariable construct, and Sanger Sequencing of
pcDNA and GP4 clones. For amplifying GP4, the forward and reverse primer were designed to feature
homologous base pairs at the beginning and end of the GP4 template. The primer design for amplifying
the pcDNA-GP4 backbone was to use a random forward and reverse primer to randomly amplify its
template for a site directed mutagenesis. Sanger Sequencing of pcDNA-GP4 and hypervariable clones
was done using a designed primer that amplifies the template downstream its stop codon. The reverse
primer of GP4 was also used in sequencing the pcDNA-GP4 clones.
All primer sets were initially converted from nanomoles to picomoles and diluted further 1 in 10 to
contain 10 picomoles/μl.
Template
Primer ID
Sequence
Molecular weight
Tm
(g/mol)
(°C)
OD
nmol
Amplicon
product
expected size
in Base pairs
GP4
gp4 forward
taagcttggtaccgagctcgCCatg
12,922.4
72.5
47.2
121.9
GCGGCGGCGATACTCTT
gp4 reverse
actgtgctggatatctgcagTCATATC
576
15,074.8
69.9
58.7
123.5
GCCAACAGGATCGCAAACAGGC
pcDNA-GP4
pcDNA_ran_f
CACAGTGCAGGGAAGC
4,940.3
53.7
22.2
138.4
backbone
pcDNA_ran_r
TAATATTCTGCAAGACCATGAAG
7,055.7
50.7
31.8
136.3
Sanger
Sequencing
TTCGTCGGTAACATTTGCGG
5.13
55.9
17.6
94.0
Sequencing
5000
576
TABLE 1. Generated primer features indicating suppliers conditions and expected amplicon size. The
yellow block and red block of GP4 primer sequences indicate the forward and reverse homologous
end designed reverse complementary to the pcDNA3.1 vector.
1.3
1.3.1
BIOINFORMATICS ANALYSIS OF GP4
DESIGN OF GP4 TEMPLATE
The GP4 template used in this project was obtained from the complete cds of a codon optimized
PRRSV 1 Olot strain provided by our collaborators at The Pirbright Institute. The total concentration
of the sample supplied was 5ng for 10 μl and was diluted 1 in 10 before use.
Description
Sequence
PRRSV-1 GP4 ccatgGCGGCGGCGATACTCTTT
(Olot strain)
Base pairs
576
Optic ID
15990
OPPF no
OPPF22390
Amino
Additional
acids
comments
2-183
Full length with
CTGTTGGCGGGAGCCCAGCAT
native
TTTATGGTATCTGAGGCATTTG
sequence fused
CCTGTAAGCCGTGTTTCTCTAC
TCACCTTTCTGATATTAAGACT
AACACGACCGCCGCGGCAGGC
TTCATGGTCTTGCAGAATATTA
ATTGTCTTAGGCCGCACGGAG
TCAGCACAGCTCAAGAGAACA
TATCATTCGGAAAGCCCTCACA
GTGCAGGGAAGCCGTAGGGAT
ACCTCAATATATAACCATTACC
GCAAATGTTACCGACGAATCAT
ATTTGTATAACGCGGACCTTCT
GATGCTGTCAGCGTGTCTCTTT
TACGCATCCGAAATGTCAGAG
AAGGGCTTCAAAGTAATCTTTG
GTAATGTTAGTGGAGTAGTTA
GTGCGTGTGTGAATTTCACTGA
TTACGTGGCACATGTAACCCAG
CATACTCAGCAGCACCACCTTG
to
leader
GFP
expression
screening.
for
TGATAGACCATATCAGGCTGCT
TCACTTCTTGACCCCATCAACA
ATGCGCTGGGCAACTACCATC
GCCTGCCTGTTTGCGATCCTGT
TGGCgatatga
TABLE 2. Nucleotide sequence of GP4 protein, codon optimised by Prof. Simon Graham and colleagues
at The Pirbright Institute. The sequence and the construct itself were obtained from Prof. Simon
Graham directly through personal communication.
1.3.2
1.3.2.1
CLONING OF GP4
AMPLIFICATION OF GP4
Amplification of the GP4 template was done using Polymerase Chain Reaction (PCR). In a sterile pcr
tube, 1.5μl of the gp4 forward primer, 1.5μl of the gp4 reverse primer, 2μl of the diluted GP4 template,
10μl of the Q5 master mix and 5μl of sterile water were added to make up a total volume of 20μl. The
reaction mixture was briefly vortexed and centrifuged before placing in a Technine Thermal cycler for
PCR under the following conditions:
Initial Denaturation at 98°C for 30 seconds
Annealing at
98°C for 30 seconds
68°C for 30 seconds
72°C for 30 seconds
Final Extension at 72°C for 2 minutes
x35 cycles
Final Hold at 10°C
(NEB Q5®High-Fidelity DNA Polymerase M0491 protocol)
1.3.2.2
DOUBLE DIGEST OF pcDNA 3.1 EXPRESSION VECTOR
Double Digest of the pcDNA 3.1 vector was done using restriction enzymes BamHI-HF and EcorI-HF.
These enzymes were selected after using the online Invitrogen tool to check for the available multiple
compatible cloning sites. The pcDNA 3.1 (+) expression vector was 5000 base pairs. For setting up the
digest, 2μl of the pcDNA 3.1 template (1 in 10 diluted), 2μl of rcut smart Buffer, 1μl of EcorI and 1μl
BamHI and 14μl of sterile water were added into a 1.5ml Eppendorf tube to make a total reaction
volume of 20μl (NEB protocol). The reaction was incubated at 37°C for 3 hours.
FIGURE 1. A screenshot showing the indicated cleavage sites of restriction enzymes BamHI and EcoRI
highlighted in yellow and green respectively on the multiple cloning sites of a pcDNA 3.1 (+) vector
Invitrogen map .
1.3.2.3
GEL ELECTROPHORESIS OF AMPLIFIED GP4 AND DIGESTED pcDNA 3.1 TEMPLATE
Gel electrophoresis of the amplified GP4 template and digested pcDNA 3.1 vector was done by loading
20μl of both samples and running them on 1% of agarose gel following previously stated protocol (see
section 3.2.1.2). The resulting gel bands were excised following standard procedure (see section 3.2.2).
1.3.2.4
EXTRACTION AND PURIFICATION OF GP4 AND pcDNA 3.1 VECTOR BANDS
The excised gel mass of GP4 was 0.56g and 0.31g for pcDNA 3.1. DNA extraction and purification of
excised gel bands was done using NEBs Monarch® DNA Gel Extraction kit following its manufacturers
protocol (NEB T1020 protocol). Final elution volume of extracted samples was 20μl.
1.3.2.5
SPECTROPHOTOMETRIC ANALYS IS OF PURIFIED GP4 AND pcDNA3.1
Spectrophotometric analysis of the GP4 and pcDNA 3.1 extracted samples was done using a Nanodrop.
2μl of DNA elution buffer was used in blanking the device before loading 2μl of each sample for
analysis.
1.3.2.6
CONFIRMATION OF EXTRACTED GP4 AND pcDNA3.1 SAMPLES
Another gel electrophoresis was set up to confirm the presence of the extracted GP4 and pcDNA 2.1
vector samples. In two separate 1.5ml Eppendorf tubes, 2μl of each extracted sample were added and
mixed with 18μl of sterile water. 20μl of both samples were loaded into separate wells of 1% of
agarose gel and run following previous protocol (see section 3.2.1.2).
1.3.2.7
GIBSON ASSEMBLY, TRANSFORMATION AND CELL PLATING
Gibson Assembly to clone GP4 into pcDNA 3.1 was done using a Gibson Assembly Kit following its
manufacturers protocol (NEB Gibson assembly protocol E5510). For optimized cloning efficiency,
0.008pmol of the purified pcDNA 3.1 sample and 0.08pmol the GP4 insert were used in the ratio 1:3
for preparing 20μl of the Gibson assembly reaction mix.
Transformation of the Gibson assembly mix was done into two tubes of NEB 5-alpha competent E. coli
cells. 10μl of the Gibson assembly mix was added directly into each tube of the competent cells and
incubated on ice for 30mins. The mixture was heat shocked in a water bath for 45 seconds at 42°C and
transferred back on ice to cool for 2 minutes completing the transformation process. A negative
control using 10μl of only the digested pcDNA sample as the insert was set up following the same
procedure. 900μl of LB broth was added to the transformation mixture and placed in a 37°C shaking
incubator for 2 hours (NEB Transformation protocol C2987H).
Under sterile conditions, 150μl was taken from the three transformation reactions and pipetted into
separate labelled LB agar plates (see section 3.2.1.4). The samples were streaked around the whole
circumference of the plates using an inoculating loop and were set to dry before incubating them at
37°C for 24 hours.
1.3.2.8
PLASMID EXTRACTION AND PURIFICATION OF pcDNA-GP4 TRANSFORMED COLONIES
Under sterile conditions, eight transformed colonies were randomly picked from the cultured plates,
and each separately inoculated into sterile universal vials containing 50ml LB broth x ampicillin. The
bacterial cultures were placed in a 37°C shaking incubator for 16 hours.
pcDNA-GP4 plasmid extraction and purification was done using of NEBs Monarch® Plasmid Miniprep
Kit following its manufacturers protocol. Final elution of the purified plasmid sample was 30μl.
1.3.2.9
SPECTROPHOTOMETRIC ANALYSIS OF PURIFIED pcDNA SAMPLE
Spectrophotometric analysis of the purified pcDNA samples was done using a Nanodrop. 2μl of the
plasmid elution buffer was used in blanking the device before loading 2μl of each sample for analysis.
From the results, the three purified samples (C, D and F) with the highest concentrations were selected
for the stages of confirming double digest of transformed pcDNA-GP4 clones and also in the
amplification of the pcDNA backbone.
1.3.3
CONFIRMATION OF PCDNA-GP4 CLONES
To confirm the presence of GP4 insert, 2μl each of purified plasmid were used in setting up a pcr
reaction for amplifying GP4 following the previous protocol (see section 3.4.2.1).
A double digest consisting of 2μl rcut smart buffer, 1μl EcoRI, 1μl BamHI, 2μl each of purified plasmid
sample (C, D and F) and 14μl of sterile water was set up in a 1.5ml Eppendorf tube and incubated at
37°C for 2 hours. Both samples were run on a single 1% agarose gel following previous protocol (see
section 3.4.2.2).
1.4
1.4.1
pcDNA-GP4 BACKBONE CONSTRUCTION
AMPLIFICATION OF pcDNA-GP4 BACKBONE
In a pcr tube, 2.5μl of pcDNA_ran_f primer, 2.5μl of pcDNA_ran_r primer, 3μl of the three selected
purified pcDNA-GP4 samples (diluted to contain 10ng), 25μl of Q5 Master Mix and 18μl of sterile water
were added and mixed to a total volume reaction of 50μl. The reaction was placed in a thermal cycler
for PCR under the following conditions:
98°C for 30 seconds
98°C for 10 seconds
61°C for 10 seconds
x42 cycles
72°C for 3 minutes
72°C for 5 minutes
1.4.2
GEL ELECTROPHORESIS OF pcDNA BACKBONE
Gel electrophoresis of the amplified pcDNA backbone template was done by loading 15μl of each
sample before running on 1% gel following previous protocol (see section 3.2.1.2). The resulting gel
bands were excised and measured following the previous conditions (see section 3.2.2).
1.4.3
pcDNA-GP4 BACKBONE EXTRACTION
DNA extraction of pcDNA-GP4 backbone gel bands was done using Monarch® DNA Gel Extraction Kit
following the manufacturers protocol (NEB T1020 protocol). The final volume of each purified sample
eluted was 20 μl. The three samples used were divided into duplicates for DpnI digestion i.e C1, C2,
D1, D2, F1 and F2.
1.4.4
DpnI DIGESTION AND SPECTROPHOTOMETRIC ANALYSIS
For DpnI digestion, 1 μl of DpnI enzyme, 1 μl of the purified sample, 5 μl of rcut Buffer were diluted to
40μl in an Eppendorf tube. The DpnI enzyme was inactivated by incubating the reaction mix in a dry
bath at 80°C for 20 minutes before transferring on sample ice before carrying out ethanol
precipitation. Ethanol precipitation was done by adding 100μl of 100% ethanol and 4μl of sodium
acetate to the 40μl of digestion mix before storing the total mixture overnight at -80°C. The samples
were retrieved and centrifuged in a 4°C incubator for 1 hour at 13000 rpm, washed with 1ml of 70%
Ethanol (-20°C stored) and set to completely air dry. The pellets were resuspended in 20μl of sterile
water and stored at -20°C (Coventry University protocol).
2μl of the RO water was used in blanking the Nanodrop before loading 2μl of the digested sample. The
sample with the highest concentration was used for cloning the hypervariable construct.
1.5
1.5.1
BIOINFROMATICS ANALYSIS OF HYPERVARIABLE CONSTRUCT
DESIGN OF HYPERVARIABLE CONSTRUCT FROM GP4
The 40 random GP4 PRRSV-1 cds exported from the NCBI database were translated into their
respective amino acids sequences using Expasy and then aligned with the muscle tool of Mega 11
software. The hypervariable region of the amino acid alignment sequence was determined by
identifying the region that displayed the most variability that was common amongst all 40 sequences.
The identified sequence was then exported as a single Fasta format file for phylogenetic analysis and
study. The DNA regions responsible for translating each of the 40 amino acid sequence of the
hypervariable regions were determined manually by cross referencing them in the Expasy tool before
creating an alignment of those regions using muscle tool of Mega 11 software.
To generate the web logo, the exported fasta file of the aligned DNA regions were imported into the
Berkely Web Logo online tool (Default settings of the tool were used in generating the logo image).
The resulting logo generated gave a representation of the 87 conserved nucleotide bases in the
hypervariable region.
To design the hypervariable construct template for amplification, each axis of the 87 conserved
nucleotide bases from the DNA logo were manually inputted into an Excel file. The 87 nucleotide bases
were extended to 100 bases by adding 13 homologous bases to each end from the original GP4
sequence. Using the IUPAC Nomenclature for nucleotide bases, each axis of the 100 nucleotide bases
were interpreted for its respective code. The resulting sequence was sent to The Pirbright institute for
the template generation.
For easy viewing, the translated amino acid sequences were also analysed using Clustal Omega.
SEQUENCE ID
SEQUENCE
Molecular
weight
(g/mol)
Hyp_var_gP4_TPI
Tm
OD
nmol Base
pair
(°C)
CTTCATGGTCTTGCAGAATATTATYRVHTGYBY 30,776.7 72.4
size
194.9
99.1
100
YCRRYYYYRHVRRYMHYRRMNRYRYMDNVBY
SVVVHVYYHHHHNVVVVRYVRYCACAGTGCAG
GGAAGC
TABLE 3. Random library of 40 GP4 Hypervariable construct. The sequence was generated individually
and sent for its generation with the aid of Prof. Simon Graham.
1.5.2
AMPLIFICATION OF HYPER VARIABLE CONSTRUCT.
In a pcr tube, 12.5μl of primer 2, 12.5μl of the Hypervariable construct and 25μl of Q5 Master mix
were added and mixed by vortexing. The reaction mix was placed in a thermal cycler under the
following pcr conditions:
98°C for 30 seconds
98°C for 2 minutes
60°C for 15 seconds
x 1 cycle
72°C for 15 min
1.5.3
GEL ELECTROPHORESIS OF HYPER VARIABLE CONSTRUCT
25μl of the amplified hypervariable construct was loaded into two well before running on 2% of
agarose gel following protocol (see section 3.2.2). The gel bands were excised and measured under
the standard condition (see section 3.2.1.2).
1.5.4
DNA EXTRACTION AND SPECTROPHOTOMETRIC ANALYSIS OF HYPERVARIABLE CONSTRUCT
DNA extraction of pcDNA-GP4 backbone gel bands was done using NEBs using Monarch® DNA Gel
Extraction Kit following its manufacturers protocol (NEB T1020 protocol). The final volume of the
sample eluted was 20μl.
Spectrophotometric analysis of the purified plasmid samples was done using a Nanodrop. 2μl of
plasmid elution buffer was used to blank the device before loading 2μl of each sample for analysis.
The sample with the highest concentration was selected for the Gibson Assembly procedure.
1.5.5
GIBSON ASSEMBLY AND TRANSFORMATION
Gibson Assembly to clone the hypervariable construct into the pcDNA-GP4 plasmid was done using a
Gibson Assembly Kit following its manufacturers protocol (NEB Gibson assembly protocol E5510). For
optimized cloning efficiency, the vector insert ratio used for this procedure was 1:5 (i.e 3.5μl of
purified pcDNA-GP4 backbone sample and 1.5μl of the purified hypervariable construct).
Transformation was done using 10 μl of the Gibson Assembly mix into three tubes of NEB 5-alpha
competent E. coli cells following the previously stated protocol (see 3.4.2.7).
Under sterile conditions, 100 μl, 150 μl and 900 μl of each transformation mix were inoculated and
streaked into separate LB agar plates. The plated dishes were incubated at 37°C for 24 hours.
1.5.6
COLONY SELECTION AND PLASMID PURIFICATION
Under sterile conditions, eight random colonies (A, B, C, D, E, F and G) were picked from the
transformed plates and grown in 50ml of LB Broth x Ampicillin. Sample G was inoculated with multiple
random colonies to serve as a multiple construct template.
pcDNA-GP4 plasmid extraction and purification was done by NEBs Monarch® Plasmid Miniprep Kit
following manufacturers instruction. Final plasmid elution of the purified sample had 30μl.
1.6
SANGER SEQUENCING OF PCDNA-GP4 CLONES AND HYPER VARIABLE CONSTRUCT
Sanger sequencing of the purified pcDNA-GP4 clones and hypervariable construct were done using
BigDye™ Terminator v3.1 Cycle Sequencing Kit following its protocols. The samples were diluted to
contain 100ng/μl of DNA and 0.8 pmol/μl of sequencing primers adding up to a total volume of 20μl.
The reactions were placed in a thermocycler under the following PCR conditions:
STEP
TIME
TEMPERATURE
Initial Denaturation
1 minute
96°C
Denaturation
10 seconds
96°C
5 seconds
96°C
Extension
4 minutes
96°C
Final Hold
∞
10°C
Annealing
X25 Cycles
TABLE 4. Sanger Sequencing conditions of pcDNA-GP4 and Hypervariable clones.
After the PCR step, 50 μl of 100% ethanol and 2μl of Sodium Acetate were added to the sequencing
mix and stored at -80°C overnight. The samples were retrieved and centrifuged for 1hour at 13000
rpm in a 4°C incubator, before being washed with 1ml of 70% Ethanol (-20°C) and centrifuging for 15
minutes and setting to air dry. The pellets were resuspended in 15μl of formamide and loaded into
the well plate of Applied Biosystems 3500 analyser for sequencing (Coventry University protocol).
1.7
PHYLOGENETIC ANALYSIS OF 40 GP4 SEQUENCES
Phylogenetic analysis was done by computing a time tree of 40 GP4 sequences using the Mega 11
software. The exported amino acid alignment was calibrated to contain the isolation year and location
of each GP4 sequence. The new alignment was used in simulating a Neighbourhood Joining estimation
phylogeny tree. The time tree analysis of the phylogeny tree was run using PRSSV-2 GP4 cds as the
outgroup and the isolation year as the sample times.
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