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Korya 1
Abstract
Green Fluorescent Proteins (GFP) are proteins that exhibit bright green fluorescence under
UV light. The objective of this project was to produce GFP that was isolated from a new
starfish species to determine whether Fluorotek Company can file a patent on this protein.
Escherichia coli cells were transformed from the starfish Amphioxus by introducing the
pGLO plasmid vector, containing the genes for GFP and ampicillin resistance (bla). The
transformed cells were grown on two plates, where one of them expressed GFP that
fluoresced under UV light. The transformation frequency of the transformed bacterial cells
was 35.0 Transformants/πœ‡g DNA. The transformed E.coli cells grown from the +pGLO
(LB/amp/ara) plate were ruptured to extract the GFP, which was then purified using
Hydrophobic Interaction Chromatography (HIC). Afterwards, the concentrations of the prepurified and the post-purified GFP were measured using a Bradford Assay. The pre-purified
sample concentration was 723.3 ΞΌg/mL, and the post-purified sample concentration was
263.7 ΞΌg/mL. SDS-PAGE gel electrophoresis was preformed using denatured and native
GFP samples to determine the molecular weights of the proteins present in both, pre and
post purified samples. The molecular weight of native GFP was calculated to be 38.23 kDa,
this value was higher than that of denatured GFP, which was calculated to be 26.1 kDa. This
molecular weight of denatured GFP was 3.2% lower than the molecular weight of GFP
extracted from Aequorea victoria, which was reported as 26.9 kDa (4). This result indicated
that the project was effective in producing a new, lighter GFP with many potential patent
applications in biotechnology and the research field.
Korya 2
Background
The Green Fluorescent Protein (GFP) was originally discovered and isolated from
the jellyfish Aequoria victora (4). This protein is made of 238 amino acids and exhibits a
bright green fluorescence when expressed to Ultraviolet light (4). In molecular biology,
GFP has been used as a reporter of expression. Also, it has been used as a proof of
concept that a gene can be expressed throughout an organism, and maintained in their
genomes (4). The main objective of this project was to isolate the gene responsible for
GFP production from a newly discovered starfish, found in the South Pacific. This gene
was placed into a plasmid vector (pGLO) to produce significant quantities of the protein
for characterization, so Fluorotek Company can file for a patent.
In this project, E. coli cells were transformed using the plasmid vector pGLO that
contains the genes for GFP production and an ampicillin resistance gene (bla) that codes
for beta-lactamase. Afterwards, GFP was produced and subsequently purified using
Hydrophobic Interaction Chromatography column and assayed for protein concentration,
then analyzed using SDS-PAGE gel electrophoresis.
Figure 1: pGLO Structure
Ara B, A, and D are genes present in E. coli that
code for the enzymes responsible for utilizing
arabinose. These genes are controlled by araC, the gene
that is bound by the cell’s DNA at the promoter (PBAD).
AraC codes for the binding protein and triggers the
transcription of Ara B, A, and D in the presence of
arabinose, resulting in the production of the enzymes.
In this project, the araC gene and the PBAD promoter were present in the in the bacterial
Korya 3
cells; however, the structural genes, BAD, were replaced with the GFP. From here, with
the presence of arabinose, RNA polymerase binding was promoted by araC, which
primed the transcription of the GFP gene, resulting in the production of GFP. Bla is also
on the same plasmid as GFP, yet it is not controlled by the ara operon. The bacterial cells
secreted beta-lactamase enzyme so that they can survive in an ampicillin environment,
which facilitated identification and selection of transformed cells.
Procedure
Two microtubes, labeled +pGLO for transforming cells and –pGLO for controlled
cells, were filled with 250 microliters of the transformation solution calcium chloride and
E. coli cells. Plasmid DNA was only added to the +pGLO tube, and then both tubes were
incubated on ice for ten minutes. Afterwards, both tubes were heat shocked into a 42˚C
waterbath for fifty seconds then placed back on ice for two minutes. Luria-Brentani (LB)
nutrient broth was added and the tubes were left to incubate. Subsequently, the
transformed and control cells were then placed into four different agar plates with their
appropriate control suspensions: -pGLO (LB), -pGLO (LB/amp), +pGLO (LB/amp), and
+pGLO (LB/amp/ara). Then, the plates were stacked and incubated for 24 hours. The
following week, growth observations on each plate were recorded under UV light and
presented in [table 1].
After observing the (+) and the (-) tubes under UV light, the (-) tubes were disposed
and the (+) liquid culture was placed in a (+) microtubes in order to be centrifuged at a
maximum speed, then the supernatant was discarded. Afterwards, 250 microleters of TE
(Tris EDTA) solution were added to the tube and the pellet was resuspended. Only one drop
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of Lysosome was then added to the resuspended pellet, mixed, and observed under UV light.
From here, the liquid was frozen in an ethanol/dry ice bath for 30 seconds, and then thawed
with hand warmth. This process was repeated five times. Then, the microtube was
centrifuged for 10 minutes at maximum speed. 150 πœ‡L were transferred to a new microtube
labeled β€œ+”, and 20 πœ‡L were transferred to another microtube that was set on ice and labeled
β€œpre-purficiation GFP”. Binding buffer was added the β€œ+” microtube and refrigerated. While
the tube was centrifuged, the hydrophobic interaction chromatography (HIC) column was
prepared by adding 2 mL of equilibrium buffer to it, then, 150 πœ‡L of the β€œ+” supernatant and
150 πœ‡L of binding buffer were added on top of the column, followed by 250 πœ‡L of the
washing buffer. As the solution was going down the bottom, 750 πœ‡L of TE were added and
the column was examined under UV light.
While the chromatography column was in process, a Bradford Assay of protein serial
dilutions were set up. Pre-Purified dilutions were 1:2 and 1:5, while Post-Purified dilutions
were 1:1, 1:2, and 1:5. The absorbance values were recorded at 595 nm using a spec20
spectrophotometer, and then the data obtained was used to create a standard curve.
Following, Laemmli buffer was added to four microtubes, two for pre-purified GFP
and other two for post-purified GFP. One tube of each kind of GFP was placed for native
protein and the other for denatured protein. Designated microtubes for denatured proteins
were placed in a hot water bath. Specific amounts of native pre-purification GFP, native postpurification GFP, heated pre-purification GFP, and heated post-purification GFP were loaded
into their designated SDS-PAGE electrophoresis gel wells and electrophoresis was
preformed. Afterwards, the gel was analyzed under UV light. The migration distances of the
protein bands of Kaleidoscope and native GFP were measured, and a standard curve was
Korya 5
created using a Kaleidoscope bands distances. The gel was stained overnight using a Bio-safe
Coomassie Blue stain, and then each band migration distance and the Rf values were
measured. GFP molecular weight was then calculated using a generated standard curve.
Results
Table 1: Transformation Results
Plate
Number of
Colonies
-pGLO (LB)
0
-pGLO (LB/amp)
+pGLO (LB/amp)
0
7
+pGLO (LB/amp/ara)
4
Bacteria Characteristics under UV light
Did not fluoresce, bacterial growth present in a thick, gel like
structure. Confluent growth. Very strong Pungent smell.
No bacterial growth, clear looking plate.
White, little drop-like, circular colonies spread all around the
dish. Did not fluoresce under UV light.
White, little drop-like, circular colonies spread all around the
dish. Bright Green fluorescent under UV light.
Calculation 1: Transformation Efficiency
DNA concentration = 0.08 πœ‡g/mL, Plasmid volume = 10 πœ‡L
DNA amount = concentration of DNA x volume of DNA
= 0.08 πœ‡g/mL x 10 πœ‡L = 0.8 πœ‡g DNA used
Fraction of DNA used =
π‘‰π‘œπ‘™π‘’π‘šπ‘’ π‘†π‘π‘Ÿπ‘’π‘Žπ‘‘ π‘œπ‘› 𝐿𝐡/π‘Žπ‘šπ‘/π‘Žπ‘Ÿπ‘Ž
π‘‡π‘œπ‘‘π‘Žπ‘™ π‘£π‘œπ‘™π‘’π‘šπ‘’ 𝑖𝑛 𝑑𝑒𝑠𝑑 𝑑𝑒𝑏𝑒
=
100 .0πœ‡L
510.0 πœ‡L
= 0.1961
The amount of DNA spread on the agar plate = Total amount of DNA x fraction of DNA
= 0.8 πœ‡g/ πœ‡L x 10 πœ‡L = 0.157 πœ‡g
+pGLO LB/amp/ara transformation frequency =
+pGLO LB/amp transformation frequency =
Average =
4+7
2
= 5.5 οƒ 
5.5
0.157
=
π‘‡π‘œπ‘‘π‘Žπ‘™ π‘›π‘’π‘šπ‘π‘’π‘Ÿ π‘œπ‘“ 𝑐𝑒𝑙𝑙𝑠 π‘œπ‘› π‘‘β„Žπ‘’ π‘π‘™π‘Žπ‘‘π‘’
π΄π‘šπ‘œπ‘’π‘›π‘‘ π‘œπ‘“ 𝐷𝑁𝐴 π‘ π‘π‘Ÿπ‘’π‘Žπ‘‘ π‘œπ‘› π‘‘β„Žπ‘’ π‘π‘™π‘Žπ‘‘π‘’
π‘‡π‘œπ‘‘π‘Žπ‘™ π‘›π‘’π‘šπ‘π‘’π‘Ÿ π‘œπ‘“ 𝑐𝑒𝑙𝑙𝑠 π‘œπ‘› π‘‘β„Žπ‘’ π‘π‘™π‘Žπ‘‘π‘’
π΄π‘šπ‘œπ‘’π‘›π‘‘ π‘œπ‘“ 𝐷𝑁𝐴 π‘ π‘π‘Ÿπ‘’π‘Žπ‘‘ π‘œπ‘› π‘‘β„Žπ‘’ π‘π‘™π‘Žπ‘‘π‘’
=
=
4
0.157
7
0.157
= 25.5
= 44.6
π‘‘π‘Ÿπ‘Žπ‘›π‘ π‘“π‘œπ‘Ÿπ‘šπ‘Žπ‘›π‘‘π‘ 
πœ‡g DNA
π‘‘π‘Ÿπ‘Žπ‘›π‘ π‘“π‘œπ‘Ÿπ‘šπ‘Žπ‘›π‘‘π‘ 
πœ‡g DNA
35.0 Transformants/ πœ‡g DNA
Comments/Observations 2: Chromatography:
Observations
*The pellet was originally green under UV light. After the freeze/thaw cycles, it became
white, while the supernatant turned into foggy green.
*Five drops of GFP were collected. Drops collected expressed a bright green color under
UV light.
Korya 6
Table 2: List of Volumes and Samples Taken During Extraction and Purification Processes
Sample
Volume extracted (πœ‡g)
Used for
Column/Binding buffer
(Purification process)
Freeze (for Pre-Pure GFP
Electrophoresis)
Bradford Assay (1:2 dilution/
pre-pure)
Bradford Assay (1:5
dilution/Pre-Pure)
Left over
Bradford Assay (1:1/PostPure)
Bradford Assay (1:2
dilution/Post-Pure)
Bradford Assay (1:5
dilution/Post-Pure)
Post-Pure GFP
Electrophoresis
Left over
Volume used (πœ‡g)
150
40
β€œ+” Supernatant
235
25
10
10
50
25
Post-Purification GFP
240
10
40
115
Table 3: BSA Standard Concentration and
Absorbances at 595 nm
Concentration
B
C
D
E
750 πœ‡g/mL
500 πœ‡g/mL
375 πœ‡g/mL
250 πœ‡g /mL
Absorbance at
595 nm
0.606
0.302
0.247
0.101
Calculations 2: Sample Concentration
calculations for the pre and post GFP samples
Graph 1: BSA Bradford Assay Standard Curve (PostPure)
Absorbance at 595 nm
Test Tube Label
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
y = 0.001x – 0.1476
RΒ² = 0.98
0.247
0.302
0.101
100
Undiluted Post-Purified GFP
Y= 0.116 οƒ  0.116=0.001X + 0.1476
X = 263.7 ΞΌg/mL
0.606
300
500
700
Pre-Purified GFP (1:2) Dilution
Y= 0.214 οƒ  0.214 = 0.001X + 0.1476
X= 361.6(*2) οƒ  723.3 ΞΌg/mL
* The absorbances of the undiluted Post Purified GFP, Pre
Purified GFP (1:2 Dillution) and the Pre-Purified (1:5
Dillution) fall within the standard curve.
Table 4: Bradford Assay Results for Pre-Pure and Post-Pure GFP

Tube Label
Absorbance at 595nm
Concentration (ΞΌg/mL)
Before Dilution Factor
Concentration (ΞΌg/mL)
After Dilution Factor
Post-Purified 1:1
Post-Purified 1:2
Post-Purified 1:5
Pre-Purified 1:2
Pre-Purified 1:5
0.116
0.057
0.024
0.214
0.128
263.7
204.7
171.7
361.7
275.7
263.7
409.3
855.0
723.3
1378
Table 5: Loading the Samples of Electrophoresis
Lane
1
2
3
4
5
6
900
BSA Protein Concentration (ΞΌg/mL)
Volume
10Β΅l
10Β΅l
10Β΅l
20Β΅l
20Β΅l
20Β΅l
Sample
Kaleidoscope standards
Heated pre-purification GFP
Heated post-purification GFP
Heated pre-purification GFP
Heated post-purification GFP
Kaleidoscope standards
Korya 7
7
8
9
10
10Β΅l
10Β΅l
20Β΅l
20Β΅l
native pre-purification GFP
native post-purification GFP
native pre-purification FGP
native post-purification GFP
Table 5: Gel Electrophoresis Migrated Distances for Denatured and Native Pre and Post Purified GFP
stds
Well 1
10 ΞΌL
KaleidOscope
standards
Distance/rf
250 18.8 / 0.37
150 22.6/ 0.44
100 25.5/ 0.50
75 30.9/ 0.61
50 35.0/ 0.69
37 40.0/ 0.79
25 42.5/ 0.85
20 45.5/ 0.90
15 48.8/ 0.97
10 50.6/ 1.0
Well 2
10 ΞΌL
Heated
Prepure
GFP
——-No data
No data
No data
No data
No data
No data
No data
No data
No data
No data
Well 3
10 ΞΌL
Heated
Postpure
GFP
distance RF/MW
0.51/113
23.9
0.56/90.4
26.2
0.63/67.8
29.1
0.77/35.6
35.6
0.83/26.3
38.7
1/12.1
46.5
Well 4
20 ΞΌL
Heated
Prepure
GFP
distance RF/MW
0.63/66.5
29.3
0.64/64.6
29.6
0.66/59.0
30.5
0.71/46.1
33.0
0.74/40.9
34.2
0.78/33.6
36.2
0.84/25.6
39.1
0.89/19.8
41.5
0.94/15.7
43.9
1/12.1
46.5
Well 5
20 ΞΌL
Heated
Postpure
GFP
distance RF/MW
0.58/83.9
25.8
0.63/65.4
28.2
0.77/35.2
34.2
0.83/26.0
37.1
1/12.1
44.5
Well 6
20 ΞΌL
KaleidOscope
standards
Distance/rf
17.2/ 0.34
21.9/ 0.43
24.3/ 0.48
29.5/ 0.57
34.2/ 0.67
38.7/ 0.76
41.1/ 0.81
44.2/ 0.87
47.2/ 0.93
51.0/ 1.0
Well 7
10 ΞΌL
Native
Prepure
GFP
distance RF/MW
0.45/ 136
23.1
0.49/ 113
25.1
0.55/ 86.2
28.1
0.60/ 67.5
30.8
0.64/ 56.3
32.8
0.68 /48.2
34.5
0.73 /37.0
37.4
0.79 /28.5
40.3
0.88 /18.2
45.2
1/10.7
51.1
Well 8
10 ΞΌL
Native
Postpure
GFP
distance RF/MW
0.56/80.8
26.0
0.63/60.3
28.9
0.73/38.1
33.2
0.75/33.3
34.9
1/10.7
46.1
Well 9
20 ΞΌL
Native
Prepure
GFP
distance RF/MW
0.47/126.0
22.1
0.52/97.1
24.8
0.59/71.6
27.9
0.63/57.7
30.1
0.65/51.8
31.2
0.69/45.6
32.5
0.72/37.8
34.4
0.79/27.6
37.6
0.84/22.1
39.9
0.86/20.0
40.9
0.90/16.6
42.8
0.97/12.1
46.0
1/10.7
47.3
Well 10
10 ΞΌL
Native
Postpure
GFP
distance RF/MW
0.5/108.2
23.5
0.55/85.3
25.9
0.60/69.3
28.0
0.72/38.6
33.9
0.76/33.0
35.5
1/10.7
46.9
The Unit for Molecular Weight is (kDa)
The Unit for RF and the distance is (mm)
Numeric Values with a yellow highlight are the suspected to be the MW of the denatured GFP
Numeric Values with a blue highlight are suspected to be the MW of the denatured GFP
Graph 3: Protein Molecular Weight
Standard Curve (Well 6)
3
Log Molecular Weight kDa
Log Molecular Weight (kDA)
Graph 2: Protein Molecular Weight
Standard Curve (Well 1)
2.5
2
1.5
1
y = -1.9997x + 3.0829
RΒ² = 0.9873
0.5
0
0.2
0.4
0.6
0.8
1
3
2.5
2
1.5
1
y = -2.0167x + 3.0446
RΒ² = 0.9937
0.5
1.2
0
0.2
0.4
0.6
Rf Value (mm)
0.8
1
1.2
Rf Value (mm)
Calculations 3: Rf and MW Calculations for heated
GFP
Calculations 4: Rf and MW Calculations for Native
GFP
Rf calculations (of the 3rd well’s GFP band)
Rf calculations (of the 8th well’s GFP band)
Rf =
π‘‘π‘–π‘ π‘‘π‘Žπ‘›π‘π‘’ π‘œπ‘“ π‘π‘Žπ‘›π‘‘ π‘œπ‘“ π‘–π‘›π‘‘π‘’π‘Ÿπ‘’π‘ π‘‘
π‘‘π‘–π‘ π‘‘π‘Žπ‘›π‘π‘’ π‘œπ‘“ π‘‘β„Žπ‘’ π‘™π‘œπ‘€π‘’π‘ π‘‘ π‘π‘Žπ‘›π‘‘
=
38.7 π‘šπ‘š
46.5 π‘šπ‘š
= 0.83 mm
MW calculations of GFP
*X= Rf (average)
*Y= Log kDa
π‘‘π‘–π‘ π‘‘π‘Žπ‘›π‘π‘’ π‘œπ‘“ π‘π‘Žπ‘›π‘‘ π‘œπ‘“ π‘–π‘›π‘‘π‘’π‘Ÿπ‘’π‘ π‘‘
π‘‘π‘–π‘ π‘‘π‘Žπ‘›π‘π‘’ π‘œπ‘“ π‘‘β„Žπ‘’ π‘™π‘œπ‘€π‘’π‘ π‘‘ π‘π‘Žπ‘›π‘‘
=
34.4 π‘šπ‘š
47.3 π‘šπ‘š
= 0.72 mm
MW calculations of GFP
*X= Rf (average)
*Y= Log kDa
0.83+0.84+0.83
Log (Y) = -1.9997 (
Rf =
3
Molecular Weight of GFP = 10
) + 3.0829 = 1.42
1.42
= 26.09 kDa
Log (Y) = -2.0167 + (
0.73+0.73+0.72+0.72
4
Molecular Weight of Native GFP = 10
) + 3.0446 = 1.58
1.58
= 38.23 kDa
Korya 8
Picture 1: Electrophoresis gel Picture (Post-Stained)
Picture 2: Electrophoresis Gel Picture under UV light
Korya 9
Discussion
The purpose of this project was to produce GFP by transforming Escherichia coli cells with a
plasmid vector that contained the GFP gene, then purify the produced GFP and analyze it so that Fluorotek
can potentially file a patent on the new protein. In this project, two microtubes containing E. coli bacteria
were prepared. One of the tubes contained pGLO plasmid (+pGLO), and the other tube contained no plasmid
(-pGLO). Calcium chloride and Luria-Bertani (LB) nutrient broth were added to both tubes. Calcium chloride
was used as the transformation solution to neutralize the negative charges of the phosphate backbone of DNA,
which allowed it to pass through the bacterial cell walls and enter the cell. LB broth was the nutritious supply
for bacteria to grow. After transformation, both +pGLO and –pGLO E.coli were plated onto their selected
agar plates (+pGLO LB/amp/ara, +pGLO LB/amp, -pGLO LB/amp, and –pGLO LB). The agar plates were
incubated for a day and the data was collected the following week [Table 1].
The -pGLO (LB) plate showed bacterial growth due to the presence of LB; however, there were no
colonies present, instead, confluent growth was observed. Bacterial cells were clumping together forming
thick gel-like structure with a strong pungent smell. This plate served as a control, which verified that the cells
remained viable after transformation.
The –pGLO (LB/amp) plate had no bacterial growth. Bacterial cells on this plate were not
transformed (lacked the pGLO plasmid), meaning that they had no resistance to ampicillin; therefore, all of
the cells on that plate died. This plate served as a control, which verified that E. coli cells lacking the pGLO
plasmid were ampicillin sensitive and proved that selective pressure was applied against non-transformed
cells.
The transformed bacterial cells containing the plasmid showed E. coli colonies even in the presence
of ampicillin, because the pGLO plasmid also contained a gene for beta-lactamase protein that confers
resistance to the antibiotic ampicillin. However, only the plate that contained arabinose should fluoresce under
UV light, because in the presence of arabinose, the GFP gene that is attached to the plasmid under ara control
Korya 10
will be transcribed, inducing GFP production. That can be indicated by the green fluoresce observed under
UV light. According to the experimental observations, both plates containing the plasmid (+pGLO) had
bacterial colonies on them. The +pGLO (LB/amp) plate had seven white bacterial colonies that were
distributed around the plate. Under UV light, the colonies did not fluoresce (same color was maintained), due
to the absence of arabinose. This plate verified that E.coli cells containing the plasmid were ampicillin
resistant.
The +pGLO (LB/amp/ara) plate on the other hand, had only four colonies that looked the same as the
+pGLO (LB/amp) colonies; yet, they expressed a bright green fluorescence under UV light due to the
presence of arabinose. This plate is considered the experimental plate because it had the appropriate growth
environment for GFP production.
The calculated transformation efficiency was 3.5 x 101 transformants/πœ‡g DNA. This is considered very
low with the expected frequency range being between 8.00 x 102 and 7.00 x 103 transformants/πœ‡g DNA. The
number of colonies on the two plates was different, which resulted in different transformation frequencies,
indicating the presence of an error. The reason for this low efficiency could have been due to multiple errors.
An operator error that could have reduced the number of colonies observed was improper mixing of the
transformed cells before transferring them to the plates. The transformed cells might have settled to the
bottom of the tube, resulting in minimal transfer of transformed cells when pipetting the supernatant.
In the next step of this project, GFP was extracted from the bacterial cells, purified through the HIC
column, and analyzed using Bradford assay. Two test tubes labeled as (-) and (+) were inoculated by each
+pGLO (LB/amp/ara) and +pGLO (LB/amp) and both tubes had the growth medium LB/amp/ara. The
microtubes were incubated to allow the E. coli cells to grow and produce GFP and the enzyme beta-lactamase.
Both tubes fluoresced under UV light due to the presence of arabinose that induced the production of GFP in
Korya 11
the cells from both, the +pGLO (LB/amp) and +pGLO (LB/amp/ara). This was preformed to demonstrate that
arabinose turns on the ara operon.
GFP extraction began by transferring two milliliters of the …
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You have to be 100% sure of the quality of your product to give a money-back guarantee. This describes us perfectly. Make sure that this guarantee is totally transparent.

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Zero-plagiarism guarantee

Each paper is composed from scratch, according to your instructions. It is then checked by our plagiarism-detection software. There is no gap where plagiarism could squeeze in.

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Free-revision policy

Thanks to our free revisions, there is no way for you to be unsatisfied. We will work on your paper until you are completely happy with the result.

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Privacy policy

Your email is safe, as we store it according to international data protection rules. Your bank details are secure, as we use only reliable payment systems.

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Fair-cooperation guarantee

By sending us your money, you buy the service we provide. Check out our terms and conditions if you prefer business talks to be laid out in official language.

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