Bioreactors: The Effect of Induction Time &

Fed-Batch Mode on the Production of Pfu

DNA Polymerase from
E. coli BL21 (DE3)

Group Members:
Amirreza Sadrmanochehrinaeini (1153336)
Nicole Mangiacotte, Patrick Morkus, Robin Ng, Tyler Patten, Shayani Joseph, Michelle Ly,
Chen Pang, Simran Saini & Wing Lee

TA: Xudong Deng

Biochem 4LL3
1

Introduction
The developments made in genetic engineering and recombinant DNA technology during
1970s made a major breakthrough in field of biochemistry and biomedicine. The techniques
manipulated in this field are basic mechanisms used within the cell itself. Advancements in the
world of recombinant DNA lead to using these basic mechanism and features to produce specific
proteins in desired amounts. In this project, the purpose of S&E Laboratories, is to design a high
throughput expression system for producing Pfu DNA polymerase. In our attempt to do this, our
technicians used Escherichia coli (E.coli) BL21 (DE3). This system was equipped with pLysS
plasmid and pET15b- Pyrococcus furiosus (Pfu) plasmid vector. Some of important properties of
E.coli BL21 (DE3) is having a lacUV5 promoter as well as a chromosomal copy of the LacI
gene. LacI gene controls the T7 RNA polymerase gene. Induction of the cells with isopropyl D-thiogalactopyranoside (IPTG) will cause expression of T7 RNA polymerase which in turn
binds to the T7 promoter on plasmid vector and induces the expression of Pfu DNA polymerase.
Another advantageous and essential feature of this bacterial system is the pLysS plasmid.
pET15b-Pfu can be toxic and unstable in expression of BL21 (DE3) without additional pLysS
plasmid. This plasmid contains T7 lysosomes which decreases the expression of Pfu before
induction with IPTG; however it doesnt interfere with the expression after the cell is induced.
The last feature of the chosen bacterial system is deficiency in protease Lon and OmpT. These
two proteases degrade regulatory proteins necessary for cell survival and T7 RNA polymerase
during purification steps, respectively. Both Lon and OmpT are specific for degradation of Pfu
that is why their absence is crucial in our system. Additionally, our vector includes a hexahistidine (His(6))-tag that is important in purification step and also contains an ampicillin
resistance (AmpR) gene as a selectable marker for plasmid survival during transgene process.
2

In order to get an optimal condition, we tried to test the effects of time of induction and
production method on expression of our protein of interest, His(6)-Pfu DNA polymerase. The
optimal time to induce the production of protein is at log-phase when production is at its
maximum rate1. The production method is also being tested, between batch to fed-batch to seek a
solution for negative impacts that the by-products may have on cell growth. So, the ultimate goal
of this project is to find an optimal high-throughput expression protocol for our system, E.coli
BL21 (DE3) by changing absorbance range between 0.5-0.7 to 0.8-1.0 and also the production
method to fed-batch.
The reason for using an altered method of production such as fed-batch is that, if E.coli is
provided with excess nutrients, along with cell growth it will produce other by-products such as
acetic acid that will affect the cell growth negatively2. By using fed-batch, these nutrients are
slowly added to the media therefore they will be used up as soon as they enter the media by
E.coli for cell growth. This would result in a higher final density compared to the normal batch
method1.
Our experiment was designed with two batch reactions as controls with temperature of
37C, pH of 7.0, agitation of 300rpm and induction at absorbance range of 0.5-0.7 O.D. the two
other reactions were conducted independently to test induction time at absorbance of 0.8-1.0
O.D. and fed-batch production. Figure 1 illustrates a summary of experiments that were
completed in this project.
In the beginning, lysogeny broth (LB) media was prepared and autoclaved. Our
technicians ran four reactions using BioFlo 110 Fermentor3. While doing so, a sample was
taken from each bioreactor consistently and was checked for optimal density value. When
reached the determined value of O.D., the solution was induced by IPTG. The results are
3

summarized in Tables 1-4. Afterward, 3 bottles of 250 mL were collected from each bioreactor
and were centrifuged to separate out the media from the cells. The cells were then lysed using
BugBuster Protein Extraction Kit4. Since, Pfu DNA polymerase is a soluble protein, it would
stay in the supernatant. They were centrifuged to separate the insoluble debris from the solution.
The supernatant was then purified using nickel-nitrilo acetate (Ni-NTA) affinity chromatography
to separate the Pfu DNAP from the cell debris. Afterward, the purified Pfu DNAP was run on a
Bradford assay in order to find the concentration of the Pfu DNA polymerase. The calculated
concentration is then used to perform SDS-PAGE. SDS-PAGE is used to identify the expressed
protein and to validate the purity of protein sample. After running Bradford and SDS-PAGE for
all the four bioreactors solutions, Western blot was conducted in order to quantify the amount of
out specific protein produced from our system. Western blot helps in detecting the target protein
from rest of the complex of proteins present in the sample. Finally PCR amplification was used
as a form of functional assay to determine if the Pfu DNA polymerase is functional. After the
PCR was ran, the agarose gel electrophoresis was performed to check the efficency of PCR
reaction and also functionality of Pfu DNA polymerase.

Results
Bradford Assay-Standard curve
Absorbance of solutions with different BSA concentrations was measured at 595nm using
a Multiskan Ascent PlateReader. For Batch #1 and #2 BSA concentrations of 1, 0.6, 0.5, 0.4, 0.3,
0.2, 0.1, 0.05 mg/mL were used (Table 1, 3); whereas for Batch #3 and #4 different
concentrations were used which can be seen in Tables 5 and 7 in appendix A. The BSA standard
curve was then generated for each batch independently, by plotting these average absorbance
values presented in corresponding Tables, as a function of the concentration using Microsoft
4

Excel (Figures 2-5). To the resulting figure a line of best fit was added. The resulting equations
and the correlation factor are shown on each figure. All of lines showed medium to very strong
correlation between absorbance and concentration of the protein. Additionally, standard
deviation of each point was used as its error bar (Figure 2-5). Using these equations, the
absorbance values were correlated to an unknown concentration of Pfu and concentration of
some fractions were calculated. For instance for batch #1, the E1 (1/5 dilution) had an average
absorbance of 0.376 (Table 2). Using the equation from Figure 2, the corresponding
concentration was calculated to be 0.835 mg/mL (Table 2). Comparing the elution fractions of
batch #1 and #2, elution samples from batch #1 had more protein concentration. Batch #1 was
induced at O.D. of 0.8-1.0 while batch #2 was induced between O.D. of 0.5-0.7 and was used as
control batch. Also by comparing elution samples batch #3 and #4 that were diluted with factor
of 1/10, it can be seen that batch#4 shows higher protein concentration than batch#4. Batch #3 is
another control that was induced at 0.5-0.7 and batch #4 was induced at 0.8-1.0 O.D. For
instance, E1 (1/10 diluted) of batch #4 has 0.54 mg/mL Pfu DNAP, whereas E1 (1/10 diluted) of
batch #3 has concentration of 0.186 mg/mL (Tables 6 and 8).
SDS-PAGE visualization in order to identify relative purity
The relative purities of fractions obtained from cell lysis were compared by running a
sample of each on SDS-PAGE. Four SDS-PAGE were performed corresponding to each batch
samples (Figures 6-9). In each gel a molecular weight marker (ladder) was loaded in order to
enable comparison between different protein sizes present in the gel. Pfu DNAP is expected to
appear around 90kDa in SDS-PAGE. Looking at SDS-PAGE of batch #1, a thin band can be
seen above 78kDa. This band is also present in W1-3 and FT. Also, a very thick and strong band
can be seen around 48kDa. A similar trend goes is present in other batches. The band above
5

78kDa and around 48kDa is present in elutions and most of times in wash samples. The elution
samples for all gels, appear to be largely contaminated or impure due to presence of other bands.
These bands result from presence of other proteins in the elution other than Pfu DNAP (Figure 69).
Visualization of Pfu DNA polymerase using Western Blot
Figures 10-13 show the results of the performed western blot on some fractions obtained
after purification. Using a primary antibody, mouse monoclonal anti-polyhistidine clone -His.
This results in clear distinction of Pfu DNA polymerase that has a His tag on it from rest of
complex proteins in the gel. Same marker as SDS-PAGE was used in western blotting in order to
be able to compare the size of present proteins. Looking at Figures 10 that is for batch #1,
although the bands are a bit bend inward, there is a distinctive band in E1-3 and W1-3 above
78kDa and around 48kDa. Same is true for figures 12 and 13. Along with the desired band, there
are lots of other bands in each fraction present that indicates presence of other proteins in this
fractions. In Figure 11, which is for batch #2, there is no indication of Pfu DNA polymerase
around 90kDa. However bands at 48kDa are very thick and dark.
Testing Functionality of Pfu DNA polymerase using PCR
The PCR reaction was carried out and the products of the reaction were ran on agarose
gel electrophoresis to separate according to their sizes. Results are shown in Figure 14 and 15.
Figure 14 shows the agarose gel electrophoresis of batch #1 and #2. As shown on the figure 14,
in batch #1 elution 1 there is a distinctive dark band that represents the Pfu DNAP. On the other
lanes, commercial Taq DNA polymerase and different dilutions of Pfu DNA polymerase are
present to compare to our Pfu DNA polymerase. A ladder is also loaded in the gel in order to
ease the comparison. The bands appear around 500 kb. Figure 15 shows the agarose gel
6

electrophoresis of batch #3 and #4 fractions. Bands appear around 250 kb in all fractions but
mostly in batch #3 wash 1 and batch #4 wash 1.

Discussion
By studying the data of Bradford assays obtained from four bioreactors solutions, a
general trend can be observed. Comparing batch #1 and #2, batch #1 seems to have more protein
concentration than batch #2. For instance when calculated, Elution 1 (1/5 Diluted) of batch #1
had 0.835 mg/mL protein concentration (Table 2); whereas batch #2 Elution 1 (undiluted) had
0.526 mg/mL protein concentration (Table 4). This shows that the optimum range to induce
bacterial growth is at range of 0.8 to 1.0 O.D. Cells on average had higher rate of growth when
induced in this range compared to 0.5-0.7 O.D in control batch. Batch #3 elution 1 (1/10 diluted)
was calculated to have 0.1858 mg/mL protein compared to 0.5427 mg/mL protein concentration
in batch #4 elution 1(1/10 diluted) (Tables 6 and 8). This shows that use of fed-batch along with
induction at later time, between ranges of 0.8-1.0 O.D. enabled the cells to grow at a faster rate.
In batch #1 and #2, the change in time of induction increased the growth for elution fraction 1 for
about ~59% in protein concentration obtained. This increase was about 192% comparing batch
#3 and fed-batch, batch #4. This shows that as hypothesized, the use of fed-batch can help the
cell to grow in faster and more efficient rate. The reason for this is that, while growing bacterial
cells produced by-products which in turn can affect their growth and slow them down and this
effect is more visible when nutrient is in excess. However when using fed-batch the nutrient is
supplied slowly and at a consistent rate. Although this might slow down the growth of cells as it
on supplies a low amount of nutrients at a time, but this effect is overcame by the fact that there
are less by-products produced.

Looking at the SDS-PAGE that were performed on samples of different bioreactors

solutions, there is one band at 48kDa that is very dark and distinct compared to other bands
present (Figures 6-9). This may be due to fragmentation of the Pfu DNA polymerase while being
purified or even at cell lysing step. While lysing the cells, benzonase nuclease was not added to
the sample until after the cell lysis was carried out. In standard protocol it should be added in the
beginning of the step. This resulted in not having DNA strands degrading properly and may have
resulted in impurities in samples used for SDS-PAGE and western blotting. Also, adding
benzonase nuclease at a later stage may have because fragmentation of some of Pfu DNA
polymerase produced in cells and the bands at 48 kDa are thought to be the fragment of this
DNA polymerase that has His tag on it. That is why it is present in both SDS-PAGE and western
blotting. There are also thin and faint bands above 78 kDa where Pfu is expected to be found.
The bands are visible in Elutions 1-3 of SDS-PAGE of batch #1 -#4 (Figure 6-9). These bands
are also present in wash fractions of SDS-PAGE. This may be due to the fact that in two
instances the resin of the column chromatography was allowed to dry out. This may have caused
the resin structure to be disturb and that is why in wash samples we also get bands both at 48
kDa and 78 kDa (Figures 6-9).
For western blots, by looking at figures 10-13, we can see that the same pattern that
was present in SDS-PAGE is also visible here. Two types of bands are present at 48kDa and
above 78kDa. This shows that the protein, or protein fragments that are present at 48 kDa contain
his tags and that is why they are able to bind onto primary antibodies. Worth noting is that in
western blot of batch #1 (Figure 10) more Pfu DNAP was obtained in wash solution rather than
elutions. This may be again due to the error in performing the purification steps. In western blot
of batch #2 (Figure 11) there are no Pfu DNAP band present in any of samples. However, the
bands at 48 kDa are strong. The reason for this should be in performing western blot. Because
8

the Pfu bands are visible in SDS-PAGE (Figure 7) of batch #2 and there must have been
something wrong with the binding of antibodies to his tag of Pfu DNAP.
After running PCR products on agarose gel electrophoresis Figures 14 and 15 were
obtained. Between batch #1 and #2 only elution fraction 1 of batch #1 had a band at 500 bp
(Figure 14). This suggests that the Pfu DNA polymerase that was produced in batch # is
functional and is able to yield same amount of pET28b-folA as commercial Taq and Pfu DNA
polymerases. For batch #2 there was no visible band around 500 bp which was expected since
the western blot of batch# 2 did not show any presence of Pfu DNAP. In gels for batch#3 and #4,
the bands appear in wash fractions and they are at 250 bp (figure 15). This suggests that the
products of PCR might have been either fragmented or partially amplified.
In future experiment, we will be performing direct PCR product sequencing in order to
make sure that the right product and right gene was amplified. Also, the number of bioreactors
will be increased in order to balance out for experimental error by averaging the results of each
condition used. Further SDS-PAGEs and western blots will be performed again to confirm the
results obtained and to try to make the results more accurate and determine precision of results.
Also, next time performing the experiment it is important to make sure that the cells are lysed
properly and that the purification step using Ni-NTA chromatography is done without mistake.
This stage is very crucial and the samples obtained are important as all of results in later steps are
based on these samples.

References
(1) Jones, K., Vulcu, F., Cornelius, R., and Heirwegh, M. Section III: Bioreactors, in Custom
courseware for Biochem4LL3 Biotechnology & Genetic Engineering, pp 6380. Campus
store and Media Production Services: McMaster University.
(2) Shuler, M. L., and Kargi, F. (2002) Bioprocess Engineering: Basic Concepts 2nd ed. Prentice
Hall, New Jersey.
(3) (2007, August 8) BioFlo 110 Modular Benchtop Fermentor. New Jersy.
(4) Novagen. BugBuster Protein Extraction Reagent.
http://wolfson.huji.ac.il/purification/PDF/Protein_
Expression_Extraction/NOVAGEN_BugBuster_protein_extraction.pdf (Accessed January
26, 2014)
(5) Thermo Scientific. http://www.seas.upenn.edu/~belab/equipment/equipment_links/
Spec20_Manual.pdf (accessed January 26, 2014)
(6) Pfu DNA Polymerase. http://www.thermoscientificbio.com/pcr-enzymes-master-mixes-

and -reagents/pfu-dna-polymerase/ (accessed January 26, 2014).

(7) Bio-Rad. A Guide to Polyacrylamide Gel Electrophoresis and Detection; Bulletin No. 6040;
http://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_6040A.pdf (Accessed
January 26, 2014)
(8) Yang, Y; Ma, H. Western Blotting and ELISA Techniques. Researcher. 2009, 1(2), 67-86.
(9) Vulcu, F. Inquiry in Biochemical Techniques: Introduction to Biochemical Research. Fall

2012.
(10) Bio-Rad Protein Assay. The Bradford Assay.: Microtiter Plate Protocol.

http://labs.fhcrc.org/hahn/Methods/biochem_meth/biorad_assay.pdf. (Accessed on Jan 10,

2014)
(11) Bio-Rad. Handcasting Polyacrylamide Gels; Bulletin No. 6201; 2011. http://www.biorad.com/webroot/web/pdf/lsr/literature/Bulletin_6201.pdf
(12) Invitrogen by Life Technologies. iBlot Dry Blotting System.
http://tools.lifetechnologies.com/content/sfs/manuals/iblotsystem_qrc.pdf (Accessed Jan 26,
2014).

10

Appendix A: Figures and Tables

Figure 1. Summary of experiments. The above figure illustrates the sequence of experiments conducted in this
project.

Table 1. Summary of information regarding BSA standard curve for Batch #1 used to create Figure 2. The
absorbance for blank sample was adjusted to 0 and all other average absorbance values were adjusted
accordingly relative absorbance of blank sample. Absorbance was measured using the Multiskan Ascent Plate
Reader (Thermo Scientific). The error values are also shown in form of standard deviation from the mean.

Average
Absorbance
Concentration
(mg/mL)
Standard
Deviation
Relative
Absorbance

BSA 1

BSA2

BSA3

BSA4

BSA5

BSA6

BSA7

BSA8

Blank
(Water)

0.8345

0.7615

0.6185

0.6605

0.5205

0.5325

0.366

0.316

0.256

0.6

0.5

0.4

0.3

0.2

0.1

0.05

0.1011

0.0134

0.0629 0.089803

0.045962

0.027577

0.5785

0.5055

0.3625

0.2645

0.2765

0.4045

0.0113 0.0057 0.001414

0.11

0.06

Table 2. Absorbance of Elution samples 1 and 2 and flowthrough and calculated concentrations. The absorbance of
elution samples was obtained using Ascent Plate Reader (Thermo Scientific). Their average and the equation from
standard curve (Figure 2) was used in order to postulate the concentrations of proteins for elutions 1-3 and
flowthrough of batch #1.

Elution 1
1/5
Dilution
Average
Absorbance
Concentration
(mg/mL)

Elution 2
1/10
1/5
1/10
Dilution Dilution Dilution

Elution
3
1/5
Dilution

1/10
Dilution

Undiluted

Flow-through

0.376

0.331

0.3385

0.3405

0.2645

0.265

0.6655

0.835

1.044

0.574

1.176

0.0591

0.125

0.570

11

Batch #1 Absorbance vs. Concentration Standard Curve

1.1
1

Absorbance

0.9
0.8

y = 0.7182x + 0.256
R = 0.8084

0.7
0.6
0.5
0.4
0.3
0.2
0

0.2

0.4

0.6

0.8

1.2

BSA Concentration (mg/mL)

Figure 2. Absorbance of Bovine serum albumin (BSA) Standards at Varying Concentrations using the Bradford
Assay Technique for Batch #1. Shown is the collected data of absorbance of BSA standards measured at 595 nm,
versus varying concentrations of BSA in a Bradford assay reaction. The linear equation represents the relationship
2
between BSA absorbance and BSA concentrations. The R value shows a strong correlation between the linear
equation and data points. Data points represent the average value of the sample sets. The error bars on the data plot
show the value of each samples standard deviation. Its important to note that the error bars for first three data
points and eight data point are present however they are very small values. Figure was generated using Microsoft
Excel. In plotting this graph, the absolute values of absorbance were used and the intercept was set to the absorbance
of blank, which in this case was water.

Table 3. Summary of information regarding BSA standard curve for Batch #2 used to create Figure 3. The
absorbance for blank sample was adjusted to 0 and all other average absorbance values were adjusted accordingly
relative absorbance of blank sample. Absorbance was measured using the Multiskan Ascent Plate Reader (Thermo
Scientific). The error values are also shown in form of standard deviation from the mean.

BSA 1
Average
Absorbance
Concentration
(mg/mL)
Standard
Deviation
Relative
Absorbance

BSA2

0.59

BSA3

BSA4

BSA5

BSA6

BSA7

BSA8

Blank

0.618

0.391

0.525

0.3835

0.456

0.322

0.296

0.2545

0.6

0.5

0.4

0.3

0.2

0.1

0.05

0.0424

0.0636

0.0226

0.000707

0.0240

0.0735

0.000707

0.3355

0.3635

0.1365 0.2705

0.129

0.2015

0.0675

0.0415

Table 4. Absorbance of Elution samples 1-3 and calculated concentrations. The absorbance of elution samples was
obtained using Ascent Plate Reader (Thermo Scientific). Their average absorbance and the equation from standard
curve (Figure 3) was used in order to postulate the concentrations of proteins for elutions 1-3 undiluted of batch #2.

Average Absorbance
Concentration (mg/mL)

Elution 1 Undiluted Elution 2 Undiluted Elution 3 Undiluted

0.4795
0.6005
0.346
0.526
0.809
0.214
12

Batch #2 Absorbance vs. Concentration Standard Curve

0.8

Absorbance

0.7
0.6
y = 0.4276x + 0.2545
R = 0.6267

0.5
0.4
0.3
0.2
0

0.2

0.4

0.6

0.8

1.2

BSA Concentration (mg/mL)

Figure 3. Absorbance of Bovine serum albumin (BSA) Standards at Varying Concentrations using the Bradford
Assay Technique for Batch #2. Shown is the collected data of absorbance of BSA standards measured at 595 nm,
versus varying concentrations of BSA in a Bradford assay reaction. The linear equation represents the relationship
2
between BSA absorbance and BSA concentrations. The R value shows a fairly good correlation between the linear
equation and data points. Data points represent the average value of the sample sets. The error bars on the data plot
show the value of each samples standard deviation. Its important to note that the error bars for all data points is
present however in some points they are very small values. Figure was generated using Microsoft Excel. In plotting
this graph, the absolute values of absorbance were used and the intercept was set to the absorbance of blank, which
in this case was water.

Table 5. Summary of information regarding BSA standard curve for Batch #3 used to create Figure 3. The
absorbance for blank sample was adjusted to 0 and all other average absorbance values were adjusted accordingly
relative absorbance of blank sample. Absorbance was measured using the Multiskan Ascent Plate Reader (Thermo
Scientific). The error values are also shown in form of standard deviation from the mean.

BSA 1
Average
Absorbance
Concentration
(mg/mL)
Standard
Deviation
Relative
Absorbance

0.7255

BSA2

BSA3

BSA4

BSA5

BSA6

Blank
(PBS)

0.6425

0.5155

0.4155

0.339

0.291

0.2585

0.75

0.5

0.25

0.125

0.0625

0.0714

0.0912

0.0233

0.027577

0.011314

0.015556

0.000707

0.467

0.384

0.257

0.157

0.0805

0.0325

13

Table 6. Absorbance of Elution samples 1-3 and calculated concentrations. The absorbance of elution samples was
obtained using Ascent Plate Reader (Thermo Scientific). Their average absorbance and the equation from standard
curve (Figure 3) was used in order to postulate the concentrations of proteins for elutions 1-3 (diluted 10 times) of
batch #3.

Average
Absorbance
Concentration
(mg/mL)

Elution 1Elution 2Elution 31/10 Dilution 1/10 Dilution 1/10 Dilution

0.26767
0.26433
0.27
0.1858

0.1182

0.2331

Absorbance

Batch #3 Absorbance vs. Concentration Standard Curve

0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0

y = 0.4934x + 0.2585
R = 0.9871

0.2

0.4

0.6

0.8

1.2

BSA Concentration (mg/mL)

Figure 4. Absorbance of Bovine serum albumin (BSA) Standards at Varying Concentrations using the Bradford
Assay Technique for Batch #3. Shown is the collected data of absorbance of BSA standards measured at 595 nm,
versus varying concentrations of BSA in a Bradford assay reaction. The linear equation represents the relationship
2
between BSA absorbance and BSA concentrations. The R value shows a very strong correlation between the linear
equation and data points. Data points represent the average value of the sample sets. The error bars on the data plot
show the value of each samples standard deviation. Its important to note that the error bars for all data points is
present however in some points they are very small values. Figure was generated using Microsoft Excel. In plotting
this graph, the absolute values of absorbance were used and the intercept was set to the absorbance of blank, which
in this case was PBS.

14

Table 7. Summary of information regarding BSA standard curve for Batch #4 used to create Figure 3. The
absorbance for blank sample was adjusted to 0 and all other average absorbance values were adjusted accordingly
relative absorbance of blank sample. Absorbance was measured using the Multiskan Ascent Plate Reader (Thermo
Scientific). The error values are also shown in form of standard deviation from the mean.

BSA 1
Average
Absorbance
Concentration
(mg/mL)
Standard
Deviation
Relative
Absorbance

0.531
1
0.1216
0.279

BSA2

BSA3

0.462

0.437

0.75

0.5

0.0764 0.0764
0.21

0.185

BSA4

BSA5

BSA6

Blank
(PBS)

0.3495 0.3115 0.2735

0.25

0.252

0.125 0.0625

0.0389 0.0304 0.0092

0.0975 0.0595 0.0215

Table 8. Absorbance of Elution samples 1-3 and calculated concentrations. The absorbance of elution samples was
obtained using Ascent Plate Reader (Thermo Scientific). Their average absorbance and the equation from standard
curve (Figure 3) was used in order to postulate the concentrations of proteins for elutions 1-3 (diluted 10 times) of
batch #4.

Elution 11/10 Dilution

Average
Absorbance
Concentration
(mg/mL)

Elution 21/10 Dilution

Elution 3- 1/10
Dilution

0.268

0.262

0.248

0.5427

0.3405

-0.1314

15

Absorbance

Batch #4Absorbance vs. Concentration Standard Curve

0.7
0.65
0.6
0.55
0.5
0.45
0.4
0.35
0.3
0.25
0.2

y = 0.2967x + 0.252
R = 0.9564

0.2

0.4

0.6

0.8

1.2

BSA Concentration (mg/mL)

Figure 6. Absorbance of Bovine serum albumin (BSA) Standards at Varying Concentrations using the Bradford
Assay Technique for Batch #4. Shown is the collected data of absorbance of BSA standards measured at 595 nm,
versus varying concentrations of BSA in a Bradford assay reaction. The linear equation represents the relationship
2
between BSA absorbance and BSA concentrations. The R value shows a very strong correlation between the linear
equation and data points. Data points represent the average value of the sample sets. The error bars on the data plot
show the value of each samples standard deviation. Its important to note that the error bars for all data points is
present however in some points they are very small values. Figure was generated using Microsoft Excel. In plotting
this graph, the absolute values of absorbance were used and the intercept was set to the absorbance of blank, which
in this case was PBS.

Figure 5.SDS-PAGE of Nickel

Column Elution Fractions for
Batch #1. Sodium dodecyl sulfate
polyacrylamide gel
electrophoresis was run for ~1
hour at 115 V after the fractions
were obtained from nickel affinity
column chromatography. A
BLUeye prestained protein ladder
(Gene DireX, Tris-Glycine 420%) was used in Lane 2 in order
to measure the sizes of proteins in
each band in different lanes. L,
refers to Ladder, E to elution, W
to wash and FT to flow-through
fractions.

16

Figure 7.SDS-PAGE of Nickel

Column Elution Fractions for Batch
#2. Sodium dodecyl sulfate
polyacrylamide gel electrophoresis
was run for ~1 hour at 115 V after
the fractions were obtained from
nickel affinity column
chromatography. A BLUeye
prestained protein ladder (Gene
DireX, Tris-Glycine 4-20%) was
used in Lane 2 in order to measure
the sizes of proteins in each band in
different lanes. L, refers to Ladder,
E to elution, W to wash and FT to
flow-through fractions. Lys, refers
to Lysate.

Figure 8.SDS-PAGE of Nickel

Column Elution Fractions for Batch
#3. Sodium dodecyl sulfate
polyacrylamide gel electrophoresis
was run for ~1 hour at 115 V after
the fractions were obtained from
nickel affinity column
chromatography. A BLUeye
prestained protein ladder (Gene
DireX, Tris-Glycine 4-20%) was
used in Lane 2 in order to measure
the sizes of proteins in each band in
different lanes. L, refers to Ladder, E
to elution, W to wash and FT to
flow-through fractions.

17

Figure 9.SDS-PAGE of Nickel Column

Elution Fractions for Batch #4. Sodium
dodecyl sulfate polyacrylamide gel
electrophoresis was run for ~1 hour at
115 V after the fractions were obtained
from nickel affinity column
chromatography. A BLUeye prestained
protein ladder (Gene DireX, TrisGlycine 4-20%) was used in Lane 2 in
order to measure the sizes of proteins in
each band in different lanes. L, refers to
Ladder, E to elution, W to wash and FT
to flow-through fractions. Lys, refers to
Lysate. The ladder for this gel was
missed while inserting the solutions into
wells of the gel. That is why the ladder
from Batch #2 SDS-PAGE experiment
was cut and used for indications of size
of substrate present in the fractions.
Figure 10. Western characterization
of batch #1. Western blot was
loaded with samples of flowthrough, wash and elution fractions.
It was run at 200V for 45min.
proteins were transferred to
nitrocellulose membrane via iBlot
Dry Blotting System (Life
Technologies). The blot was then
developed with primary/secondary
antibodies and a color development
solution. Prestained ladder
(Genedirex) was used in Lane 3.
Lanes 4-6 are elution fractions and
Lanes 7-9 are washes. A sample of
flow-through is also loaded on Lane
10.

Figure 11. Western characterization

of batch #2. Western blot was
loaded with samples of flowthrough, wash and elution fractions.
It was run at 200V for 45min.
proteins were transferred to
nitrocellulose membrane via iBlot
Dry Blotting System (Life
Technologies). The blot was then
developed with primary/secondary
antibodies and a color development
solution. Prestained ladder
(Genedirex) was used in Lane 1.
Lanes 2-4 are elution fractions and
Lanes 5-7 are washes. A sample of
flow-through is also loaded on Lane
8.

18

Figure 12. Western

characterization of batch #3.
Western blot was loaded with
samples of flow-through, wash and
elution fractions. It was run at 200V
for 45min. proteins were transferred
to nitrocellulose membrane via iBlot
Dry Blotting System (Life
Technologies). The blot was then
developed with primary/secondary
antibodies and a color development
solution. Prestained ladder
(Genedirex) was used in Lane 1.
Lanes 3-5 are elution fractions and
Lanes 7-9 are washes. A sample of
flow-through is also loaded on Lane
10. Two lanes between marker and
eltuions and elutiosn and washes
were not used.
Figure 13 Western
characterization of batch #4.
Western blot was loaded with
samples of flow-through, wash
and elution fractions. It was run at
200V for 45min. proteins were
transferred to nitrocellulose
membrane via iBlot Dry Blotting
System (Life Technologies). The
blot was then developed with
primary/secondary antibodies and
a color development solution.
Prestained ladder (Genedirex) was
used in Lane 3. Lanes 8-10 are
elution fractions and Lanes 5-7 are
washes. A sample of flow-through
is also loaded on Lane 4. Lysate
sample was also loaded on Lane 2.

19

Figure 14.Funtional assay

PCR amplification
Figureusing
15.Funtional
assay usingon Pfu
DNA polymerase
from
batch #1
PCR amplification
on Pfu
DNA
and #2.
PCR
products
were
polymerase
from
batch
#3 and
#4. run
on agarose
gelrun
electrophoresis
PCR products
were
on agarose at
100V for ~45atminutes.
gel electrophoresis
100V forThe gene
amplified
pET28b-folA
~45 minutes.
Thewas
gene
amplified
which is around
500
bp. The
was pET28b-folA
which
is around
is showed
500 bp.folA
Theproduced
folA produced
is with
the
name
of
DNAP
on
the side
showed with the name of DNAP
figure.
The GeneRuler
1kb
on the of
side
of figure.
The
ladder1kb
was
loaded
onloaded
lane 13 as
GeneRuler
ladder
was
Lanes Lanes
2-5 are1
on lanea 2reference.
as a reference.
controls
loaded with
and 3-5positive
are positive
controls
DNA DNA
polymerases
loadedcommercial
with commercial
Taq and
Pfu.
Commercial
Pfu
polymerases
Taq
and
Pfu.
DNA polymerase
was assayed
Commercial
Pfu DNA polymerase
at various
concentrations. Lane
was assayed
at various
6 is negative
that lacks a
concentrations.
Lanecontrol
6 is negative
polymerase.
controlDNA
that lacks
a DNA Lanes 7-9 are
products
produced
polymerase.
Lanes
7-9 areby proteins
in batch#1
Lanes
products
producedfractions.
by proteins
in 1012fractions.
are products
from
batch
batch#3
Lanes
10-12
are#2
samples.
products
from batch #4 samples.

20

Appendix B: Sample Calculations

Batch #1-E1 (1/5 diluted):
Average absorbance=y=0.376
Equation of standard curve: y = 0.7182x + 0.256
Therefore: x=y/0.7182 - 0.356447
X= 0.8354 mg/mL
Note: all other calculations were done in the same way using Microsoft Excel 2010.

21