bioreactor optimization

8/10/2019 Bioreactor Optimization

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Bioreactors: The Effect of Induction Time &

Fed-Batch Mode on the Production of Pfu

DNA Polymerase fromE. col i 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


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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 producingPfu DNA polymerase. In our attempt to do this, our

technicians usedEscherichia 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 ofPfu DNA polymerase.

Another advantageous and essential feature of this bacterial system is the pLysS plasmid.

pET15b-Pfu canbe 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 hexa-

histidine (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.


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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


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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 thePfu DNAP from the cell debris. Afterward, the purifiedPfu 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 ofPfu 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


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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 PfuDNAP, 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. PfuDNAP 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


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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 thanPfuDNAP (Figure 6-

9).

Visualization of PfuDNA 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 PfuDNA 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 PfuDNA polymerase

around 90kDa. However bands at 48kDa are very thick and dark.

Testing Functionality of PfuDNA 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 thePfu DNAP. On the other

lanes, commercial Taq DNA polymerase and different dilutions of Pfu DNA polymerase are

present to compare to our PfuDNA 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


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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.


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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 thePfuDNA 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) morePfu 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


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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 ofPfu 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-folAas 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 ofPfuDNAP. 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.


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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 January26, 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.bio-

rad.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).
http://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_6201.pdfhttp://tools.lifetechnologies.com/content/sfs/manuals/iblotsystem_qrc.pdfhttp://www.google.com/url?q=http%3A%2F%2Ftools.lifetechnologies.com%2Fcontent%2Fsfs%2Fmanuals%2Fiblotsystem_qrc.pdf&sa=D&sntz=1&usg=AFQjCNG4oYN04hUfXiJznspqtPtv4rhTiQhttp://www.google.com/url?q=http%3A%2F%2Ftools.lifetechnologies.com%2Fcontent%2Fsfs%2Fmanuals%2Fiblotsystem_qrc.pdf&sa=D&sntz=1&usg=AFQjCNG4oYN04hUfXiJznspqtPtv4rhTiQhttp://tools.lifetechnologies.com/content/sfs/manuals/iblotsystem_qrc.pdfhttp://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_6201.pdf


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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. Theabsorbance 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 BSA2 BSA3 BSA4 BSA5 BSA6 BSA7 BSA8Blank

(Water)

Average

Absorbance0.8345 0.7615 0.6185 0.6605 0.5205 0.5325 0.366 0.316 0.256

Concentration

(mg/mL)1 0.6 0.5 0.4 0.3 0.2 0.1 0.05 0

Standard

Deviation0.1011 0.0134 0.0629 0.089803 0.045962 0.027577 0.0113 0.0057 0.001414

RelativeAbsorbance

0.5785 0.5055 0.3625 0.4045 0.2645 0.2765 0.11 0.06 0

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 Elution 2Elution

3Flow-through

1/5

Dilution

1/10

Dilution

1/5

Dilution

1/10

Dilution

1/5

Dilution

1/10

DilutionUndiluted

Average

Absorbance0.376 0.331 0.3385 0.3405 0.2645 0.265 0.6655

Concentration

(mg/mL)0.835 1.044 0.574 1.176 0.0591 0.125 0.570


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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

between BSA absorbance and BSA concentrations. The R2

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 (ThermoScientific). The error values are also shown in form of standard deviation from the mean.

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.

y = 0.7182x + 0.256

R = 0.8084

0.2

0.3

0.4

0.5

0.6

0.70.8

0.9

1

1.1

0 0.2 0.4 0.6 0.8 1 1.2

Absorbance

BSA Concentration (mg/mL)

Batch #1 Absorbance vs. Concentration Standard Curve

BSA 1 BSA2 BSA3 BSA4 BSA5 BSA6 BSA7 BSA8 Blank

Average

Absorbance0.59 0.618 0.391 0.525 0.3835 0.456 0.322 0.296 0.2545

Concentration

(mg/mL)1 0.6 0.5 0.4 0.3 0.2 0.1 0.05 0

Standard

Deviation0.0424 0.0636 0.0226 0 0.000707 0 0.0240 0.0735 0.000707

Relative

Absorbance0.3355 0.3635 0.1365 0.2705 0.129 0.2015 0.0675 0.0415 0

Elution 1 Undiluted Elution 2 Undiluted Elution 3 Undiluted

AverageAbsorbance 0.4795 0.6005 0.346

Concentration (mg/mL) 0.526 0.809 0.214


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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


value shows a fairly good correlation between the linear


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. Theabsorbance 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 BSA2 BSA3 BSA4 BSA5 BSA6Blank

(PBS)

Average

Absorbance0.7255 0.6425 0.5155 0.4155 0.339 0.291 0.2585

Concentration

(mg/mL)1 0.75 0.5 0.25 0.125 0.0625 0

Standard

Deviation

0.0714 0.0912 0.0233 0.027577 0.011314 0.015556 0.000707

Relative

Absorbance0.467 0.384 0.257 0.157 0.0805 0.0325 0

y = 0.4276x + 0.2545

R = 0.6267

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0 0.2 0.4 0.6 0.8 1 1.2

Absorbance




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curve (Figure 3) was used in order to postulate the concentrations of proteins for elutions 1-3 (diluted 10 times) of

batch #3.

Elution 1-

1/10 Dilution

Elution 2-

1/10 Dilution

Elution 3-

1/10 Dilution

Average

Absorbance

0.26767 0.26433 0.27

Concentration

(mg/mL)

0.1858 0.1182 0.2331


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

between BSA absorbance and BSA concentrations. The R2value shows a very strong correlation between the linearequation and data points. Data points represent the average value of the sample sets. The error bars on the data plot




in this case was PBS.

y = 0.4934x + 0.2585

R = 0.9871

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

0 0.2 0.4 0.6 0.8 1 1.2

Absorbance




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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 BSA2 BSA3 BSA4 BSA5 BSA6 Blank

(PBS)

Average

Absorbance

0.531 0.462 0.437 0.3495 0.3115 0.2735 0.252

Concentration

(mg/mL)

1 0.75 0.5 0.25 0.125 0.0625 0

Standard

Deviation

0.1216 0.0764 0.0764 0.0389 0.0304 0.0092 0

Relative

Absorbance

0.279 0.21 0.185 0.0975 0.0595 0.0215 0



curve (Figure 3) was used in order to postulate the concentrations of proteins for elutions 1-3 (diluted 10 times) of

batch #4.

Elution 1-

1/10 Dilution

Elution 2-

1/10 Dilution

Elution 3- 1/10

Dilution

Average

Absorbance0.268 0.262 0.248

Concentration

(mg/mL)0.5427 0.3405 -0.1314


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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


value shows a very strong correlation between the linear





in this case was PBS.

y = 0.2967x + 0.252

R = 0.9564

0.2

0.25

0.3

0.35

0.4

0.45

0.5

0.55

0.6

0.65

0.7

0 0.2 0.4 0.6 0.8 1 1.2

Absorbance


Batch #4Absorbance vs. Concentration Standard Curve

Figure 5.SDS-PAGE of Nickel

Column Elution Fractions for

Batch #1. Sodium dodecyl sulfatepolyacrylamide gel

electrophoresis was run for ~1

hourat 115V 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.


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Column Elution Fractions for Batch

#2. Sodium dodecyl sulfate

polyacrylamide gel electrophoresis

was run for ~1 hourat 115V after

the fractions were obtained from

nickel affinity columnchromatography. 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.


Column Elution Fractions for Batch

#3. Sodium dodecyl sulfate

polyacrylamide gel electrophoresis

was run for ~1 hourat 115V 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 indifferent lanes. L, refers to Ladder, E

to elution, W to wash and FT to

flow-through fractions.


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Figure 11.Western characterization

of batch #2. 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 iBlotDry 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.

Figure 9.SDS-PAGE of Nickel Column

Elution Fractions for Batch #4. Sodium

dodecyl sulfate polyacrylamide gel

electrophoresis was run for ~1 hourat

115V after the fractions were obtained

from nickel affinity column

chromatography. A BLUeye prestainedprotein 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. 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

Figure 10.Western characterization

of batch #1. 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





(Genedirex) was used in Lane 3.Lanes 4-6 are elution fractions and



10.


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Figure 12.Western

characterization of batch #3.

Western blot was loaded with

samples of flow-through, wash andelution fractions. It was run at 200V

for 45min. proteins were transferred

to nitrocellulose membrane via iBlot

Dry Blotting System (Life





(Genedirex) was used in Lane 1.

Lanes 3-5 are elution fractions and



10. Two lanes between marker andeltuions and elutiosn and washes

were not used.

Figure 13Western

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 withprimary/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.


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Figure 14.Funtional assay

using PCR amplification on Pfu

DNA polymerase from batch #1

and #2. PCR products were run

on agarose gel electrophoresis at

100V for ~45 minutes. The geneamplified was pET28b-folA

which is around 500 bp. The

folAproduced is showed with

the name of DNAP on the side

of figure. The GeneRuler 1kb

ladder was loaded on lane 13 as

a reference. Lanes 2-5 are

positive controls loaded with

commercial DNA polymerases

Taq andPfu. CommercialPfu

DNA polymerase was assayed

at various concentrations. Lane

6 is negative control that lacks a

DNA polymerase. Lanes 7-9 are

products produced by proteins

in batch#1 fractions. Lanes 10-

12 are products from batch #2

samples.

Figure 15.Funtional assay using

PCR amplification on Pfu DNA

polymerase from batch #3 and #4.

PCR products were run on agarose

gel electrophoresis at 100V for~45 minutes. The gene amplified

was pET28b-folAwhich is around

500 bp. ThefolAproduced is

showed with the name of DNAP

on the side of figure. The

GeneRuler 1kb ladder was loaded

on lane 2 as a reference. Lanes 1

and 3-5 are positive controls

loaded with commercial DNA

polymerases Taq andPfu.

CommercialPfu DNA polymerase

was assayed at various

concentrations. Lane 6 is negativecontrol that lacks a DNA

polymerase. Lanes 7-9 are

products produced by proteins in

batch#3 fractions. Lanes 10-12 are

products from batch #4 samples.


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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.

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