CELLULAR(UPTAKE(OFPOLYPHENOLSIN(A(BACTERIAL( … ·...

COLUMBUS STATE UNIVERSITY

CELLULAR UPTAKE OF POLYPHENOLS IN A BACTERIAL PROTEIN EXPRESSION SYSTEM

A THESIS SUBMITTED TO

HONORS COLLEGE

IN PARTIAL FULFILLMENT OF THE

REQUIREMENTS FOR THE HONORS IN THE DEGREE OF

BACHELOR OF SCIENCE

DEPARTMENT OF CHEMISTRY

COLLEGE OF LETTERS AND SCIENCES

BY

B. KAMERON GRIFFIN

ABSTRACT

The biosynthesis of single chain insulin analogs is prohibitively inefficient due to

their propensity to form non-‐specific aggregates and ordered fibrils. It is well recognized

that certain polyphenol compounds are inhibitors of fibril formation in vitro and in

eukaryotic cells. However, there has been no systematic exploration of their effect in

bacterial expression systems. It was determined that Escherichia coli (BL21) cells do not

absorb phenol red (PR) under normal culture conditions. Additionally, it was observed that

both heat shocking competent cells in the presence of PR, and treatment of bacterial cell

pellets with a dimethyl sulfoxide (DMSO)/PR solution produced cell lysates that strongly

absorbed light at 555 nm. This is indicative of PR uptake.

DEDICATION

To Miss P. Griffin-‐ for your love and support.

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ACKNOWLEDMENTS

I would like to thank Dr. Jonathan Meyers for your mentorship, the development of

the project, and commitment to the research. Additionally, thank you for your motivation

and willingness to answer my many questions. Thank you to my other committee

members, Dr. Monica Frazier and Dr. Daniel Holley, for your time, knowledge and input.

I would also like to thank the other student members of the Meyers lab-‐both

undergraduate and graduate, with special thanks to Emily Fairchild, for help with data

collection. Additionally I would like to thank the Department of Chemistry for funding and

laboratory space, and the Columbus State University SRACE grant for additional funding.

Finally, I would like to thank my friends and family for your love and support.

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TABLE OF CONTENTS

Abstract ....................................................................................................................................................... iii

Dedication ................................................................................................................................................... iv

Acknowledments ......................................................................................................................................... v

Tables and Figures ..................................................................................................................................... viii

Introduction and background ...................................................................................................................... 1

Diabetes .................................................................................................................................................. 2

Insulin and fibril formation ................................................................................................................... 3

Eukaryotic and prokaryotic cells .............................................................................................................. 6

Polyphenols ............................................................................................................................................ 6

Proposal .................................................................................................................................................. 8

Uptake of phenol red .................................................................................................................................. 9

Introduction .......................................................................................................................................... 10

Methods and Materials .......................................................................................................................... 10

Transformed E. coli .......................................................................................................................... 10

Isosbestic point of phenol red ......................................................................................................... 11

Stability of phenol red in Luria broth ............................................................................................. 11

Baseline optical density measurements of e. coli BL21 ................................................................. 11

Antibiotic effect of phenol red ......................................................................................................... 11

Cellular uptake of phenol red .......................................................................................................... 12

Preparation of lysis buffers ............................................................................................................. 12

EDTA assay ....................................................................................................................................... 12

DMSO assay ....................................................................................................................................... 14

Results and discussion ......................................................................................................................... 15

Predicted changes in the absorbance of phenol red in E. coli culture .......................................... 15

Concentration of phenol red ............................................................................................................ 16

Isosbestic point of phenol red ......................................................................................................... 16

Stability of phenol red in Luria broth ............................................................................................. 18

Baseline optical density measurements of phenol red .................................................................. 18

Antibiotic effect of phenol red ......................................................................................................... 21

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Cellular uptake of phenol red ............................................................................................................ 21

EDTA assay ......................................................................................................................................... 22

DMSO assay ....................................................................................................................................... 24

Conclusion and future work ................................................................................................................... 25

Bibliography ........................................................................................................................................... 26

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TABLES AND FIGURES

Figure 1: The primary structure of proinsulin..............................................................................................................3

Figure 2: Insulin family hormones and receptors........................................................................................................4

Figure 3: Examples of commercially available polyphenol compounds that have been shown to inhibit fibril formation in vitro.............................................................................................................................................6

Figure 4: Thin section of the cell envelope of E. Coli K-‐12 after conventional embedding........................7

Figure 5: Structure of phenol red……………………………………………………………………………………………….10

Figure 6: Concentrations of phenol red and EDTA used in EDTA assay..........................................................13

Table 1: PR and EDTA solutions .......................................................................................................................................14

Table 2: Concentration of Phenol red data ..................................................................................................................16

Figure 7: Isosbestic point of Phenol red .......................................................................................................................18

Table 3: Absorbance data for isosbestic point ...........................................................................................................19

Figure 8: Profile of phenol red in Luria broth.............................................................................................................20

Figure 9: Optical Density of early stage E. Coli(BL21).............................................................................................20

Figure 10: Optical density of E. Coli(BL21) Raw Data................ ............................................................................21

Figure 11: Optical density of early stage E. Coli(BL21) ..........................................................................................21

Figure 12: E. Coli(BL21) uptake of Phenol red in LB media..................................................................................23

Table 4: EDTA absorbance results ..................................................................................................................................24

Figure 13: Change in absorbance of E. Coli with PR and DMSO vs Time.........................................................25

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INTRODUCTION AND BACKGROUND

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DIABETES

Diabetes Mellitus is currently the seventh leading cause of death in the United

States, and comes with a shocking financial burden. In 2010, over 230,000 death

certificates listed diabetes as a contributor to mortality. This number is assumed to be

severely under reported however, as only about 35% of people with diabetes who died had

diabetes listed as a cause of death. Additionally, the financial burden of diabetes is

astounding, with $176 billion in direct medical costs in 2012, and $69 billion in indirect

costs (1,2).

The two most common forms of Diabetes Mellitus are type I and type II. Type I

diabetes is typically diagnosed in children and occurs when the pancreas does not

synthesize enough insulin to properly regulate blood glucose levels. This form of diabetes

requires treatment with exogenous insulin, and is sometimes called insulin-‐dependent

diabetes.

Type II diabetes accounts for over 90% of all diagnosed cases. There are two

primary causes of Type II diabetes. The first occurs when the body does not use insulin

properly, which is known as insulin resistance. At the onset of type II diabetes, the

pancreas synthesizes and secretes enough insulin to overcome the body’s inability to use it

properly. However, as the disease progresses and the need for insulin rises, beta cells lose

the ability to produce sufficient amounts of insulin to compensate. This causes glucose

levels to rise and leads to systemic complications and other health problems (3).

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INSULIN AND FIBRIL FORMATION

Insulin was discovered in 1921 and was the first peptide-‐based therapeutic

approved by the FDA. Insulin is a globular protein that is rich in secondary and tertiary

structure and is derived from a single chain proinsulin precursor. Active insulin is created

by excision of the c-‐peptide from proinsulin, and is composed of two chains: a 21 amino

acid A chain attached by two disulfide bonds to a 30 amino acid B chain (Figure 1).

FIGURE 1: THE PRIMARY STRUCTURE OF PROINSULIN. The residues of the active two-‐chain hormone are in black. The dibasic cleavage recognition sites are in yellow. The excised c-‐peptide is in white, and the red arrows show the cleavage location (4).

There are many therapeutics used to treat type II diabetes; however, most patients

eventually become insulin dependent (3). The progressive nature of the disease and its

increasing prevalence generated global insulin sales approaching $17 billion in 2011 (5).

Although insulin is a lifesaving drug of incalculable value, it does have one major drawback

as a therapeutic. Studies show that insulin therapy can cause an increased risk of a variety

of cancers, including pancreatic, kidney and stomach (6). While the underlying factors are

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still unclear, there is strong support that the off target activation of growth factor receptors

by exogenous insulin may be a contributing factor (7).

The members of the insulin superfamily of peptide hormones, including insulin,

insulin-‐like growth factor-‐1 (IGF-‐1) and insulin-‐like growth factor-‐2 (IGF-‐2), have different

roles in the body; however, they are able to activate each other’s receptors. The presence of

insulin can activate the insulin receptor, but it can also activate the growth factor receptor.

If the growth factor receptor becomes too active, it can stimulate cell growth, which in turn

may cause cancer (Figure 2). Some insulin analogs have a greater affinity for the IGF-‐1

receptor than for the insulin receptor, and have been shown to cause cancer in rats.

FIGURE 2: INSULIN FAMILY HORMONES AND RECEPTORS. Native insulin mediates mitogenic effects by weakly activating several growth factor receptors. Some insulin analogs have been shown to be carcinogenic due to increased activation of the IGF-‐1R (Red arrow).

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It has been proposed that insulin analogs with novel c-‐peptide connecters (single

chain insulin, SCI) could reduce or eliminate this risk by increasing the selectivity for the

insulin receptor over the associated growth factor receptors. Unfortunately, SCI with

connecting peptides longer than eight residues are very difficult to synthesize by chemical

methods due to their unfavorable physical properties and increased length. As a result, SCI

have been traditionally been biosynthesized in bacterial expression systems. However, this

has also proven to be very inefficient due to irreversible non-‐specific aggregation and fibril

formation in the cellular environment (8). Native insulin is prone to fibril formation, and

this propensity is most likely exacerbated by unfavorable changes in the secondary

structure of the insulin backbone caused by the introduction of a nonnative c-‐peptide and

the high intracellular concentrations associated with biosynthesis. These factors greatly

reduce the ability to isolate the active monomeric forms of SCI, and have been identified as

a major hurdle in the exploration of SCI as superior therapeutics.

In addition to being problematic for biosynthesis, fibril formation plays an active

role in diseases, such as Type II Diabetes. The formation of fibrils in diabetes produces an

islet of amyloid, a name given due to the formation’s appearance. The amyloid is a solid,

insoluble complex that consists of proteins in β-‐pleated sheets. In Type II Diabetes, the

existence of these amyloid aggregations is associated with cell death (9). Additionally, Islet

amyloid polypeptide (IAPP) is a 37-‐residue hormone peptide that is secreted with insulin,

and has recently been shown to have a connection with inhibition of insulin action and the

loss of β-‐cells in Type II Diabetes (10).

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POLYPHENOLS

There is previous research that shows the naturally occurring class of compounds,

known as polyphenols (Figure 3), is effective at inhibiting the formation of higher order

aggregates. Research shows that many polyphenols inhibit fibril formation of various

proteins. However, the polyphenol of specific interest here is phenol red, as the dye was

shown to inhibit aggregation of insulin. Phenol red was also used in vitro in eukaryotic cells

and decreased the formation of aggregates and later fibrils (11).

FIGURE 3: EXAMPLES OF COMMERCIALLY AVAILABLE POLYPHENOL COMPOUNDS THAT HAVE BEEN SHOWN TO INHIBIT FIBRIL FORMATION IN VITRO. A: The synthetic dye Congo red. B: The naturally derived compound curcumin.

Another polyphenol, Congo red, has been shown to absorb in gram negative

bacteria. Upon overnight incubation, the cytosol of the bacteria appeared to be pale pink

instead of deep red upon visual examination, suggesting the cellular uptake of the dye (12).

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EUKARYOTIC AND PROKARYOTIC CELLS

Eukaryotic and prokaryotic cells each contain a plasma membrane that regulates

the passage of materials into and out of the cell. Each type of membrane is composed of a

phospholipid bilayer and proteins. The primary membrane function is the same in both

prokaryotic and eukaryotic cells. Small, nonpolar molecules can diffuse freely through

plasma membranes to an area of lower concentration through simple diffusion. Conversely,

large or polar molecules cannot typically participate in simple diffusion across the plasma

membrane, and therefore, must undergo facilitated diffusion with the assistance of

transport proteins (13).

FIGURE 4: THIN SECTION OF THE CELL ENVELOPE OF E. COLI K-‐12 AFTER CONVENTIONAL EMBEDDING. The periplasmic space is empty of substance, and the peptidoglycan layer (PG), outer membrane (OM), and plasma membrane (PM) can be seen. Bar 5 100 nm. (14)

In addition to a plasma membrane, prokaryotic cells also contain a cell wall-‐ a rigid

structural layer. In bacterial cells, the cell wall is composed of cross-‐linked peptidoglycan,

which can increase the vulnerability of the cell to certain antibiotics. There are two broad

classes of bacterial cells, as determined by the structure of their cell walls. Gram-‐positive

bacteria, such as Staphylococcus aureus, consist of one plasma membrane that is

surrounded by a thick cell wall. Whereas, gram-‐negative bacteria, such as E. coli, have a

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dual membrane system, in which there are two plasma membranes with a much thinner

cell wall in between (Figure 4). This extra cell wall acts as an addition layer through which

large molecules must undergo facilitated diffusion.

PROPOSAL

While polyphenols have been shown to inhibit development of fibrils in vitro and in

eukaryotic cells, the use of polyphenols as an aggregation inhibitor in bacteria remains

unexplored. However, a paper published in 1989 shows minor proof that gram-‐negative

bacteria will absorb Congo red (15). In addition to incubation, heat shock and membrane-‐

modifying agents will be explored as a method of facilitating diffusion. If bacteria can

absorb the polyphenols, then it seems reasonable that the polyphenols could effectively

inhibit the formation of insulin aggregates in the bacterium. However, the amount of

polyphenol that can successfully be absorbed has not previously been reported. Therefore

the main question to be answered is, “Is the intercellular concentration of polyphenol high

enough to inhibit the formation of aggregates?”

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UPTAKE OF PHENOL RED

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INTRODUCTION

Phenolsulfonphthalein, also known as Phenol red, is a synthetic polyphenol

consisting of two phenol groups (Figure 5). This dye is typically used in laboratories as a

pH indicator, due to its gradual transition from yellow to red in the pH range of 6.8 to 8.2.

Phenol red has been shown to inhibit the formation of fibrils by insulin and islet amyloid

polypeptide (IAPP) in vitro. Additionally, the dye has been shown to decrease IAPP

cytotoxicity toward mammalian β-‐cells (10).

METHODS AND MATERIALS

Transformed E. coli

In order to replicate conditions that exist during recombinant protein biosynthesis,

all experiments were conducted using E. coli BL21 (donated by Dr. Rachel Whitaker,

Coastal Carolina University) transformed with a custom expression vector encoding for the

rat hormone preptin. The pD444-‐NHT plasmid (DNA 2.0) imparts resistance to the

antibiotic kanamycin and allowed cells to be grown under selection.

FIGURE 5: STRUCTURE OF PHENOL RED.

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Isosbestic point of phenol red

Three solutions of phenol red were made by diluting a stock solution of phenol red

in PBS (0.02 mM, pH 7.66) with equal volumes of strong acid (HCl), strong base (NaOH), or

buffer (PBS) to produce a set of samples with a range of pH values and colors. The

absorbance spectrum from 400 nm to 600 nm, in 1 nm increments, was taken for each

solution on a Thermo Fisher Genesys 10s UV-‐Vis spectrophotometer using a 700-‐μL quartz

cuvette (Thor Labs). The spectra were compared and the actual isosbestic point was

determined by repeated measurements at 478 and 479 nm.

Stability of phenol red in Luria broth

Varying concentrations of phenol red were incubated in LB with shaking at 37℃.

The absorbance of the LB/Phenol red solution was monitored at 479 nm for up to 24 hours.

Baseline optical density measurements of e. coli BL21

Starter cultures of transformed E. coli(BL21) were grown overnight in LB with

shaking at 37℃. Overnight starter cultures were used to inoculate larger cultures. Cell

growth was monitored at 750 nm and 478 nm. LB was used for all dilutions and to blank

the spectrophotometer.

Antibiotic effect of phenol red

In order to determine the suitability of phenol red for use with E. coli, transformed

cells were cultured in LB spiked with various concentrations of polyphenol. Bacterial

growth was monitored at 750 nm instead of the traditional 600 nm to avoid complications

caused by the strong absorbance of phenol red at wavelengths below 650 nm.

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Cellular uptake of phenol red

Starter cultures (4mL) of E. coli(BL21) were incubated overnight in the presence of

kanamycin. Aliquots of the overnight culture were used to inoculate multiple fresh 4 mL

cultures spiked with various concentrations of phenol red. Cultures were incubated at 37℃

with shaking. Cell growth was monitored at 478 nm and 750 nm using fresh LB media as a

blank.

Preparation of lysis buffers

Two lysis buffers were prepared for use in EDTA and DMSO assays. The first lysis

buffer was prepared with 50mM glucose, 25mM Tris-‐HCl (pH 8.0, Sigma) and 10mM EDTA

(pH 8.0, Sigma). The second lysis buffer was prepared 0.2 N NaOH (freshly diluted from a

10 N stock) and 1% (w/v) SDS (Sigma). The buffers were then autoclaved and stored at

room temperature.

EDTA assay

A 50 mL culture was inoculated with E.Coli(BL21) cells and incubated until it

reached early exponential stage (OD600= 0.355). The cells were aliquoted (1 mL) into

microcentrifuge tubes and centrifuged (17,000xg) for 10 minutes at 4℃ to collect the cells.

The cell pellets were resuspended in calcium chloride (100mM, Sigma Aldrich), and

incubated on ice for 10 minutes. The cells were pelleted, resuspended in calcium chloride

(100mM), and incubated on ice for one hour. After incubation the cells were collected by

centrifugation (17,000xg) at 4℃ for 10 minutes. The cells were then resuspended in a

combination of ice-‐cold glycerol (10mM, Sigma) and Tris-‐HCl (pH 8, Sigma). The competent

cells were collected by centrifugation (5,000xg) for ten minutes at 4℃.

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Luria broth was added to each microcentrifuge tube. At a steady pace, PR (5.3 mM in

PBS) was added to each tube prior to the addition of varying concentrations of EDTA

(Figure 6 & Table 1). The tubes were then heat shocked at 45℃ for approximately one

minute and incubated on ice for five minutes. The samples were then centrifuged (9,000xg)

at 4℃ for 5 minutes. The absorbance of each sample’s supernatant was taken at 478 and

750nm.

The cell pellets were then washed with PBS and treated with equal amounts of the

two lysis buffers (200μL each), incubated at room temperature for 15 minutes and frozen.

After one week at -‐20℃, the samples were thawed and centrifuged (13,000xg) at 4℃ for 10

minutes to remove the cellular debris. Absorbance of the supernatant was taken at 555nm

with water used as a blank.

An aliquot (100 µL) of each culture was removed prior to cell lysis and spread on

kanamycin plates to assure that EDTA treatment did not kill the cells.

FIGURE 6: CONCENTRATION OF PR AND EDTA USED IN EDTA EXPERIMENT. The concentration of EDTA (blue) was held constant while concentration of PR (red) was increased in samples B,C,&D. In samples E,F, G,& H the concentration of PR was held constant while the concentration of EDTA was decreased.

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TABLE 1: PR AND EDTA SOLUTIONS. Eight trials were run with varying concentrations of PR and EDTA as shown. Trial A acted as a control-‐ with only PBS and LB.

Trial PR (mM) EDTA (mM)

A 0 0

B 0.03 2

C 0.3 2

D 3 2

E 3 1

F 3 2

G 3 3

H 3 4

DMSO assay

A 50 mL culture was inoculated with E.Coli (BL21) cells and incubated until the cells

were aliquoted (1 mL) into microcentrifuge tubes. Cells were collected by centrifugation at

8,000xg for 10 minutes at 4℃. The cell pellets were resuspended in PR/DMSO solution (0.5

mL, 97μM). One sample of cells was resuspended in PBS to serve as a control. The tubes

were then incubated for various times (15, 30, 45, 60mins). After incubation the cells were

spun down at 8,000xg for 10mins at 4℃. The pellets were then washed with ice cold PBS

three times. The pellets were then treated with equal amounts of the two lysis buffers

(200μL), incubated at room temperature for 15 minutes and then frozen at -‐20℃ for two

days. The samples thawed and centrifuged (8,000xg) at 4℃ for 10 minutes. Absorbance of

the supernatant was taken at both 555 and 750nm, with PBS used as a blank.

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RESULTS AND DISCUSSION

Predicted changes in the absorbance of phenol red in E. coli culture

Precise quantification of chromophore concentration in a growing cell culture is

extremely difficult due to a number of complicating factors including variable total volume,

the rapid increase and variable nature of cellular volumes, attenuation of chromophore

absorbance by interaction with cellular components, and nonspecific light scattering by

cellular membranes.

Previous publications have attempted to quantify intracellular concentrations of

chromophores by taking into account nonspecific light scattering by cellular membranes.

However, these publications did not address the changes that result from increasing cell

density in an active culture.

Using approximate values from BioNumbers, a database of useful biological

numbers hosted by the Systems Biology department in Harvard, the approximate changes

in absorbance were calculated (16). It is universally accepted that an 𝑂𝐷!"" !" of 1

represents a cellular concentration of 8𝑥10! -‐ 1𝑥10! cells/mL of culture. Additionally, the

predicated average volume of an E. coli cell is 1𝑥10!!" mL. Using these values, a culture

with an 𝑂𝐷!"" !" = 1 would have approximately 0.001 mL of cell volume per 1 mL of

culture volume. Using an initial concentration of 500 mM in the culture media, a target

intracellular concentration of 100 𝜇𝑀, and accounting for changes to the culture media

volume caused by increasing cellular concentrations, the calculated change in

chromophore concentration would be undetectable using a UV-‐Vis spectrophotometer.

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However, the target chromophore concentration in the cell lysate would be in the

detectable range after processing.

Concentration of phenol red

The phenol red stock solution concentration was determined by taking the

absorbance at 555nm (Table 2) using the published molar absorption coefficient of 31.62

mM-‐1cm-‐1. This resulted in an average phenol red concentration of 0.022 ± 0.003 mM.

TABLE 2:CONCENTRATION OF PHENOL RED DATA: Recorded at 555nm. All absorbances were corrected to include the dilution factor shown.

Stock Solution (mL)

PBS (mL) Abs555 nm

Measured Conc. (mM)

Dilution Factor

Corrected Conc. (mM)

1.000 0.000 0.700 0.022 1.000 0.022 0.500 0.500 0.356 0.011 2.000 0.023 0.400 0.600 0.273 0.009 2.500 0.022 0.300 0.700 0.207 0.007 3.333 0.022

Isosbestic point of phenol red

Phenol red is used as an indicator due to its ability to change color as a result of

varying pH. While useful for visually assessing the pH of cell culture media, this

characteristic of phenol red complicates absorbance based concentration measurements. In

spectroscopy, an isosbestic point refers to the wavelength at which the absorbance by a

mixed solution remains unchanged as the equilibrium of the two components changes. The

isosbestic point of a substance does not depend on the physical characteristics, such as

color. Therefore, at the isosbestic point the absorbance of the dye does not change simply

as an effect of the color change due to pH differences (Figure 7).

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Brown and Campbell (17) found the isosbestic point of phenol red to be 478 nm.

After repeated measurements within a narrow range of 470, 478 and 479 nm, it was

determined that there was also less variance on our instrument at a wavelength of 478 nm

(Table 3).

FIGURE 7: ISOSBESTIC POINT OF PHENOL RED. The isosbestic point occurs around 478nm, the point where all the data lines intersect.

TABLE 3: ABSORBANCE DATA FOR ISOSBESTIC POINT: Absorbance recorded at varying wavelengths and pH values to determine isosbestic point of 478nm.

Abs at 470nm Abs at 478nm Abs at 479nm

Low pH 0.355 0.329 0.305 pH= 7.66 0.325 0.296 0.291 High pH 0.22 0.288 0.297

Average STD 0.31±0.06 0.30±0.02 0.30±0.003

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Stability of phenol red in Luria broth

Phenol red has long been used as a pH indicator in cell culture media. However, its

stability in LB media used for bacterial culture was unknown. No significant change in the

absorbance spectrum was noted (Figure 8) confirming phenol red’s stability in LB.

FIGURE 8: PROFILE OF PHENOL RED INCUBATED IN LURIA BROTH AT 37℃. The small deviations are consistent with error introduced during dilution of the samples for absorbance measurements. The absorbance profile was stable beyond 24 hrs. (data not shown).

Baseline optical density measurements of phenol red

Phenol red has a strong absorbance spectrum centered at 555 nm. This spectrum

overlaps with the traditional wavelength for monitoring bacterial cell growth (600 nm).

Previous work has shown that there is a linear relationship between light scattering and E.

coli cell density at low concentrations across a wide range of wavelengths (Figure 9). This

allows for correlation of optical density values measured at various wavelengths. However,

concentrations resulting in an absorbance above 0.4 results in nonlinear graphs, due to

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deviations from ideal at both wavelengths (Figure 10). Therefore, it was important to

maintain an absorbance below 0.4, with dilutions if necessary.

A correlation factor (OD 478 nm = 1.8085 * OD 750 nm + 0.0313) was determined

(Figure 11).

FIGURE 9: OPTICAL DENSITY OF EARLY STAGE E. COLI (BL21). The light scattering by E. coli is linear at low concentrations across the spectrum. A culture of transformed E. Coli (BL21) was monitored at 478nm (yellow), 600nm (blue), and 750nm (red).

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y = 1.6056x + 0.0643 R² = 0.991

0

0.2

0.4

0.6

0.8

1

1.2

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7

Absorbance (478 nm)

Absorbance (750 nm)

Optical Density of E.coli (BL21) at 478 nm vs 750 nm (Raw Data)

FIGURE 10: OPTICAL DENSITY OF E.COLI (BL21) RAW DATA. Absorbance above 0.4 results in nonlinear graphs, so dilutions were used in subsequent experiments.

FIGURE 11: OPTICAL DENSITY OF E. COLI (BL21). A strong linear relationship was determined for OD values less than 0.4 for 750nm and 0.8 at 478 nm. This correlation factor was used in subsequent experiments.

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Antibiotic effect of phenol red

Although polyphenol compounds have been found to prevent aggregation in

eukaryotic cells, many of these have documented antibiotic effects. This limits their utility

in bacterial cells used for protein expression. Therefore, phenol red was tested for

antibiotic effects by growing E. coli (BL21) cells in LB media spiked with varying

concentrations of phenol red. Growth curves were similar for cultures with and without

phenol red. Phenol red failed to exhibit antibiotic effects for up to 24 hours and

concentration up to 200 μM (data not shown).

Cellular uptake of phenol red

Eqn 1: 𝑶𝑫𝟒𝟕𝟖 𝒏𝒎,𝑬.𝒄𝒐𝒍𝒊 = 𝟏.𝟖𝟎𝟖𝟓 𝑶𝑫𝟕𝟓𝟎 𝒏𝒎,𝑬.𝒄𝒐𝒍𝒊 + 𝟎.𝟎𝟑𝟏𝟑

Eqn 2: 𝑨𝒃𝒔𝟒𝟕𝟖 𝒏𝒎,𝑷𝒉𝒆𝒏𝒐𝒍 𝑹𝒆𝒅 = 𝑨𝒃𝒔𝒐𝒃𝒔𝒆𝒓𝒗𝒆𝒅 @ 𝟒𝟕𝟖 𝒏𝒎 − 𝑶𝑫𝟒𝟕𝟖 𝒏𝒎,𝑬.𝒄𝒐𝒍𝒊

After it was determined that phenol red exhibited no apparent antibiotic effects, E coli’s

ability to absorb phenol red was explored. Cells were grown in LB spiked with phenol red and

the absorbance at 750 and 478nm were monitored to account for the increase in light

scattering as a result of the increasing cell concentration. The absorbance of phenol red in the

growth media was determined by using Equations 1 and 2. No uptake of phenol red was

detected (Figure 12). Furthermore, the absorbance at 478 nm of the phenol red spiked LB

media after centrifugation was unchanged after incubation, which indicates that the phenol red

concentration also remained unchanged. Additionally, cells grown in the presence of phenol

red gave a cell pellet identical in color to cells grown in LB only. This is a strong indicator that

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the cells did not absorb an appreciable amount of the dye because concentrations as low as 25

µM have a red color detectable by the naked eye. After multiple trials, it was determined that a

cell membrane modifier or competent cells should be explored.

EDTA assay

A common method for introducing foreign materials into bacterial cells is to make

the outer membrane more permeable by treating them with high salt concentrations and

cells treated in this manner are said to be competent. Additionally, EDTA has been shown

to further increase bacterial cell permeability by a poorly understood mechanism. EDTA is

also known to initiate the release of some membrane lipids in a dose dependent manner.

y = 0.0029x + 0.3196 R² = 0.9868

y = 0.0029x + 0.223 R² = 0.971

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9

0 20 40 60 80 100 120 140 160 180

Absorbance at 478nm

Time (mins)

E. Coli(BL21) Uptake of Phenol Red in LB Media

E. coli, Phenol Red and LB E. coli and LB Corrected Phenol Red Absorbance

FIGURE 12: E. COLI (BL21) UPTAKE OF PHENOL RED IN LB MEDIA. A representative growth curve for transformed E. coli(BL21) cells grown under selection in the presence of phenol red. The total absorbance of LB, Phenol red, and E. coli (blue) shows an identical slope as the absorbance of LB and E. Coli (purple). The difference between the two lines represents the solution concentration of phenol red, which remains unchanged (green).

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Consequently, prolonged exposure to high concentrations of the chelating agent is lethal.

Previous studies have shown that treatment of competent cells with EDTA can increase the

cellular permeability of compounds that would not otherwise cross the membrane (18,19).

Transformed cells were treated following the protocol in the methods and materials

section. These competent cells were then treated with varying concentrations of PR and

EDTA. An aliquot of each sample was plated to determine if they were still viable after

treatment. Each sample produced an overgrown plate after overnight incubation at 37℃ on

LB/kanamyacin plates confirming that the EDTA treatment does not result in cell death.

Also, upon addition of the two lysis buffers to the washed cell pellets, some samples

showed a distinct pink color. Unfortunately, it is unknown if this color change is a result of

remaining supernatant or from phenol red in the lysed cells, because the samples were

only washed once with PBS.

TABLE 4: EDTA ABSORBANCE RESULTS. There is no significant change in any of the tubes, suggesting no absorption of PR.

Tube Absorbance (555 nm) Tube Absorbance (555 nm)

A 0.0073±0.002 E 0.0620±0.03

B 0.021±0.02 F 0.0397±0.02

C 0.0365±0.01 G 0.0527±0.04

D 0.0176±0.005 H 0.0350±0.03

P a g e | 24

DMSO assay

DMSO is also known to increase membrane permeability through another poorly

understood mechanism. DMSO is thought to participate in three mechanisms to help

facilitate diffusion: membrane loosening, the formation of pores, and bilayer collapse (20).

Additionally, there is evidence of DMSO creating transient water pores in the cell

membrane, which could in turn facilitate diffusion across the plasma membrane (21).

In a similar manner to the EDTA assay, the competent cells were treated with a

concentration of DMSO and Phenol red. However, the time of incubation was varied over a

time of one hour. After the three PBS washes, the cell pellets were all a whitish color.

However, upon addition of the second lysis buffer, the samples were a bright fuchsia in

color, suggesting cellular uptake of phenol red. In addition, the absorbance of the samples

that were incubated for 30 minutes or more show an increase in absorbance at 555nm,

further suggesting the presence of phenol red in these samples (Figure 13).

FIGURE 13: CHANGE IN ABSORBANCE OF E. COLI WITH PR AND DMSO VS TIME. Absorbance at 555nm (blue) increases at the 30minute mark and beyond. Absorbance at 750nm (purple) shows no significant increase over time.

P a g e | 25

CONCLUSION AND FUTURE WORK

In conclusion, we are fairly positive that phenol red cannot permeate the cell

membrane of E. coli (BL21) without some form of facilitator of active transport. However,

upon treatment of competent cells with DMSO, there shows an increase in absorbance,

which would suggest the cellular uptake of phenol red. Furthermore, the white cell pellets

turned a bright fuchsia color upon the addition of the second lysis buffer.

Further research is needed to ensure the competent cells are still viable after

treatment with DMSO. An aliquot of each sample should be removed and plated on an

LB/Kanamycin plate and cell growth must be monitored with incubation to ensure that the

DMSO treatment does not result in cell death.

Additionally, the absorbance of the cells treated with DMSO showed no significant

absorbance difference upon incubation past the 30-‐minute mark. More research is required

to determine whether the dye goes directly into the cells or if there is some sort of time

dependence.

Another possible area of future research includes the testing of other polyphenols to

determine if they show an increase in ability to transport into the cell. One polyphenol of

specific interest could be Congo red, as it has been shown to absorb in gram-‐negative

bacteria and to inhibit the formation of aggregates (15).

P a g e | 26

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