The diffusion of antibiotics through the biofilm pores produced by Pseudomonas
aeruginosa using a synthetic neutrophil extracellular trap
Team Nanobots
Mark Ly, Fahima Nakitende, and Shannon Wesley
Theme: Nanoscience
Subtheme: Nanoparticle systems for studying biomolecules
Keywords: Pseudomonas aeruginosa biofilm, copper-oxide nanowire bundles, neutrophil
extracellular trap, cystic fibrosis, ciprofloxacin/tobramycin
1
SUMMARY
We intend to study Pseudomonas aeruginosa bacterial infections using nanowires to
deliver antibiotics across P. aeruginosa’s biofilm for the treatment of chronic infections. Past
research studied 40 patients suffering from cystic fibrosis disease, and determined 77% were
infected by P. aeruginosa even though 71% of them were healthy (Neter 1974; Diaz et al. 1970;
Diaz and Neter 1970). Thus, the antibiotic treatment was ineffective against P. aeruginosa. So,
we intend to penetrate through the biofilm produced by P. aeruginosa using copper-oxide
nanowire bundles (CuO NWBs) to help deliver antibiotics and destroy the bacteria residing
inside the biofilm.
P. aeruginosa bacterial infections form very thick biofilms which guard them against any
immunity and prevent antibiotics from reaching the pathogen for destruction (Costerton et al.
1999; Nickel et al. 1985). The biofilm structure of P. aeruginosa is important because it consists
of pores and channels that will allow us to use diffusion as a way to penetrate through the biofilm
(Costerton et al. 1999; Stewart 1996).
Previous studies used ciprofloxacin and tobramycin antibiotics to treat P. aeruginosa
biofilm infections which were found ineffective as a result of a lack of oxygen and low metabolic
activity that was created inside the biofilm (Walter et al. 2002). To solve this problem, we
propose to organize copper-oxide nanowires into nanowire bundles to form a synthetic mesh-like
net. This will provide a large surface area for antibiotic attachment as well as prevent the
obstruction of the biofilm diffusion channels thanks to their nanometer size.
Neutrophil extracellular traps (NETs) were observed to be effective in binding and
destroying bacterial cells, but bacterial toxins can kill them (Brinkman 2004: Tirouvanziam
2
2007). Therefore, we will formulate a NET but one that is synthetic (synNET) to overcome this
disadvantage.
The aim of our study is to determine the effectiveness of ciprofloxacin and tobramycin
coupled CuO NWBs against Pseudomonas aeruginosa biofilm growth. We hypothesize that
CuO NWBs carrying antibiotics will mimic the NET and penetrate through P. aeruginosa
biofilm to kill the pathogen from the inside. If this happens successfully, we expect to see no
growth on the antibiotic/CuO NWBs agar plates.
We will follow Harrison’s et al. (2006) procedure to grow P. aeruginosa biofilms every
three days to avoid biofilm degradation, use ciprofloxacin and tobramycin antibiotics, follow the
methods outlined by Li et al. (2010) to synthesize CuO NWBs, and use electrostatic interactions
to couple these antibiotics to the CuO NWBs. We will prepare four treatments each in 24 out of
96 wells and expose them to our P. aeruginosa biofilms. Our treatments will consist of: our
control (no antibiotic/no CuO NWBs), antibiotics only, CuO NWBs only, and antibiotic/ CuO
NWBs.
From this experiment, we will determine the mean amount of P. aeruginosa biofilm
growth after being exposed to each treatment by using a one-way fixed effect ANOVA statistical
test to compare the mean colony forming units (CFUs) for our three different treatments. We
expect to see 100% growth of P. aeruginosa biofilms for the control and antibiotics treatment,
limited growth for CuO NWBs treatment, and 0% growth for the treatment of antibiotic/CuO
NWBs. If our experimental results correlate with the above expected results, then our synNETs
can be used as an alternative therapy for P. aeruginosa biofilm infections. Also for future
studies, determination of the optimal concentration of CuO NWBs without antibiotics needed to
effectively kill P. aeruginosa biofilm growth would be necessary.
3
INTRODUCTION AND BACKGROUND
We propose to study the control of Pseudomonas aeruginosa infections by using
nanowires to deliver antibiotics across the Pseudomonas aeruginosa’s biofilm to treat chronic
infections. According to previous research, among 40 patients suffering from cystic fibrosis,
77% were infected by Pseudomonas aeruginosa, even though 71% of these patients exhibited a
normal immune response (Neter 1974; Diaz et al. 1970; Diaz and Neter 1970). Consequently,
conventional antibiotic therapy was ineffective against the Pseudomonas aeruginosa infection.
Thus, we will use copper-oxide nanowire bundles (CuO NWB) to penetrate the biofilm produced
by Pseudomonas aeruginosa to deliver antibiotics and kill the bacteria residing inside the
biofilm.
Cystic fibrosis is an inherited disease that causes a thick, sticky mucus build up in the
lungs, which is an ideal environment for Pseudomonas aeruginosa biofilms to fluorish.
Pseudomonas aeruginosa infections form biofilms to defend themselves against human
immunity and conventional antibiotic therapy (Costerton et al. 1999; Nickel et al. 1985). This is
important because a Pseudomonas aeruginosa’s biofilm forms a 200 µm barrier of sugars and
carbohydrates to prevent antibiotics from reaching the pathogens (Hanlon et al. 2001; Anwar and
Costerton 1992; Costerton et al. 1987; Govan and Deretic 1996).
Earlier studies have used diffusion as a way to enter the pathogen’s biofilm layer to kill
it, which is the method we propose to use in this study. This is because Pseudomonas
aeruginosa’s biofilm consists of numerous pores and channels, which can be used for the
diffusion of nanowires (Costerton et al. 1999; Stewart 1996).
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Nanowires have been used in various biological applications due to their size and
selectivity (Li et al. 2010; Bao et al. 2008; Lu et al. 2007). These wires have a diameter of the
nanometer scale, which can have different shapes and properties depending on the material they
are made of. And, we can arrange these nanowires into nanowire bundles to form a synthetic
mesh-like net that will increase the available surface area for antibiotic attachment.
Metal ions have been used by Harrison et al. (2005) to kill the pathogen by diffusing
metal ions through the Pseudomonas aeruginos’s biofilm. In addition, the results showed that a
high concentration and long exposure time of various metal ions were able to completely
eliminate the biofilm. And, the lengthy exposure time was a result of cationic binding of the
metal ions to the biofilm that restricted the rate of penetration. Harrison et al. (2005) also
determined that 40 mM of lead, 120 mM of zinc, 140 mM of cobalt and nickel, and 300 mM of
aluminum metal ions were required to destroy a biofilm, whereas only 30 mM of copper was
required to have the same outcome. Thus, we decided to make our nanowires out of copper
because copper was effective at low concentrations and non toxic to biological systems.
Therefore, we will use electrostatics to compose CuO NWB antibiotic carriers. We predict the
antibiotics will occupy the positive charge on the CuO NWBs and will not adhere to the surface
of the biofilm during diffusion.
Previous research performed by Walters et al. (2002) demonstrated tobramycin and
ciprofloxacin eventually penetrated the biofilm, but failed to kill Pseudomonas aeruginosa once
inside the biofilm. As indicated by Figure 1, both ciprofloxacin and tobramycin did not increase
Pseudomonas aeruginosa’s mortality rate over a 100 hour exposure time.
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Figure 1. Killing of P. aeruginosa in biofilms in exposure to ciprofloxacin (A) and killing
of P. aeruginosa in biofilms in exposure to tobramycin (B). Filled squares were the treatment
and the unfilled were the controls (Walter et al. 2002)
Thus, the ineffectiveness of these antibiotics was not because of poor penetration, but
oxygen limitation and low metabolic activity inside the biofilm. A lack of oxygen within the
biofilm either prevented Pseudomonas aeruginosa growth or the action of the antibiotics;
whereas, free swimming bacterial cells were susceptible to tobramycin and ciprofloxacin. For
these reasons, we decided to use these two antibiotics in conjunction with our CuO NWBs to kill
the pathogen inside the biofilm. These bundles will not block the Pseudomonas aeruginosa
biofilm pores because of their nanometer size. Therefore, oxygen transport will be restored,
metabolic activity will continue as normal, and we will be able to spread the antibiotics over a
larger area. So, by combining these antibiotics to our CuO NWBs we are creating a synthetic
neutrophil extracellular trap (synNET).
6
According to Brinkmann (2004), white blood cells in a
normal immune response form a neutrophil extracellular trap (NET)
by secreting proteins and chromatin. This NET traps the pathogen
and prevents damage to the adjacent tissues at the site of infection.
NETs bind to bacterial cells, prevent the spread of infection, and kill
the pathogen using antimicrobial agents (toxins, chemicals, or
antibodies) (Weinrauch et al. 2002). As a result of these above
advantageous properties, we will use a bundle of nanowires to
imitate a NET and trap the pathogen.
Figure 2. Neutrophil extracellular
trap (NET).
According to Tirouvanziam (2007), NETs are rapidly killed by toxins released by the
bacterial cells making them ineffective. This is why patients with cystic fibrosis are
continuously suffering from recurring episodes of Pseudomonas aeruginosa infection. So, could
CuO NWBs carrying antibiotics diffuse through the porous structure of Pseudomonas
aeruginosa’s biofilm and act as a synNET?
We hypothesize that CuO NWBs will imitate a NET and carry antibiotics through the
biofilm of Pseudomonas aeruginosa to kill the pathogen from the inside. If this synNET carrying
antibiotics diffuses through the biofilm successfully and if the antibiotics detach from the
synNET inside the biofilm, we predict no growth on the antibiotic/CuO NWB agar plates.
7
PROPOSED RESEARCH
The aim of our study is to determine the effectiveness of ciprofloxacin and tobramycin
coupled CuO NWBs against Pseudomonas aeruginosa biofilm growth.
We will grow Pseudomonas aeruginosa biofilms every three days to avoid biofilm
degradation which usually occurs at about 48 hours and to ensure a constant 24 hour aged
biofilm for testing (D. Storey, pers. Commun.). Also, we will synthesize one set of CuO NWBs
for each antibiotic.
Pseudomonas aeruginosa Biofilm Synthesis
We will follow Harrison’s et al. (2006) procedure shown in Figure 1 to grow
Pseudomonas aeruginosa biofilms using the Calgary Biofilm Device (CBD) with the exception
of using the scanning electron microscopy method.
Figure 1. The experimental design to synthesize Pseudomonas aeruginosa biofilms, using LuriaBurtani Broth (LB) and the Calgary Biofilm Device (CBD) system; and examination of the
biofilm’s structure using confocal laser scanning microscopy (CLSM) equipment (Harrison et
al., 2006).
8
A confocal laser scanning microscope (CLSM) and its 3D imaging properties will be
used to examine the biofilm’s structure and confirm the presence of pores. Only biofilm
structures containing pores will be used in this research study to make sure diffusion transport is
accessible.
Ciprofloxacin and Tobramycin
We will purchase 100 mg of deprotonated (negatively charged) ciprofloxacin and
tobramycin from Sigma-Aldrich.
Walter’s et al. (2002) procedure will be used to test ciprofloxacin and tobramycin against
Pseudomonas aeruginosa biofilms as our control. This will ensure that these two antibiotics are
ineffective against Pseudomonas aeruginosa biofilms individually.
Synthesis of CuO NWBs
We will follow the methods outlined by Li et al. (2010) to synthesize the CuO NWBs.
We will create a one dimensional nanowire and combine it with an aqueous solution of CuCl2.
This composite solution will be vacuum filtered and dried in an oven to produce our nanowires
composition. The initial template will be removed with a solution of NaOH and the copper-oxide
nanowire bundles will dry a final time. Creating our CuO NWBs will take 24 hours to complete.
We will determine the structure and correct chemical composition of our CuO NWBs by using
transmission electron microscopy (TEM) and X-ray diffraction (XRD). This will guarantee our
CuO NWBs are self assembled correctly.
Synthesis of synNETs
To attach our antibiotics, we will use electrostatic interactions (CuO NWBs positive
charge with the antibiotics negative charge) to couple with our CuO NWBs. A solution of
9
ciprofloxacin/tobramycin will be cast onto the CuO NWBs separately and then slowly
evaporated to produce our synNETs. These synNETs will be stored in a refrigerator (4℃).
We will follow Li’s et al. (2010) characterization method to characterize our synNET.
Fourier transform-IR (FT-IR) spectra will be performed using a Nicolet Avatar mass
spectrometer from the University of Calgary’s Chemical Instrumentation Facility to compare
three different peaks: the CuO NWBs individually, the antibiotic individually, and the antibiotic
coupled with our CuO NWBs. If our antibiotics coupled with our CuO NWBs successfully, we
expect to see a spectra that combines both individual peaks: CuO NWBs and antibiotic.
One factor that could affect our results is the strength of the electrostatic interaction
between the antibiotics and the CuO NWBs. If the interaction is too weak then the CuO NWB
may attach to the biofilm layer instead of the antibiotic, and the CuO NWB will not deliver the
antibiotic across the biofilm layer.
Controls and Test Samples
Pseudomonas aeruginosa biofilms in 96 welled MBEC plates will be randomly exposed to each
parameter listed in Table 1 below:
Table 1. Experimental group parameters tested with Pseudomonas aeruginosa biofilms.
TREATMENT
A
Control
B
Antibiotic only
C
Nanowire only
D
Nanowire + Antibiotic
EXPERIMENTAL TREATMENT
PARAMETERS
No antibiotic
No copper-oxide nanowire
Antibiotic
No copper-oxide nanowire
No antibiotic
Copper-oxide nanowire
Antibiotic fused
Copper-oxide nanowire
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EXPECTED
RESULT
Growth
Growth
Limited growth
(copper-oxide toxicity)
No growth
(antibiotic penetration)
To prepare each of our test group parameters in Table 1 above, we will follow the
procedures outlined in Table 2 below:
Table 2. Experimental treatment group preparations.
TREATMENT
PREPARATION PROCEDURE
A
Control
Add 200 µL of 0.9% NaCl to 24 wells of a 96-well
MBEC plate of a randomly selected region (A,B,C,
and D) (Fig. 2)
B
Antibiotic only
Add 200 µL of antibiotic to 24 wells of a 96-well
MBEC plate of a randomly selected region (A,B,C,
and D) (Fig. 2)
C
Nanowire only
Add CuO NWBs to 24 wells of a 96-well MBEC
plate containing 200 µL of 0.9% NaCl of a randomly
selected region (A,B,C, and D) (Fig. 2)
D
Nanowire + Antibiotic
Add 200 µL of diluted antibiotic fused copper-oxide
nanowires to 24 wells of a 96-well microtiter plate of
randomly selected regions (A,B,C, and D) (Fig. 2)
Figure 2. A MBEC plate illustrating the regions of the treatment parameters: control, antibiotic
only, nanowire only, and antibiotic coupled CuO NWBs. Each treatment will consist of 24 out
of the 96 wells and will be rotated between regions A, B, C, and D (Ceri et al. 1999).
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Experimental Procedure
The first day we will grow Pseudomonas aeruginosa biofilms and synthesize CuO NWBs.
Then, we will routinely repeat the following 3 day outline for the duration of our 8 month
experimentation with 2 days of intermittent data collection.
Day 1
-
Confirm Pseudomonas aeruginosa biofilm growth using CLSM and
collect A treatment results
Confirm the presence of copper on the CuO NWBs using TEM & XRD
Synthesize ciprofloxacin and tobramycin coupled CuO NWBs
Confirm proper coupling using FT-IR
Start B treatment and incubate 24 hours (35℃)
Day 2
-
Sonicate biofilm pegs, start C treatment, and incubate 24 hours (35℃)
Sonicate biofilm pegs, start D treatment, and incubate 24 hours (35℃)
Collect B treatment results
Day 3
-
Collect C treatment results
Collect D treatment results
Grow Pseudomonas aeruginosa biofilms
-
Sample Size
We expect limited growth once we penetrate the biofilm layer of Pseudomonas
aeruginosa with our synNET. To determine the sample size (N) we will use a chi-squared
logistical model to determine the noncentrality parameter (λ) using the formula N = λ / w2. The
effect size (w) is the effect we are interested in detecting. With our growth and no growth model
we expect to see a medium effect size of 0.3. With 14 degrees of freedom in our experimental
design, we obtained a noncentrality parameter of 27.2. The sample size that is required for our
experiment is 303. Thus, we will need at least 13 MBEC plates to get the required sample size.
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Analysis and Interpretation
Harrison’s (2006) viable cell counting procedure will be used to determine the mean
amount of Pseudomonas aeruginosa biofilm growth after being exposed to three different
treatments (Fig. X).
Figure 3. Diagram of experimental procedure used for group tests B, C, and D (Table 1). Step D
above will incubate with antibiotics for group tests B and D and with copper oxide nanowires for
group tests C and D (Herrmann, 2010). Cross out G
From our collected data, a one-way fixed effect ANOVA statistical test will be performed
to compare the mean colony forming units (CFUs) for our three different treatments. The
ANOVA statistical test will determine any growth differences between our antibiotic only, CuO
NWBs only, and synNETs.
Our expected results from the ANOVA statistical test should reveal 100% growth in the
control and antibiotic only treatments, less than 100% growth in the CuO NWBs only treatment,
and 0% growth in the antibiotic coupled CuO NWBs treatment. If our experimental results
correlate with our expected results presented above, then our synNETs can be used as an
alternative therapy for Pseudomonas aeruginosa biofilm infections. In addition, future studies
13
could be conducted to determine the optimum concentration of CuO NWBs (without using
antibiotic coupling) needed to effectively kill Pseudomonas aeruginosa biofilm growth.
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LITERATURE CITED
Anwar H, Costerton JW. 1992. Effective use of antibiotics in the treatment of biofilm-associated
infections. American Society for Microbiology News. 58: 665–668.
Bao SJ, Li CM, Zang JF, Cui XQ, Qiao Y, Guo J. 2008. New nanostructured TiO2 for direct
electrochemistry and glucose sensor applications. Advanced Functional Materials. 18: 591-599.
Brinkmann V, Reichard U, Goosmann C, Fauler B, UhlemannY, Weiss SD, Weinrauch Y,
Zychlinsky A. 2004. Neutrophil extracellular traps kill bacteria. American Association for the
Advancement of Science. 303: 1532-1535.
Ceri H, Olson ME, Stremick C, Read RR, Morck D, Buret A. 1999. The Calgary biofilm device:
New technology for rapid determination of antibiotic susceptibilities of bacterial biofilms.
Journal of Clinical Microbiology. 37:1771-1776.
Costerton JW, Cheng KJ, Geesey GG, Ladd TI, Nickel JC, Dasgupta M, Marrie TJ. 1987.
Bacterial biofilms in nature and disease. Annual Reviews of Microbiology. 41: 435–464.
Costerton JW, Stewart PS, Greenberg EP. 1999. Bacterial biofilms: A common cause of
persistent infections. Science. 284: 1318-22.
Diaz F, Mosovich LL, Neter E. 1970. Serogroups of Pseudomonas aeruginosa and the immune
response of patients with cystic fibrosis. Journal of Infectious Diseases. 121: 269-274.
Diaz F, Neter E. 1970. Pseudomonas aeruginosa: Serogroups and antibody response in patients
with neoplastic diseases. American Journal of the Medical Sciences. 259: 340-345.
Govan JRW, Deretic V. 1996. Microbial pathogenesis in cystic fibrosis: mucoid Pseudomonas
aeruginosa and Burkholderia cepacia. Microbiological Reviews. 60: 539–574.
Hanlon WG, Denyer Ps, Olliff JC, Ibrahim JL. 2001. Reduction in exopolysaccharide viscosity
as an aid to bacteriophage penetration through Pseudomonas aeruginosa biofilms. American
Society for Microbiology. 67: 2746-53.
Harrison JJ, Turner RJ, Ceri H. 2005. Persister cells, the biofilm matrix and tolerance to metal
cations in biofilm and planktonic Pseudomonas aeruginosa. Biofilm Research Group. University
of Calgary. 7: 981-94.
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Harrison JJ, Ceri H, Yerly J, Stremick CA, Hu Y, Martinuzzi R, Turner RJ. 2006. The use of
microscopy and three-dimensional visualization to evaluate the structure of microbial biofilms
cultivated in the Calgary biofilm device. Biological Procedures Online. 8:194-215.
Herrmann G, Yang L, Wu H, Song Z, Wang H, Hoiby N, Ulrich M, Molin S, Riethmuller J,
Doring G. 2010. Colistin-tobramycin combinations are superior to monotherapy concerning the
killing of biofilm Pseudomonas aeruginosa. The Journal of Infectious Diseases. 202:1585-1592.
Li Y, Zhang Q, Li J. 2010. Direct electrochemistry of hemoglobin immobilized in CuO nanowire
bundles. Talanta. 83: 162-66.
Lu X, Zou G, Li J. 2007. Hemoglobin entrapped within a layered spongy Co3O4 based
nanocomposite featuring direct electron transfer and peroxidase activity. Journal of Materials
Chemistry. 17: 1427-1432.
Neter E. 1974. Pseudomonas aeruginosa infection and humoral antibody response of patients
with cystic fibrosis. The Journal of infectious diseases. 130: 132-133.
Nickel JC, Ruseska I, Wright JB, Costerton JW. 1985. Tobramycin resistance of Pseudomonas
aeruginosa cells growing as a biofilm on urinary catheter material. Journal of Antimicrobial
Agents and Chemotherapy. 27: 619-624.
Stewart PS. 1996. Theoretical aspects of antibiotic diffusion into microbial biofilms. Journal of
Antimicrobial Agents and Chemotherapy. 40: 2517-2522.
Tirouvanziam.R, Gernez Y, Conrad KC, Moss BR, Schrijver I, Dunn EC, Davies AZ,
Herzenberg AL, Herzenberg AL. 2007. Profound functional and signaling changes in viable
inflammatory neutrophills. National Academy of Science. 105: 4335-4339.
Walters CM, Roe F, Bugnicourt A, Franklin MJ, Stewart SP. 2003. Contributions of antibiotic
penetration, oxygen limitation, and low metabolic activity to tolerance of Pseudomonas
aeruginosa biofilms to ciprofloxacin and tobramyacin. American Society for Microbiology. 47:
317-23.
Weinrauch Y, Drujan D, Shapiro SD, Weiss J, Zychlinsky A. 2002. Neutrophil elastase targets
virulence factors of enterobacteria. Nature. 417: 91-94.
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FAHIMA NAKITENDE
DEPARTMENT OF SCIENCE
UNIVERSITY OF CALGARY
fahmashadia@hotmail.com
Objective: To be a Researcher in Nanomedicine
HIGHLIGHTS OF QUALIFICATIONS AND SKILLS
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Undergraduate scientist, 1st concentration in Biology
Research experience in nanoscience and nanomedicine in a Natural science project
course, Science 501
Interest in learning and discovering new ideas in science
Cooperative and team work skills
Able to interpret and analyze data
Sufficient knowledge about computers and mathematical skills
Ability to communicate both written and orally
Easily adaptable to various working environments
Open minded , Voluntary and non judgmental
Hard working and positive attitude
EDUCATION
B.S.c. Major Natural Science: 1st concentration in Biology, 2nd concentration in Math University of
Calgary, 2007
VOLUNTEER AND EMPLOYMENT HISTORY
May 2006-present Wal-Mart Health and Safety team associate, Calgary, AB
2009-present
Volunteer one on one with Aphasia patients to help them improve on their
speech (CHAT, Community Accessible Rehabilitation), South Calgary health centre, AB
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Volunteer in chat group among Aphasia patients, Community
Accessible Rehabilitation, South Calgary Health Centre, AB
2006-present
Voluntary member of SIMS (Students interested in medical school),
University of Calgary
2006-present
Voluntary performer and a member of ASA (African students association),
University of Calgary
18
SHANNON WESLEY
610 7th Street NW
High River, AB T1V 2C8
(403) 652-1679
sbwesley@ucalgary.ca
Objective: Nanoscience and Biofilm Researcher, Nanotechnology and Medicine
QUALIFICATIONS
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Work experience in a 3-year research study about oil & gas emission effects on cattle
10 years of work experience in veterinary medicine, food safety, pathology, and disease
Microbiology research experience as an Animal Health Technologist and scientist
Comfortable training new employees, working in large groups, and peer mentoring
EDUCATION
Diploma in Animal Health Technology – Olds College, Olds AB, 2001
Bachelor of Natural Science – University of Calgary, Calgary AB, exp. 2012
RELEVANT SKILLS AND EXPERIENCE
Research Experience
 Western Interprovincial Scientific Studies Association (WISSA) research study (2002-2005)
- data entry using Microsoft Access databases
- organ and tissue sample collection from aborted or stillborn calves
 University of Calgary Microbiology laboratory (2010) – microorganism growth and identification
Communication and Leadership Experience
 Peer mentor for Dr. Lisa Bryce – COMS 363 Professional and Technical Communication (2011)
- responding constructively to students’ ungraded oral and written assignments
- advising, guiding, and conversing with students individually about course material
- cyber mentoring (online communication with students)
- facilitating class discussions
Professional Involvement
 Alberta Association of Animal Health Technologists (AAAHT) – 10 year Active Service Award
 Arts Peer Mentorship Program (Winter 2011) – University of Calgary
 Biology Students’ Association – University of Calgary
 Chemistry Students’ Chapter – University of Calgary
EMPLOYMENT HISTORY
May 2005 – present
June 2002 – May 2005
June 2001 – May 2005
Canadian Food Inspection Agency – EG03 Meat Hygiene Inspector
Supervisor: Dr. Constance Taylor (403) 652-8413
University of Saskatchewan – Research Data Entry Clerk
Supervisor: Dr. Richard Kennedy (403) 627-5481
Pincher Creek Veterinary Clinic – Animal Health Technologist
Supervisor: Dr. Charles Zachar (403) 627-3900
REFERENCES AVAILABLE UPON REQUEST
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BUDGET
Summer students: We plan on having 2 summer students for the first year at a rate of $5K/student each
year with a total of $10K. We then plan on hiring 2 graduate students of $13k/student for the second year
for a total of $26K. Over the 2 year period the gross total cost for personal is expect to be $36K without
considering USRA grants and TA positions.
Publication costs: We plan on publishing a paper in the American Society for Microbiology. The cost
associated with publishing this will be $3549.
Equipment costs: We will need to purchase materials to create our own Pseudomonas Aeruginosa
biofilms. The lab of Dr. Douglas Storey at the University of Calgary will provide the bacterial strain. 630
LB Agar plates ($2393.20), 100 mg of Tobramycin and 100 mg of ciprofloxacin (Total $198.90), will be
purchased from Sigma-Aldrich. There will be a rental fee associated with the use of the X-Ray
Diffraction microscope ($15/hour) in the department of Geology at the University of Calgary. Chemicals
for creating the nanowires bundles will be provided by the Department of Chemistry at the University of
Calgary. We will use the Nicolet Avatar FT-IR Mass spectrometer at the University of Calgary Chemical
instrumentation facility. We will require an 3D Gyratory Rocker ($1114.99), an incubator ($2291.94),
and a refrigerator ($150.00).
Personal: We plan to hire a lab technician to set up and maintain our equipment during the 2-year period
costing $40K/year for a total cost of $80K.
Traveling Costs: We expect to present our findings at the Alberta Nanotech showcase ($25/registration).
We plan to send 2 people to the international conference on Nanotechnology, which should be a total of
($3K).
The total cost over 2 years will be $129 198.03.
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TIMELINE
We expect to take 8 months to do our experimental proceed which consists of making,
observing, and examining our nanowires bundles, biofilms, and our synthetic net. Testing of our
hypothesis will also be done in this 8 month period. The next 4 months will be decided to data
collection and analysis of our results. The following year, we will spend 4-5 months interpreting
our results. The remainder of our time will be spent writing our paper and attending conferences
to promote our findings.
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