1. Scientific Manuscript 2014
Abstract
Fully characterized mycobacteriophages may be
utilized as potential treatments for diseases
caused by bacterial infections. Isolation and
characterization of novel mycobacteriophages
could lead to advances in the rising field of
phage therapy. Using Mycobacterium
smegmatis, a novel mycobacteriophage was
isolated and purified from tropical soil collected
in Puerto Rico. This serves as evidence that
Puerto Rico’s tropical environment is adequate
for the growth of phage populations.
1. Introduction
One of the most challenging problems facing
scientists today is the constant resistance of
bacterial infections to current antibiotics.
Bacteria can grow resistant through several
mechanism; some develop the ability to
neutralize the antibiotic before it can do harm,
others can rapidly pump the antibiotic out, and
still others can change the antibiotic attack site
so it cannot affect the function of the bacteria.
Because of this, researchers have had to find
new alternatives to fight the ever stronger
resistance of bacterial infections. In recent years,
bacteriophages have emerged as the ideal
alternative and answer to this problem.
Bacteriophages (phages) are bacterial viruses
that infect, disrupt and lyse bacterial cells
resulting in cell death. As an example,
mycobacteriophages are a specific group of
viruses that only infect bacteria from the
mycobacteria genus. Common bacteria from
this genus include Mycobacterium smegmatis,
which is harmless, and others like
Mycobacterium tuberculosis and
Mycobacterium leprae; both of which cause
deadly bacterial infections. Most of these
bacteria are constantly growing resistant to
antibiotics, but in the future fully characterized
mycobacteriophages may be utilized as potential
treatments for these and other diseases.
By utilizing Mycobacterium smegmatis as a
rapidly growing host (Endersena et al. 2013), it
is possible to isolate and characterize a whole
variety of mycobacteriophages found in the
environment. In this manner, researchers are
able to use genomics and proteomics to study
the complete genetic information (genomes),
and the structure and function of the phage’s
proteins (proteome). These studies help
scientists determine the unique genes that each
phage may possess, giving clues and ultimately
an indication into their possible antimicrobial
properties.
Phages are estimated to be the most abundant
biological entities on Earth (Suttle, 2005;
Wommack & Colwell, 2000). They are found on
a variety of ecosystems; in other words they are
easily found everywhere. Puerto Rico is
characterized as being one of the most diverse,
in terms of climate and types of environments.
The question is if this diversity and tropical
environment is sufficiently adequate for
bacteriophages to thrive. It is clear that our
environment has a whole diversity of microbes,
hence we can hypothesize that that there is a
whole variety of bacteriophages that can be
found on the tropical soils of Puerto Rico which
can be successfully isolated and purified. In this
study, we describe the isolation and purification
of a novel mycobacteriophage: Incognito.
2. Materials and Methods
2.1. Sample Collection and Preparation
Isolation and Purification of Novel Mycobacteriophage: Incognito
Paola G. Caballero León1
, Anthony Hernández Rivera2
1
Department of Chemistry, RISE Program, University of Puerto Rico at Cayey
2
Department of Biology, RISE Program, University of Puerto Rico at Cayey

2. Scientific Manuscript 2014
A soil sample was collected and analyzed for the
presence of mycobacteriophages. Using aseptic
techniques, 10 mL of AD Suplement Smeg
Master Mix were mixed with 1 mL of M.
smegmatis bacteria and 0.5 g of the soil sample
in a labeled tube. This enrichment was
incubated at 37°C for 24 hours and then
centrifuged for 15 minutes, at room
temperature, and 3,000 rpm. Following this
enrichment procedure, 1 mL of the supernatant
was obtained, placed inside a microtube and
centrifuged a second time at 10,000 rpm for 10
minutes, to obtain the phage filtrate. Without
disturbing the pellet that formed, 500 μL of the
supernatant were removed and placed inside a
clean microtube for further processing.
2.2. Isolation of Phage
A small agar plate for the culture of
M. smegmatis bacteria was used for the streak
protocol. A sterilized wooden stick was inserted
inside the filtrate and was then used to streak
about one-third of the agar plate. A new stick
was used to streak the adjacent area of the first
streak, overlapping the original streaked area
once. The third quadrant was streaked in the
same manner. After the streak protocol, 2 mL of
LB Top Agar for M. smegmatis were obtained,
mixed with 0.25 mL of
M. smegmatis bacteria and dispensed over the
streaked agar plate. This plate was then
incubated at 37°C for 24 hours and then
examined for the presence of plaques.
2.3. Purification of Phage
One plaque was aseptically obtained from the
plate with a micropipette tip, added to a
microtube that contained 25 μL of phage
buffer, and vortexed to ensure maximum
contact of the phage with the buffer. This
isolated phage sample was then used to
perform the streak protocol on a new
M. smegmatis agar plate and obtain the first
purification of the phage. Single plaque
purification was repeated three times to obtain
a purified population of the phage.
From the third purification, one plaque was
aseptically obtained and mixed with 10 mL of
AD Supplement Smeg Master Mix and 1 mL of
M. smegmatis bacteria. This second enrichment
was then filtered and a Medium Titer Phage
Lysate (MTPL) was obtained, which is a
concentrated liquid sample of the phage. A spot
test of eight 10 μL serial dilutions of the titer
lysate was performed onto a prepared agar
plate with the LB top agar and
M. smegmatis bacteria already added and
solidified.
2.4. Characterization of Phage
2.4.1. Proteomics
Polyacrylamide gel electrophoresis was
carried out in order to isolate, separate, and
visualize the mycobacteriophage capsid
proteins. Initially, 20 μL of the MTPL were
obtained, centrifuged and mixed with 25 μL
of Beta-mercaptoethanol (BME).
Subsequently, the sample was boiled for 2
minutes and then cooled down for an
additional 2 minutes, in order to completely
denaturalize the protein. Afterwards, 17 μL of
this sample were loaded to one of the wells
on the gel. The electrophoresis was carried
out at 200 volts for 30 minutes. The gel was
washed with water for anothert 30 minutes.
2.4.2. Electron Microscopy
Electron Microscopy grids were placed,
shiny side up, on the very edge of a double
sided tape. Afterwards, 10 μL of the phage
sample were added to the grid. Once the
sample had settled and attached onto the
grid for at least 2 minutes, the excess fluid
was wicked off with filter paper. This same
procedure was repeated two more times,
the first with 10 μL of water, and the

3. Scientific Manuscript 2014
second with 10 μL of 1% uranyl acetate to
stain the phage sample.
3. Results
3.1. Soil Samples
A total of 11 soil samples were collected from all
over the island of Puerto Rico in search of a
phage. The enrichment process and streak
protocol were performed with each soil sample
until phage plaques were obtained. The
environmental data for each soil sample is
reported in Table 1.
3.2. Isolation of Phage
The soil sample #9 was taken from underneath
a plantain tree at a depth of approximately 10
cm, in a rural area of Hatillo, PR. The
coordinates of the location were 18.473341, -
66.785332 (Fig 1). The soil sample was
saturated. After completing the streak protocol,
the filtrate of this sample proved to contain a
mycobacteriophage due to presence of the four
observed turbid plaques (Fig 2).
3.3. Purification of Phage
The isolation of the phage was followed by the
single plaque purification process. The
purifications were repeated three times (Fig. 3)
to obtain a pure population of the phage. Due
to the turbid nature of the plaques, edited
pictures of this process are provided (Fig. 4) to
facilitate the identification of the plaques.
Table 1. Soil Samples
Fig. 2 Positive
phage results-
four turbid pl
Fig. 1 Localization
of soil sample #9

4. Scientific Manuscript 2014
The spot test served as an empirical test to
analyze the concentration of phage in the MTPL
(Fig. 5). We observed complete lysis in dilutions
1 through 4. Three isolated turbid plaques were
present in dilution 5. Complete infection of M.
smegmatis will be carried out with these five
dilutions in order to obtain a web pattern and
subsequently, the high titer phage lysate
(HTPL). With this data, the concentration of
phage was calculated to be 3 x 107
Plaque
Forming Units (PFU) per milliliter (PFU/mL).
3.4. Characterization of Phage
3.4.1. Proteomics
Using gel electrophoresis, the capsid
protein content of the
mycobacteriophage was isolated and
separated. Our phage sample was
loaded onto wedge #3 of the
polyacrylamide gel (Fig. 6). The other
wedges contain phage samples that
belong to other researchers.
4. Discussion
Our phage was found among a series of 11 soil
samples collected from various areas in the
island of Puerto Rico. It can be classified as a
mycobacteriophage because it infected M.
smegmatis, a member of the Mycobacteria
genus. This novel mycobacteriophage was
named Incognito due to its elusive nature.
Identifying its plaques for the first time was
difficult because of its turbidity. There were
weeks where the phage seemed to disappear,
causing doubts of its presence. By successfully
isolating Incognito, we have proven that soils in
Puerto Rico are adequate for the growth of
phage populations. Our main objective was to
purify and completely characterize Incognito to
see if its genes and proteins hold any unique
characteristics.
Fig. 4
Enhanced and
edited pictures
of the
purification
process
Fig. 3 Single
plaque
purification
process and
results
Fig. 5 Spot test- Empirical test
Fig. 6 Polyacrylamide gel electrophoresis

5. Scientific Manuscript 2014
Temperate phages have the ability to carry out
either the lysogenic or the lytic cycle of
reproduction and they usually form turbid and
cloudy plaques (Science Education Alliance).
The observed plaques fit this description; which
led us to conclude that Incognito is most likely
a temperate phage. For this reason, edited
pictures of the purification were provided.
The spot test that was carried out served as an
empirical test to determine the concentration of
the phage. Apart from the circles of complete
lysis in grids 1 through 4, there were clear lytic
plaques all over the plate. This is evidence of
bacterial contamination. Nonetheless, the
contamination can be ignored because the
objective was to simply observe in which
dilutions was Incognito present. Since three
turbid plaques were observed in dilution of 10-5
,
we used that number as the PFU (plaque
forming units) and divided by 0.01 mL, which
was the volume added of the dilution. The
concentration of our phage in the MTPL was
3 x 107
PFU/mL.
The capsid proteins of our phage were isolated
and separated by using polyacrylamide gel
electrophoresis. After image analysis, we can
observe significant similarities between the
capsid protein content of Incognito (well #3)
and that of our respective peers (wells #2, #6,
and #7). Further characterization will be carried
out. By doing this we strive to contribute to the
global scientific community in the ever more
important development of phage therapy as the
future alternative for treating all kinds of
pathogenic bacteria.
Acknowledgements
We would like to thank the RISE and Howard
Hughes Programs for this great experience in
which we gained invaluable research and
laboratory experience. Special thanks to our
RISE lab technician Giovanni Cruz and Teaching
Assistants (TAs) Joseph Perez and Gustavo
Martínez. We would also like to give special
acknowledgements to Dr. Michael Rubin and
Dr. Edwin Vazquez for their guidance and
constant support.
Literature Cited
Endersena L, Coffeya A, Neveb H, MaAuliffec O,
Rossc RP, O’Mahonya JM. [2013]. Isolation and
characterisation of six novel
mycobacteriophages and investigation of their
antimicrobial potential in milk. International
Dairy Journal. 28(1): 8-14
Suttle CA. [2005] Viruses in the sea. Nature.
437: 356-361
Wommack KE, Colwell RR. [2000]
Virioplankton: viruses in acquatic ecosystems.
Microbiology and Molecular Biology Reviews,
64: 69–114
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Mycobacteriophages Proteomics: From
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