1. Cluster Classification of Mycobacteriophages Isolated from Tropical
Soils of Puerto Rico
Nicole Colón¹, Alberto Cintron¹, Carolina Montañez¹, Luzmari Reyes¹
University of Puerto Rico, Cayey campus¹
Abstract: Mycobacteriophages have been studied through time for a number of reasons. They have been
used as model systems for the study of biological processes, such as phage infection and the dogma
central. This study aims to analyze different unsequenced mycobacteriophages and classify them into
their respective clusters using PCR and Gel Electrophoresis. Each group of researchers received a specific
Mycobacteriophage. The bacteriophages’ DNA were amplified using the PCR method, and were later
analyzed by running an electrophoresis gel.
Introduction:
For many decades bacteriophages have been
know as viruses that infect bacteria. Each
“Mycobacteriophages are viruses that infect
Bacteriophage is composed of a head and a tail
bacteria belonging to the mycobacteria
(figure 1.1). The head, stores the genetic
genus”(Rubin 2012). Mycobacteriophages can
material, while the tail is used to inject the
be found in a variety of soils. They can be
genetic material into the host bacterial cell.
classified as harmless bacteria or disease causing
Phages can be classified according to their
agents, such as tuberculosis. The size of a phage
morphology and nucleic acids. The International
depends on the average number of phage
Comittee on Taxonomy on Viruses has study
particles liberated when an infected bacterium is
and classified over 2,475 species in 2011.
lysed.

2. Bacteriophages can be isolated by enriching soil For many years, Mycobacteriophages
samples in a nutrient media containing the have been studied and analyzed in order to
bacterial host. Doing this will let the phage understand biological processes. Around 2,400
reproduce, increase in number, and form Mycobacteriophages have been analyzed and
plaques. The plaques represent the cycles of characterized. “Over 70 universities and collages
infection and cell lysis which identify the phage. around the United States, have isolated, purified,
After identification, the plaques are purified for and characterized Mycobacteriophages from soil
further characterization. samples” (Rubin 2012). This experiment
analyzes and examines specific phages in order
The Mycobacteriophages are classified into
to assign them into clusters.
clustesr using specific primers. Table 1 shows
the Mycobacteriophage Clusters in Phages data
base. For this experiment we only used from
cluster A to cluster I because the rest of the Head
cluster didn’t have design primers. Tail
Figure 1.1 Shows the structure of a
bacteriophage.

3. specific forward and reverse primers were also
incorporated. In addition, 12µl of the PCR
Master Mix, which contained Taq Polymerase,
Buffer, Nucleotides, Mg2+, were added. The
tubes were placed in a thermocycler for
amplification. Once amplified, the
electrophoresis method was performed. The
agarose gel was prepared using 200mL of TAE
buffer and 4g of agarose. After that, 2µl of
loading dye were added to the reagents. The
Table 1 Shows the Mycobacteriophage Clusters
In Phagesdb. wells of the agarose gel were loaded and they
Materials and Methods: were left to run for one hour at 80 volts. This
whole procedure was repeated with five gels of
During this experiment, four specific
different Mycobacteriophages, including the
Mycobacteriophages genomic DNA were
control gel.
assigned. Different Genomic DNA were
designated from Mycobacteriophages classified
as Phagus_Maximus, Suave, Bloo and Wilie.
Results:
The preparation of the phage DNA’s and the
forward and reverse primers were previously The gel was analyzed and photographs were
made. Test tubes were labeled from 1 to 15. To taken using a gel documentation system. The
each tube a certain amount of each reagent was bands of some of the Mycobacteriophages
added. First, 5µl of Nano Pure PCR Grade indicated that they were part of a specific
cluster. The thicker upper bands are bright,
Water (H2O) was added. Later, 5µl of each
because they contain the genome, and the ones
Mycobacteriophage genomic DNA was added to
that ran towards the bottom of the wells are
each tube. Following this step, 1µl of the

4. the primer replications of a specific region of the
genome. Figure 1.2 shows the controls on an
agarose gel. In the control gel amplification of
Colbert and Puhltonio genomic DNAs resulted
in PCR products using B1 cluster specific
primers. Thus these phages are verified as
belonging to Cluster B1 and their size pair base
is 700. As well, Ghost and LRRHood resulted in
PCR products as belonging to Cluster C1. And
their size is 400 base pair. Also for Pumpkin,
which DNA resulted, as being part of Cluster E
and its size was 800 base pair. Figure 1.3 the
Figure 1.2 Shows the Control Gel.
middle contains the results of the experimental
agarose gels. It contains the
Mycobacteriophages known as Bloo and Suave.
In the experimental gel neither Wilie nor Bloo
showed an amplified PCR product. The final gel
was named as class gel containing two
Mycobactheriophage. Figure 1.3 left bottom
contains the Mycobactheriophage Suave, which
did not show any amplified PCR product. While
in Figure 1.3, top left, contains the Figure 1.3 Shows the three gels made during
this Research Experience. From left to right are
Mycobactheriophage Phagus_Maximus, whose the Class Gel #4, Experimental Gel and Control
Gel.
amplification of its genomic DNA resulted in a
Discussion:
PCR product about 500 base pair using B2 This study provides information about
clusters specific primers. the classification of each Mycobacteriophage.

5. The conclusion during this experiment was that • Mycobacteriophage Database.
only Phagus_Maximus could be classified as [unknown]. http://phagesdb.org/
belonging to cluster B2. The results of the other • Ross, Robert. 2012. General
Mycobactheriophages were not clear; therefore Botany Study Guide. Department
of Biology UPR Cayey. Puerto
we cannot arrive to any conclusions. The Rico pp xxvii, xxviii, xxix.
Mycobacteriophage named Wilie had
• Rubin. M, 2012. Experimental
ambiguous results, because it did not show any Classification of
bands. Wilie had a low amount of DNA, that’s Mycobacteriophages: Theoretical
Background on Important
the reason why there weren’t any amplify
Concepts and Techniques.
products on the gel. In order to classify this
Mycobacteriophage it will be necessary to
• Simmons, Michael J., Snustad, D.
Peter. 2012. Principles of
prepare the phage with a greater amount of DNA Genetics. John Wiley & Sons, Inc.
and if is necessary test the phage with a new set New Jersey pp. 165, 167, 168.
of designed cluster primers. The
Mycobacteriophages classified as Wilie, Suave
and Bloo did not showed any amplified PCR
product. In order for them to be classified is
necessary to design new PCR cluster primers
from J trough Q to test on them.
Acknowledgements:
Special thanks to: our mentor Dr.Rubin, the
TA’s, Melisa Medina and Valeria Rivera,
Yadira Ortiz and RISE. Also to Dr. Eneida
Díaz, and Dr. Elena González.
References:
• Hatfull, Graham F., Cresawn,
Steven E., Hendrix, Roger, W.
2008. Comparative Genomics of
the Mycobacteriophages: Insights
into Bacteriophage Evolution.
Research in Microbiology Volume
159, Issue 5. P. 332-339.