1. Cluster Classification of Mycobacteriophages Isolated from Tropical
Soils of Puerto Rico
Nicole Colón¹, Alberto Cintron¹, Carolina Montañez¹, Luzmarie 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 “Mycobacteriophages are viruses that
For many decades bacteriophages have infect bacteria belonging to the mycobacteria
been know as viruses that infect bacteria. Each genus”(Rubin 2012). Mycobacteriophages can
Bacteriophage is composed of a head and a tail be found in a variety of soils. They can be
(figure 1.1). The head, stores the genetic classified as harmless bacteria or disease causing
material, while the tail is used to inject the agents, such as tuberculosis. The size of a phage
genetic material into the host bacterial cell. depends on the average number of phage
Phages can be classified according to their particles liberated when an infected bacterium is
morphology and nucleic acids. The International lysed.
Comittee on Taxonomy on Viruses has study
and classified over 2,475 species in 2011.

2. Bacteriophages can be isolated by enriching soil
samples in a nutrient media containing the
bacterial host. Doing this will let the phage Head
reproduce, increase in number, and form Tail
plaques. The plaques represent the cycles of
infection and cell lysis, which identify the
Figure 1.1 Shows the structure of a
phage. After identification, the plaques are bacteriophage.
purified for further characterization.
The Mycobacteriophages are classified into
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
cluster didn’t have design primers.
For many years, Mycobacteriophages
have been studied and analyzed in order to
understand biological processes. Around 2,400 Table 1 Shows the Mycobacteriophage Clusters
In Phagesdb.
Mycobacteriophages have been analyzed and
characterized. “Over 70 universities and collages Materials and Methods
around the United States, have isolated, purified, During this experiment, four specific
and characterized Mycobacteriophages from soil Mycobacteriophages genomic DNA were
samples” (Rubin 2012). This experiment assigned. Different Genomic DNA were
analyzes and examines specific phages in order designated from Mycobacteriophages classified
to assign them into clusters. as Phagus_Maximus, Suave, Bloo and Wilie.
The preparation of the phage DNA’s and the

3. forward and reverse primers were previously The gel was analyzed and photographs
made. Test tubes were labeled from 1 to 15. To were taken using a gel documentation system.
each tube a certain amount of each reagent was The bands of some of the Mycobacteriophages
added. First, 5µl of Nano Pure PCR Grade indicated that they were part of a specific
Water (H2O) was added. Later, 5µl of each cluster. The thicker upper bands are bright,
Mycobacteriophage genomic DNA was added to because they contain the genome, and the ones
each tube. Following this step, 1µl of the that ran towards the bottom of the wells are
specific forward and reverse primers were also the primer replications of a specific region of the
genome. Figure 1.2 shows the controls on an
incorporated. In addition, 12µl of the PCR
agarose gel. In the control gel amplification of
Master Mix, which contained Taq Polymerase,
Colbert and Puhltonio genomic DNAs resulted
Buffer, Nucleotides, Mg2+, were added. The
in PCR products using B1 cluster specific
tubes were placed in a thermocycler for
primers. Thus these phages are verified as
amplification. Once amplified, the
belonging to Cluster B1 and their size pair base
electrophoresis method was performed. The
is 700. As well, Ghost and LRRHood resulted in
agarose gel was prepared using 200mL of TAE
PCR products as belonging to Cluster C1. And
buffer and 4g of agarose. After that, 2µl of
their size is 400 base pair. Also for Pumpkin,
loading dye were added to the reagents. The
which DNA resulted, as being part of Cluster E
wells of the agarose gel were loaded and they
and its size was 800 base pair. Figure 1.3 the
were left to run for one hour at 80 volts. This
middle contains the results of the experimental
whole procedure was repeated with five gels of
agarose gels. It contains the
different Mycobacteriophages, including the
Mycobacteriophages known as Bloo and Suave.
control gel.
In the experimental gel neither Wilie nor Bloo
showed an amplified PCR product. The final gel
Results was named as class gel containing two
Mycobactheriophage. Figure 1.3 left bottom

4. contains the Mycobactheriophage Suave, which
did not show any amplified PCR product. While
in Figure 1.3, top left, contains the
Mycobactheriophage Phagus_Maximus, whose
amplification of its genomic DNA resulted in a
PCR product about 500 base pair using B2
clusters specific primers.
Figure 1.3 Shows the three gels made during
this Research Experience. From left to right are
the Class Gel #4, Experimental Gel and Control
Gel.
Discussion
This study provides information about
the classification of each Mycobacteriophage.
The conclusion during this experiment was that
only Phagus_Maximus could be classified as
belonging to cluster B2. The results of the other
Mycobactheriophages were not clear; therefore
we cannot arrive to any conclusions. The
Mycobacteriophage named Wilie had
ambiguous results, because it did not show any
bands. Wilie had a low amount of DNA, that’s
Figure 1.2 Shows the Control Gel. the reason why there weren’t any amplify
products on the gel. In order to classify this
Mycobacteriophage it will be necessary to
prepare the phage with a greater amount of DNA
and if is necessary test the phage with a new set
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

5. 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.
Mycobacteriophage Database. [unknown]. http://phagesdb.org/
Ross, Robert. 2012. General Botany Study Guide. Department of Biology UPR Cayey.
Puerto Rico pp xxvii, xxviii, xxix.
Rubin. M, 2012. Experimental Classification of Mycobacteriophages: Theoretical
Background on Important Concepts and Techniques.
Simmons, Michael J., Snustad, D. Peter. 2012. Principles of Genetics. John Wiley &
Sons, Inc. New Jersey pp. 165, 167, 168.