Knowledge engineering: from people to machines and back
Report of phages presentation
1. “Isolation and Characterization of
Mycobacteriophages Isolated
From Tropical Soils of Puerto Rico”
Michael Rubin, PhD
Giovanni Cruz, Laboratory Technician
Carla J. Figueroa, Crystal K. Colón (undergraduate students)
2. Introduction
• Bacteriophages, more commonly known as phages, are the
viruses of bacteria. They are sequences of genes that extract
the lysis from growing bacterial cultures, often killing them.
This means that phages can only infect members that are from
the domain Bacteria. They have many abilities that include
having RNA or DNA genomes, infecting their host in ways that
they can or can’t kill them and even taking different
morphologies. Their sizes range from 100 to 200 nm and they
can replicate and propagate, but only inside their host. They
also aren’t susceptible to antibiotics and as a consequence
they are a pervasive class of virus that has become the most
abundant life-form on earth.
3. Introduction
• The most common way to study phages is to collect soil
samples. These samples undergo a process that includes
enrichments, harvesting, plating, purification, empirical tests,
web pattern, and a high Titer phage lysate (HTPL) with
excellent aseptic techniques. At the end of the process the
obtained solution is ready for DNA purification and eventually
it will be characterized. Most of the phages from which their
DNA’s are characterized are named and inserted in a genome
banc where they are shared with the world.
5. Soil Sample Collection and
Environmental data
• The soil was collected using a wrapped spoon
and was immediately cast into a 15-mL conic
sealed tube.
• Environmental data was recorded; time of
sampling, location, air temperature, depth,
moisture content, and features of the site.
6. Enrichment
• With a clean spatula 0.5 grams of the sample were weighted
and then added to a 50 mL tube.
• Inside the flask were; 8mL of sterile H2O, 1mL of sterile 10x
7H9/glycerol broth, 1mL of AD supplement, 0.1mL of 100 mM
CaC12, and 1mL of late long stationary phase of M.smegmatis
culture.
• The flask was incubated at 37˚ C shaking at 220 rpm, for 24
hours.
7. Harvesting and Preparing the
Enriched Sample
• The flask was balanced and spin at 3,000 rpm in a centrifuge
for 10 minutes to pellet particulate matter.
• The liquid from the centrifuge sample was poured into a fresh
50 mL conical tube without any solid residue.
• Then enrichment sample was filtered sterilized.
8. Plaque Streak Protocol
• A sterile wooden stick was removed from the package.
• After opening the tube with the centrifuged filtered
enrichment it was gently streaked across the top third of the
agar plate.
• This was repeated three times from the streaked area to the
unstreak side.
• 4.5 mL of Top Agar were added to 0.5 mL aliquot of M.
smegmatis.
• At the end it was dispensed from the most dilute point to the
most concentrated areas by gently titling the plate.
9. Plaque Streak Control
• After the Top Agar was hard, the plates were incubated at
37˚ C for 24 hours.
11. Crystal K. Colón
Cidra,P.R.18˚8’ 26’’N 66˚8’ 10’’ W
Barranquitas, P.R.18˚11’ 22.65’’N 66˚19’25.07’’ W
Cayey, P.R.18˚7’30.12’’N 66˚ 7’8.01’’ W
Cidra, P.R.18˚8’ 24.13’’N 66˚8’ 10.09’’ W
12. Carla J. Figueroa
Cayey, PR (18.11235 N 66.15500 W)
Caguas, PR
(18.16853 N
65.07780 W)
Las Piedras, PR
(18.20509 N
65.87780 W)
Las Piedras, PR
(18.20554 N
65.87694 W)
15. Results
Location Plaques?
Cidra,P.R.
18˚8’ 26’’N
66˚8’ 10’’ W
No
Cayey, P.R.
18˚7’30.12’’N
66˚ 7’8.01’’ W
No
Cidra, P.R.
18˚8’ 24.13’’N
66˚8’ 10.09’’ W
No
Cidra, P.R.
18˚8’ 26.99’’N
66˚8’ 11.12’’ W
No
Barranquitas, P.R.
18˚11’ 22.65’’N
66˚19’ 25.07’’ W
No
Location Plaques?
Las Piedras, PR No
Las Piedras, PR
18.20554 N
65.87694 W
No
Caguas, PR
18.16853 N
65.07780 W
No
Las Piedras, PR No
Las Piedras, PR
18.20509 N
65.87780 W
No
Las Piedras, PR
18.20657 N
65.87817 W
No
16. Discussion
• None of the soil samples collected had any presence of
phages. This can be explained by different factors that include
the temperature, time, moisture, and location.
• The average temperature was 84 oF. The highest temperature
was 91. 4 oF and the lowest was 80 oF. This is a normal range
for our island’s air temperature meaning that temperature
may probably not be the main reason for the absence of
phages in the samples.
17. Discussion
• The time at which samples were collected varied during the
day. Some were collected during the morning, the earliest at
11:16 am, and the rest during the afternoon and the evening,
the latest at 6:37 pm.
• The moisture content of the samples varied with location and
with the depth at which the sample was extracted. The
average depth of the samples was 1.17 in. This means that the
samples were fairly superficial where there was a constant
circulation of liquid, solid or gaseous debris, animals, etc.
18. Discussion
• The soil samples were collected from 5 different cities or
towns of Puerto Rico. 25% were obtained from Cidra (3
samples), 8% from Barranquitas (1 sample), 8% from Caguas
(1 sample), 17% from Cayey (2 samples) and 42% from Las
Piedras (5 samples), for a total of 12 samples. None of the
samples were taken from the same spot or nearby area. The
samples from Las Piedras were picked from the same
neighborhood but from different environments. Samples were
from under fruit trees, from horse and cow stables, from a
swap’s shore, from plain areas. Some were urban, like the
samples from Cayey, and others, like the one from
Barranquitas are more rural.
