BIOL220W Section 19
ISOLATION AND PURIFICATION OF THE NOVEL
BACTERIOPHAGE gk32: NEW INSIGHT IN
TREATMENT OF TUBERCULOSIS
Grant Kovich
Instructor: Dr. Graham Thomas
TA: Mansi Khanna
Kovich
2
Introduction
It is difficult to imagine planet Earth without bacteria; they fill the interior of our bodies
and blanket our exterior, they are found in bodies of water, and also in the soil beneath our feet.
Although they may not seem as such, bacteria grow and reproduce just like any other organism.
With that being said, bacteria are just as susceptible to disease as are mammals. Bacteriophages
are viruses that infect bacteria, causing them to lyse and die (Duckworth and Gulig, 2002).
Discovered in 1915 by Felix D’Herelle and Frederick Twort, phages were used to treat bacterial
diseases throughout the twentieth century (G. Thomas Lecture, 21 Jan 2011). However, with the
discovery of antibiotics, phage therapy was quickly replaced by antibiotics that could slow the
growth of or kill bacteria. Antibiotic therapy appeared to be a medical miracle, in which any
bacterial disease could be treated and cured, but scientists did not realize how the bacteria that
caused disease were evolving right before their eyes. Due to the rapid life cycle of bacteria, new
generations can be produced anywhere from a few hours to overnight, which can lead to genetic
mutations within bacterial DNA. These mutations gave bacteria resistance to antibiotics, which
prevented them from being affected by the antibiotics. Scientists and doctors alike were now
faced with a problem—their cure all for multiple diseases had disappeared, and a way to combat
bacterial viruses needed to be found.
As a result of this, a recent emphasis has been placed on bacteriophage therapy. There
are more than 1031 different varieties of phage, and there are an estimated 1025 infections per
second (G. Thomas Lecture, 21 Jan 2011). A typical bacteriophage features a head, collar, tail,
tail fibers, and a base plate, all made of protein. Located inside the phage head is DNA (Figure
1). During infection, phage will attach to the host cell via tail fibers, and DNA will be injected
Kovich
3
from the head through the collar, tail, and base plate into the host organism. Phage therapy offers
a promising
Figure 1. Typical structure of a bacteriophage
(Farabee, 2011).
alternative to antibiotics because they have a large range of hosts, exhibit host specificity, and
can replicate within their host. Bacteriophages can have one of two different life cycles, lytic or
temperate. A phage with a lytic life cycle will first attach to the host’s cell wall and insert viral
DNA. Once the DNA is present in the host cell, DNA replication and protein synthesis allows
for new virus particles to be produced. The host cell will then lyse, releasing new virus particles
that can infect other cells (Figure 2). In comparison, a phage with a temperate life cycle will
remain in a lysogenic state without viral reproduction or cell death until it is induced into a lytic
life cycle. Induction can be spontaneous or caused by external factors such as UV radiation (G.
Thomas Lecture, 21 Jan 2011). Once the temperate phage is induced, it carries out a similar
cycle to that of a lytic phage (Figure 3).
Kovich
Figure 2. The lytic life cycle of a bacteriophage (Todar, 2009).
Figure 3. The lysogenic life cycle of a bacteriophage (Todar, 2009).
4
Kovich
5
In this study, the effect of bacteriophage therapy is being measured on Mycobacterium
smegmatis, a non-pathogenic strain of Mycobacterium tuberculosis. Tuberculosis (TB) is a
prime example of why bacteriophage therapy is being used today—it was easily treated by
antibiotics until the bacterium because resistant—which is causing more and more cases of TB
worldwide every year. M. tuberculosis is a rod-shaped bacterium, which can causes either
Latent TB Infection or TB disease. Latent TB disease occurs when the bacteria are kept under
control by the body’s immune system. This can eventually develop into TB disease, which
occurs when the immune system can no longer combat the TB infection. Although there are
various drugs used to treat TB disease, it is easy for the bacteria to become resistant to them,
which still causes illness to the host (G. Thomas Lecture, 04 Feb 2011). I believe that the only
way to truly combat TB disease is through bacteriophage therapy, and because of that, I am
searching to find a cure for TB. The cure will come solely from one of the numerous
bacteriophage located in the soil on the campus of The Pennsylvania State University. I hope to
locate a phage and consider it my own unique cure to combat the growing problem of TB.
Materials and Methods
Sampling
In order to collect as pure of a sample as possible, sterility was monitored through the
entirety of the sampling process. Samples were collected using a small shovel and a 15mL
sterile conical tube. All samples were collected at least six inches beneath the surface; even
deeper if conditions were favorable. The tubes remained sealed until sample insertion occurred,
in which the sample filled one-third to one-half of the tube. At the sampling site, GPS
coordinates were recorded, along with the current temperature, precipitation, date, time, moisture
of the sample and approximate depth at which the sample was recovered. Any noticeable
Kovich
6
geographic features relative to the sample’s location were recorded as well. Google Earth™ was
used to plot the GPS coordinates of each sample on a map after sampling.
Initial Sample Extraction and Plating
After obtaining soil samples, phage needed to be extracted from the sample. This was
performed by flooding each sample with enough phage buffer to fill the conical tube using a
10mL sterile pipette and pipettor. The sample was then vortexed and left to settle for twenty
minutes. When the sample finished settling, 1mL of the supernatant was extracted using a sterile
1mL syringe. This was then passed through a 0.22μm filter into a microcentrifuge tube. Once
phage was extracted from each sample, it was then plated to determine if plaques would form.
50μL of sample filtrate was added to a bacterial culture tube containing 0.5mL of Μ. Smegmatis,
vortexed, and left to infect for 20 minutes. When infection was complete, 4.5mL of top agar was
added to the culture tube, and it was plated on an agar plate, then left to incubate overnight at
37°C.
Spot Assay
In order to determine whether or not putative plaques contained phage, a spot assay was
performed. It is easy to mistake an air bubble for a plaque, so by performing this assay, we were
able to determine a true plaque from something that was not a plaque. 100μL of phage buffer
was placed in microcentrifuge tubes corresponding to the number of putative plaques, which
were previously labeled on each plate. The morphologies and other characteristics of each
plaque were recorded. Each plaque was retrieved from its plate by touching the tip of a
micropipette tip and depositing the plaque in its corresponding microcentrifuge tube. The tubes
were then vortexed in order to distribute the phage evenly among the phage buffer. A grid was
then drawn on the reverse of a standard agar plate, with enough spaces for each potential plaque.
Kovich
7
4.5mL of top agar was added to 0.5mL of M. smegmatis and plated on the labeled agar plate.
After solidifying for at least 10 minutes, 5μL of each putative phage was transferred to its
corresponding location on the labeled plate. The plates were then left to incubate overnight at
37°C.
The Phage-Titer Assay
This assay was performed to ensure that a pure phage was being produced, and was thus
repeated several times until completed purification was obtained. 100μL of phage buffer was
transferred into microcentrifuge tubes, one per plaque to be tested. Plaques were then taken from
agar plates using a sterile micropipette tip and depositing the plaque in its corresponding tube.
The tubes were then vortexed, and labeled the 100 dilutions. Four microcentrifuge tubes for each
plaque were then designated 10-1, 10-2, 10-3, 10-4 respectively for the serial dilution series, each
containing 90μL of phage buffer. 10μL of the 100 tube was added to the 10-1 tube and vortexed.
10μL of the 10-1 was then added to the 10-2 tube and vortexed. The process was repeated until
the serial dilution series was completed. 50μL of each dilution tube was then added to 0.5mL of
M. smegmatis, vortexed, and allowed to infect for 20 minutes. 4.5mL of top agar was then added
to each culture tube, and plated on a standard agar plate. The plates were left to incubate at 37°C
overnight.
Harvest of a Plate Lysate
Once the phage sample has gone through enough rounds of purification, it can be used to
purify higher numbers of filter-sterilized phage. The plate from the most recent phage-titer assay
was used, in which we were certain that only one type of pure phage was present. The plate was
flooded with 4.5mL of phage buffer and was stored overnight at 4°C overnight. The phage
buffer located on the plate containing phage was aspirated through a 5mL syringe. The resultant
Kovich
8
liquid was then passed through a 0.22μm filter into a sterile 15mL conical tube. Serial dilutions
from 10-1 to 10-10 were then set up using microcentrifuge tubes, in a process similar to that
described during the phage-titer assay. Each dilution had a corresponding plate as well, which
had 4.5mL of top agar mixed with 0.5mL of M. smegmatis solidified on top of bottom agar.
50μL of each dilution was then pipetted onto the corresponding plate and the plates were
incubated at 37°C overnight. The titer of each plate was then calculated, and a low titer stock of
the phage sample was successfully produced.
Results
Sampling
Soil Sampling Results – January 19, 2011
Sample
Number
Sample 1
Time
Precipitation
Light Rain
Temperature
(°C)
1.67
Soil
Moisture
Moist
12:23pm
Sample 2
12:30pm
Cloudy
1.67
Moist
Sample 3
12:51pm
Cloudy
1.67
Dry
Sample 4
12:58pm
Cloudy
1.67
Sample 5
1:04pm
Cloudy
1.67
Very
Moist
Dry
Location
Features
40°47’58.46’’N
77°51’26.11’’W
40°47’50.53’’N
77°51’33.81’’W
40°48’17.36’’N
77°51’40.05’’W
40°48’06.14’’N
77°51’50.58’’W
40°47’53.55’’N
77°51’53.60’’W
Near sidewalk
(~5cm deep)
Under tree
(~4cm deep)
Near sidewalk
(~5cm deep)
Near sidewalk
(~4cm deep)
Near sidewalk
(~6cm deep)
Figure 4. Results of first round of soil sampling.
No plaques were found from these soil samples. This may be because of poor conditions, not
sampling deep enough, or non-sterile sampling technique. Since no phage were found, I
attempted to resample approximately one week later.
Kovich
9
Soil Sampling Results – January 27, 2011
Sample
Number
Sample 1
Time
Precipitation
9:21am
Light Snow
Temperature
(°C)
-2.78
Sample 2
9:26am
Light Snow
-2.78
Sample 3
9:29am
Light Snow
-2.78
Sample 4
9:31am
Overcast
-2.22
Sample 5
9:33am
Overcast
-2.22
Soil
Moisture
Slightly
Moist
Slightly
Moist
Very
Moist
Slightly
Moist
Very
Moist
Location
Features
40°47’57.00’’N
77°51’51.01’’W
40°47’59.99’’N
77°51’49.13’’W
40°47’57.25’’N
77°51’44.35’’W
40°47’59.49’’N
77°51’42.39’’W
40°48’02.54’’N
77°51’38.37’’W
Near sidewalk
(~3cm deep)
Garden
(~5cm deep)
Near door
(~3cm deep)
Near sidewalk
(~2cm deep)
Garden
(~5cm deep)
Figure 5. Results from second round of soil sampling.
There were potential plaques found from these soil samples. Although the weather conditions
were worse than before, perhaps the fact that the soil was more moist allowed for better phage
capture.
Spot Assay
Results of Spot Assay After Sampling – February 4, 2011
Spot Name Plate Number Sample Number Phage Morphology
A
Plate 1
Sample 1
Turbid
B
Plate 1
Sample 2
Lytic
C
Plate 2
Sample 1
Turbid
D
Plate 2
Sample 2
Lytic
E
Plate 2
Sample 3
Turbid
F
Plate 5
Sample 1
Lytic
G
Plate 4
Sample 2
Lytic
H
Plate 4
Sample 1
Lytic
I
Plate 5
Sample 2
Lytic
J
Plate 5
Sample 3
Lytic
K
Plate 5
Sample 4
Turbid
L
Plate 5
Sample 5
Turbid
M
Plate 5
Sample 6
Turbid
Figure 6. Results from spot assay following second round of soil sampling.
Upon completion of the spot test, there wasn’t any growth. Time constraints prevented me from
resampling a third trial and performing another spot assay. Due to this, I had to move forward
with a phage that was isolated by someone else.
Kovich 10
The Phage-Titer Assay
Phage-Titer Assay Results
Date
Dilution
PFU Counted
Titer (PFU/mL)
-2
10
402
8.04x105
-3
2/11/11
10
47
9.4x105
10-4
6
1.2x105
2/18/11*
N/A
N/A
N/A
2/25/11*
N/A
N/A
N/A
3/4/11**
N/A
N/A
N/A
100
LAWN
LAWN
3/11/11
10-1
18
3.5x105
-2
10
6
1.2x105
3/18/11*
N/A
N/A
N/A
0
10
LAWN
LAWN
10-1
408
8.16x106
3/25/11
10-2
41
8.2x105
-3
10
8
1.6x105
100
LAWN
LAWN
10-1
467
9.34x106
4/1/11
10-2
56
1.12x106
-3
10
3
6.0x104
100
LAWN
LAWN
-1
10
574
1.148x107
4/8/11
-2
10
126
2.52x106
10-3
3
6.0x104
Figure 7. Results of Phage-Titer Assays over the course of the semester’s purification process.
*Due to the similarities in plaque morphology and the natural bubbles that form when the top
agar is setting, there is no data on this date due to the plating of a bubble rather than a plaque.
**There was an error with the bacterial samples used to infect the bacteriophage, therefore there
is no data on this date.
Although there were errors in technique that caused a bubble to be plated instead of a plaque, it
can be seen from the data in the table that the plaque went through four rounds of purification to
ensure that it was a one type of phage with a single morphology. Since I successfully purified
my phage, I could move on to preparing my low titer stock.
Kovich 11
Harvest of a Plate Lysate
Results of Serial Dilution from Low Titer Stock
Date
Dilution
PFU Counted
-4
10
12
10-5
N/A
4/15/11
10-6
N/A
-7
10
N/A
4/22/11
10-4
37
Figure 8. Results of serial dilution from low titer stock.
Titer (PFU/mL)
2.4x105
N/A
N/A
N/A
7.4x105
In plating my low titer stock, I only saw results from my 10-4 dilution the first time through. As
a result, I decided to perform a spot test to ensure that there were not any errors in technique. I
plated two spots of 100, 10-1, 10-2, and 10-3, along with one spot of 10-4. All spots were
completely full of plaques, except for the 10-4 spot, in which I counted the number of plaques
and calculated its titer.
References
Bruns, Peter J. NGRI Phage Laboratory Manual. Howard Hughes Medical Institute, 2010. Print.
Farabee, Michael J. "Biological Diversity: Viruses." Michigan State University. 05 Jan. 2000.
Web. 18 Apr. 2011.
<https://s10.lite.msu.edu/res/msu/botonl/b_online/library/onlinebio/BioBookDiversity_1.
html>.
Duckworth, Donna and Gulig, Paul. "Bacteriophages." BioDrugs: clinical immunotherapeutics,
biopharmaceuticals, and gene therapy 16.1 (2002): 57-62. Online.
Thomas, Graham. Lecture Notes
Todar, Kenneth. "Bacteriophage." The Microbial World. University of Wisconsin-Madison,
2009. Web. 18 Apr. 2011.
<http://www.textbookofbacteriology.net/themicrobialworld/Phage.html>.