Purification, Isolation, and DNA Extraction of Microbacterium foliorum
Phage Rencha
School of Natural and Health Sciences, Seton Hill University, Greensburg PA 15601
Introduction
Bacteriophages, viruses that infect a bacterial host with their
DNA in order to replicate, are one of the most abundant organisms
on the Earth (1). Despite this fact, they have not been as intensively
studied as have many of the other organisms of the biosphere. What
little research that has been conducted on them has found that they
are useful for many reasons; most importantly fighting off drug-
resistant bacteria (2). More and more, antibiotics are being used to
treat bacterial infections, and more and more these bacteria are
becoming immune to the antibiotics (2).
The bacteria used to isolate the phage, Microbacterium foliorum,
is a common type of actinobacteria. A majority of the phages that
infect this bacteria, like Rencha, belong to Cluster EA (3). It is
important to study these phages because we do not know all that
much information about them, and they could be useful in many
different ways, including the treatment of drug-resistant bacteria,
and many more (2).
In this experiment, bacteriophage phage Rencha was purified, its
DNA was isolated, and a restriction enzyme digestion and gel
electrophoresis were performed. Serial dilutions were used to purify
the phage, done multiple times to ensure accuracy, and once
isolated, the phage DNA was extracted using various reagents.
Materials and Methods
Culture conditions of Gordonia terrae and Microbacterium
foliorum. Cultures were maintained on Peptone-Yeast-Calcium
(PYC) media (1.5% peptone; 0.5% yeast extract; 4.5 mM CaCl2; 10
μg/mL cycloheximide; with 1.5% agar for solid media). Liquid
cultures were grown to saturation with shaking at 250 rpm at 30°C
for approximately 2-4 days and stored for a maximum of 14 days at
4°C before use. Phage buffer was prepared as 10 mM Tris (pH 7.5),
10 mM MgSO4, 68 mM NaCl, 10% glycerol, and 4.5 mM CaCl2.
PYC Top Agar was prepared as 1X PYC liquid media with 0.3%
agar.
Phage purification. A pipette tip containing positive phage was
swirled into a micro-tube containing 100 uL of phage buffer. 180 uL
of phage buffer was added to four micro-tubes. 20 uL from the
micro-tube containing the enrichment sample (100) was serially
diluted to 10-4. Six tubes of 250 uL M. foliorum were retrieved,
adding 10 uL of phage buffer to the negative control and 10 uL of
each dilution to the corresponding tube of M. foliorum. After 10
min, 3 mL of top agar was pipetted into each tube, and evenly
dispensed onto a plate. The plates were then incubated for 48 hrs at
30°C.
DNA isolation. 4 mL of phage lysate and 10 uL of nuclease mix
were transferred to a 15 mL conical tube, then incubated at 37°C.
After 10 min, 1 mL of lysate was added to four micro-tubes. 25 uL
of a 2M ZnCl2 solution was added to each tube and incubated at
37°C for 10 min. The tubes were centrifuged for 1 min at 10,000rpm
until a pellet formed, then resuspended in 250 uL of TES buffer. The
resuspended pellets were combined into two micro-tubes and
incubated at 60°C for 15 minutes. 60 uL of 3M CH3CO2K was added
to each tube and incubated on ice at -20°C for ~10 min before being
centrifuged for 1 min at 12,000rmp. 1 mL of C3H8O was added to the
supernatant, then incubated on ice at -20°C for 15 min before being
centrifuged for 20 min at 13,000rpm and decanted. The pellet was
washed with 1 mL of 70% EtOH, then centrifuged for 5 min at
13,000rmp and decanted. The pellet air dried and was resuspended
in 100 uL of ddH2O.
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Jordan Barzensky, Faith Dent, Lauren Kosslow, and Lauren Villone
Restriction enzyme digestion and gel electrophoresis. A
calculated amount of DNA, restriction enzymes, buffer, and ddH 2O
were combined in six micro-tubes before being incubated at 37°C for ● The concentration of DNA isolated is 201 ng/uL. 150 ng/uL
above the minimum concentration of DNA required for
2 hrs before being placed in a freezer for ~48 hrs. A gel
electrophoresis was performed by loading samples into the wells, then sequencing (3), Rencha is high enough in quality and
quantity for genome annotation (Figure 2). In the restriction
electrophoresing at 120V until the samples are out of the wells, then at
80V for ~60 min. digest gel, zero of the seven enzymes produce any cuts in
the Rencha DNA sequences (Figure 2).
Results ● Rencha most nearly matches the EH cluster with an 83.3%
similarity score in cut sites (Table 2). This cluster consists of
2 sequenced phages, Floof and Percival. Floof and Percival
Table 1. Identifying information for soil samples #51-58
and Rencha, a Microbacterium foliorum phage grown on consist of an average of 47,932 bp and are both lytic phages
PYCa plates at 30◦C. that infect Microbacterium (4). The next three closest
sequenced phages to Rencha are Appa, KaiHaiDragon, and
KayPaulus share a 66.6% similarity score and also exhibit
Sample GPS Bacterial Isolation Date Bacteriop only lytic life cycles (Table 2). The lytic life cycle of Rencha
Name Coordinat Host Method Collecte hage
describes the bacteriophage’s ability to lyse Microbacterium
es d Isolation
cells in ideal conditions.
#51 40.004727 Microbact Enrichment 2/25/18 No ● DNA sequencing and genome annotations are the next step
, erium
-79.58680 foliorum in studying the Rencha bacteriophage. Since the phage and
6 Figure 2. The restriction digest gel of Rencha DNA introduced to its nearest relatives are virulent, they have the potential to
multiple enzymes. The enzymes in the photo are as follows: 1 = infect targeted bacterial cells and show usefulness in
#52 40.309662 Microbact Enrichment 2/28/18 No
, erium 1kb ladder, 2 = uncut DNA, 3 = Bam HI, 4 = Cla I, 5 = Eco RI, 6 = medical research involving phage therapy..
-79.55635 foliorum Hae III, 7 = Hind III, and 8 = Sal I. The concentration of the
4 Rencha DNA used was 201 ng/ul.
#53 40.310204 Microbact Enrichment 2/25/18 No
, erium Future Directions
-78.87753 foliorum
9 Table 2. Information from the Phage Enzyme Tool website
demonstrating the cluster assignment of Microbacterium Phage • Laboratory DNA sequencing is necessary for Rencha to be
#54 40.310105 Microbact Enrichment 2/25/18 No
, erium Rencha. studied and identified genomically. With a very high
-78.87754 foliorum concentration of 201 ng/uL, Rencha is pure enough to be
8 studied with different restriction enzymes in another gel
#55 40.308535 Microbact Enrichment 3/1/18 No electrophoresis. To observe the expanse of Rencha’s
, erium infection capabilities, Gordonia terrae can be exposed to the
-79.55557 foliorum
2
phage in a similar laboratory purification protocol.
#56 40.308502 Microbact Enrichment 2/27/18 No
, erium
-79.55394 foliorum Acknowledgements
1
#57 40.313655 Gordonia Enrichment 2/26/18 No We would like to thank Dr. Kristen Butela, our research advisor, the
, terrae Seton Hill Division of Natural and Health Sciences, and the HHMI
-79.57842 SEA-PHAGES program for their help and contributions to this
2
research endeavor.
#58 40.313802 Gordonia Enrichment 2/26/18 No
, terrae
-79.57842
2 References
Rencha 40.308622 Microbact Enrichment 2017 Yes
, erium
-79.55588 foliorum 1. Kasman, L. M., & Whitten, R. A. (2018, February 26).
9
Bacteriophages. Retrieved May 9, 2018, from
https://www.ncbi.nlm.nih.gov/pubmed/29630237
Conclusions
2. LaVergne, S., Hamilton, T., Biswas, B., Kumaraswamy, M.,
Summarize the main “take home” points of your work here, Schooley, R. T., & Wooten, D. (2018, April). Phage Therapy for
discussing each figure/table in order a Multidrug-Resistant Acinetobacter baumannii
● From all eight soil samples collected in various areas of
Craniectomy Site Infection. Retrieved May 9, 2018, from
western Pennsylvania in February 2018, zero phage is found https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5905571/
with the Gordonia Terrae host or Microbacterium foliorum
host (Table 1). Rencha, a known M. foliorum phage, is used 3. Poxleitner, M., Pope, W., Jacobs-Sera, D., Sivanathan, V., &
for enrichment isolation and is grown on PYCa plates at Hatfull, G. (2018). Phage Discovery Guide. Retrieved May 3,
30◦C. With three sequences of serial dilutions and plaque 2018, from https://
assays, the Rencha phage is purified to a high concentration seaphagesphagediscoveryguide.helpdocsonline.com/ copyright
Figure 1. The morphology of plaques of a pure culture of of 3.66 x 108pfu/mL for DNA isolation and analysis.
Microbacterium phage Rencha. The plaques were cultured on ● The plaques of Rencha are on average 1 millimeter in 4. The Actinobacteriophage Database | Cluster EH Phages. (2017).
PYCa plates at 4◦C for 48 hours. diameter with several interspersed smaller plaques (Figure 1). Retrieved May 3, 2018, from http://phagesdb.org/clusters/EH/
The plaques are clear and transparent, indicating lysing of the
Microbacterium cells and the presence of a lytic, virulent
phage (Figure 1).