This document describes the isolation and analysis of two mycobacteriophages, Lilliputian and Pearbear. Through electron microscopy, restriction enzyme analysis, and comparison to existing phage data, Lilliputian was determined to likely be a member of subcluster A4. Pearbear was found to likely be a member of subcluster B1. Both phages were discovered by infecting Mycobacterium Smegmatis bacteria. Further DNA sequencing and analysis could confirm the proposed subcluster classifications of Lilliputian and Pearbear. The goal is to increase understanding of bacteriophage diversity and evolution through isolating and characterizing new mycobacteriophages.

1. Abstract
Methods
Direct
Plating
Spot
Plating
Plaque
Purification
via Serial
Dilution
High
Titer
Lysate
DNA
Extraction
EM
Grid
DNA
Sequencing
Further
Analysis/
Annotate
DNA
Enrichment
Isolate
Soil
Sample
Figure 1: Virus
infecting bacterial host.
1.Webbed Plate
2.Flood
3.Harvest
4.Titer
Results
Figure 2b Plaques produced by mycobacteriophage
Lilliputian. The plaques are turbid, and extremely small
with a diameter of approximately one millimeter. The
plates were incubated at 30 degrees Celsius for 48 hours.
Figure 2:
Figure 3b Electron micrograph of
mycobacteriophage Lilliputian. The average
tail length is approx. 208 nm. The head is
positively stained and isometric with an
average diameter of approx. 72.5 nm.
Figure 4b Restriction digest of the DNA of
mycobacteriophage Lilliputian. We used an agarose
gel and incubated the digests first at 37⁰C for 30
minutes, then 4⁰C overnight. The sole enzyme that
digested the DNA for Lilliputian was HaeIII.
Conclusion and Future Direction References
By Katie Milliken and Peri Shohnen
Figure 3: Figure 4:
Based upon comparison of the evidence listed below with information on phagesdb.org, mycobacteriophage
Lilliiputian appears to be a member of subcluster A4.
1) The EM showed a positively stained capsid- similar to members of A4
2) The only enzyme to digest was HaeIII - extremely similar to members of A4
For Lilliputian, the next steps would be to:
• redo the electron micrograph
• sequence the DNA
•compare to other sequenced DNA and confirm the cluster/ subcluster
Additionally through comparison, Pearbear appears to be a member of subcluster B1.
1) The EM showed phages with long flexible tails and an isometric head- like that of subcluster
2) The enzymes with digest Pearbear were HaeIII and ClaI
For Pearbear, the next steps would be to:
• redo the electron micrograph
• sequence the DNA
•compare to other sequenced DNA and confirm the cluster/ subcluster
•http://oceanworld.tamu.edu/resources
/oceanography-book/microbialweb.htm
•http://www.phagesdb.org
•http://www.hypertextbookshop.com/bi
ofilmbook/v005/r002/Contents/02_Lab
_Exercises/02_Protocols_and_Method
s/02_Student_Version/03_Drop_Plate
_Method.html
•SEA-PAHGES Lab Manuel and
Resource Information
Bacteriophages are highly diverse and adundant organisms.
Because of this, we utilize bacteriophages for evolutionary research
and molecular biology. Our goal was to indentify and analyze
mycobacteriophages and compare them to existing samples.
Lilliputian and Pearbear were discovered through infection of
mycobacteria M. Smegmatis. Through analysis it was determined
that phage Lilliputian is likely to be a member of subcluster A4, and
phage Pearbear is likely to be a member of B1. Through DNA
sequencing and further analysis, these suggestions can be
confirmed.
A Voyage into Mycobacteriophage Isolation and Analysis
Introduction
Bacteriophages are viruses that infect bacterium. In particular, we are
isolating mycobacteriophages that infect the host Mycobacterium
Smegmatis. The purpose for isolating bacteriophages is three-fold.
1.They are a rich resource for understanding molecular biology
because they are abundant and highly genetically diverse.
2.They contribute to bacterial pathogenesis.
3.They are useful for mapping evolutionary pathways.
Mycobacteriophages specifically have the potential to serve as
therapeutic agents for drug resistant bacteria, such as tuberculosis. The
goal of this project is to define the diversity of the bacteriophage
population and to understand the evolutionary mechanisms that shape it.
The immediate goal is to collect and analyze specific mycobacteriophage
data and compare them.
Figure 5: Serial Dilution.
Figure 3a: Electron micrograph of phage Pearbear.
Pearbear has an isometric head and a flexible tail.
The average head diameter is approx. 60nm but
due to positive staining, an accurate measurement
could not be made. The average tail length is
approx. 267nm.
Enzymes:
DNA Ladder
BamHI
ClaI
Hind III
HaeIII
EcoRI
Figure 2a Plaques formed by
mycobacteriophage Pearbear. Pearbear
produces plaques that are clear and
approximately 1 mm in diameter. Pearbear
infects M. smegmatis. This plate was incubated
at 30⁰C for 48 hours.
Figure 4a: Restriction digest of Pearbear. The digests were
run in a .7% agarose gel alongside a DNA ladder at 30⁰C
for 30 minutes. The gel shows that enzymes ClaI and
HeaIII cut Pearbear’s DNA, suggesting that Pearbear
Belongs to subcluster B1.
Ladder BamHI ClaI HindIII HeaIII EcoRI