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Poster Final: LG, EL, VR, VS
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1
Phicolas
and
Viva
are
Highly
Lytic
Siphoviridae
Mycobacteriophages,
Viva
Belonging
to
the
F
Cluster
Lauren
Gray,
Erica
Landreth,
Valeria
Rodriguez,
Victoria
Saldivar
Department
of
Molecular,
Cellular,
and
Developmental
Biology,
University
of
Colorado,
Boulder,
Spring
2018
Abstract
Lytic
phages
have
been
explored
at
an
increasing
rate,
but
more
work
is
necessary
to
further
categorize
specific
uses.
As
determined
through
electron
microscopy
and
restriction
digestion
analysis,
Viva
and
Phicolas
are
lytic,
siphoviridae
phages
sampled
in
Colorado,
with
Viva
belonging
to
cluster
F.
With
possible
applications
ranging
from
medicine
to
controlling
food
borne
illness,
further
exploration
of
these
phages’
host
ranges
could
yield
incredibly
impactful
results.
Introduction
Bacteriophages
are
viruses
that
infect
bacteria.
They
can
undergo
two
different
life
cycles:
lytic
and
lysogenic.
In
the
lytic
cycle,
a
phage
injects
it
DNA
into
the
host
cell,
and
hijacks
its
DNA
manufacturing
resources
in
order
to
create
more
phages.
As
proteins
are
translated,
the
phages
are
assembled,
lyse
the
cell,
and
move
on
to
infect
other
cells.
In
the
lysogenic
cycle,
the
phage
DNA
is
integrated
into
the
host
chromosome,
and
is
replicated
as
the
cell
divides.₃
Phages’
ability
to
kill
bacterial
cells
provide
many
possible
uses,
ranging
from
controlling
food-borne
illness
to
treating
disease
and
infection.
Conclusion
●
Viva
and
Phicolas
are
highly
lytic
siphoviridae
mycobacteriophages,
Viva
belongs
to
the
F
cluster
and
the
F1
subcluster.
This
particular
cluster
is
very
common
in
Colorado.
●
The
clear
plaques
indicate
that
Viva
and
Phicolas
are
lytic
phages
because
there
is
a
distinct
edge
between
the
area
where
bacteria
have
been
cleared
and
the
rest
of
the
bacterial
lawn.
●
Viva
and
Phicolas
are
both
highly
infectious
phage
and
by
having
them
in
the
database
it
helps
contribute
to
the
current
advancements
being
made
in
phage
therapy
today.
●
Potential
for
environmentally-specific
uses,
as
phage
can
adapt
to
severe
environments
like
the
mountains.₂
Future
Directions
●
Determine
the
cluster
of
Phicolas
via
restriction
digestion
analysis
and
PCR
●
Confirm
Viva’s
F1
subcluster
using
PCR
●
Further
narrow
down
specific
uses:
spot
testing
to
find
host
range,
test
environmental
variables
in
the
style
of
Fister
et
al.
(2016).₂
Acknowledgments
This
research
was
made
possible
by
the
HHMI
SEA
Phages
Program,
the
University
of
Colorado
at
Boulder,
Dr.
Nancy
Guild,
Dr.
Christy
Fillman,
Megan
Greening,
David
Engelhardt,
and
Andrew
Folta.
References
1.
Doss
J,
Culbertson
K,
Hahn
D,
Camacho
J,
Barekzi
N.
A
Review
of
Phage
Therapy
against
Bacterial
Pathogens
of
Aquatic
and
Terrestrial
Organisms.
Viruses.
2017.
2.
Fister
S,
Robben
C,
Witte
AK,
Schoder
D,
Wagner
M,
Rossmanith
P.
Influence
of
Environmental
Factors
on
Phage-Bacteria
Interaction
and
on
the
Efficacy
and
Infectivity
of
Phage
P100.
Frontiers
in
Microbiology.
2016;7:1152.
3.
Fillman,
C.
Phage
Genomics
Lab
I
Laboratory
Manual.
Macmillan
Learning
Curriculum
Solutions
.
2018.
P12
Results
Figure
3:
Electron
Microscopy
Both
Viva
and
Phicolas
are
siphoviridae
with
circular
heads
and
long
tails.
Figure
4-
Agarose
Gel
using
Restriction
Enzymes:
This
agar
ose
gel
shows
the
Uncut
DNA
and
fragments
made
by
the
restriction
enzymes.
BamHI,
ClaI,
and
EcoRIl
have
clear
bands
while
HaeIII,
HindIII,
and
SalI
have
smeared
fragments
which
indicates
numerous
of
cuts
that
are
similar
in
fragment
size.
Figure
1:
Phage
Life
cycles:
Lytic
phages
replicate
within
the
cell
and
then
perform
lysis,
while
temperate
phages
integrate
into
the
host
chromosome
and
replicate
with
the
cell.₁
Phicolas
Viva
Phicolas
Viva
Figure
2:
Titer
Assay
Both
VIva
and
Phicolas
have
clear
plaques
indicating
lytic
phages.
Phicolas
produces
4mm
plaques
and
Viva
has
3mm
plaques.
The
titers
of
the
page
lysates
are
6.8
x
10
9
pfu
and
4.6
x
10
9
pfu,
respectively.
Viva
2
High
Titer
:
6.8
x
10
9
Mycobacterium
Morphotype
:
Siphoviridae
Morphology
:
Lytic
(3mm
in
diameter)
Cluster
:
F
Sub-cluster
:
F1
Tail
Length
:
197
-
207
nm
(Long
and
straight)
Tail
Head
:
60nm
-
70nm
(Round
and
average
size)
Archiving
-
Time:
2:10pm
-
City
:
Boulder
-
Temperature
:
7℃
-
GPS
coordinates
:
40.0087
Latitude
-105.2690
Longitude
-
Location
:
Outside
of
Porter
Bioscience
buildings
in
a
dry
patch
in
the
middle
of
the
lawn.
Isolated
from
an
Enrichment
Procedure
Future
Direction:
PCR
to
confirm
cluster
and
sub-cluster
Figure
5
–
Electron
Micrograph
of
Viva:
The
physical
structure
of
the
phage
was
seen
using
electron
microscopy
and
the
long
straight
tails
and
the
circular
heads
are
considered
to
be
characteristics
of
the
lytic
mycobacterium
siphoviridae
family.
F
igure
3
–
20
–
200
Plaque
Plate
that
will
Determine
the
High
Titer:
This
20-200
plate
was
used
to
count
the
plaques
present
and
determined
the
titer
of
our
phage.
This
plate
was
used
because
it
had
the
right
amount
of
plaques
for
determining
phage
concentration.
After
calculation,
our
titer
we
received
a
titer
of
6.8
x
10
9
pfu/mL
Figure
4-
Agarose
Gel
using
Restriction
Enzymes:
This
agarose
gel
shows
the
Uncut
DNA
and
all
the
fragments
that
were
made
by
the
restriction
enzymes
used.
BamHI,
ClaI,
and
EcoRI
while
HaeIII,
HindII,
and
SalI
have
smeared
fragments
which
means
that
they
have
numerous
of
cuts
that
are
similar
in
fragment
size.
Conclusions:
Viva
is
a
highly
lytic
siphoviridae
mycobacteriophage
that
belongs
to
the
F
cluster
and
the
F1
subcluster.
This
particular
cluster
is
very
common
in
Colorado.
3
Phage
Name:
Phicolas
Mycobacterium
Click to add new slide
Phicolas
and
Viva
are
Highly
Lytic
Siphoviridae
Mycobacteriophages,
Viva
Belonging
to
the
F
Cluster
Lauren
Gray,
Erica
Landreth,
Valeria
Rodriguez,
Victoria
Saldivar
Department
of
Molecular,
Cellular,
and
Developmental
Biology,
University
of
Colorado,
Boulder,
Spring
2018
Abstract
Lytic
phages
have
been
explored
at
an
increasing
rate,
but
more
work
is
necessary
to
further
categorize
specific
uses.
As
determined
through
electron
microscopy
and
restriction
digestion
analysis,
Viva
and
Phicolas
are
lytic,
siphoviridae
phages
sampled
in
Colorado,
with
Viva
belonging
to
cluster
F.
With
possible
applications
ranging
from
medicine
to
controlling
food
borne
illness,
further
exploration
of
these
phages’
host
ranges
could
yield
incredibly
impactful
results.
Introduction
Bacteriophages
are
viruses
that
infect
bacteria.
They
can
undergo
two
different
life
cycles:
lytic
and
lysogenic.
In
the
lytic
cycle,
a
phage
injects
it
DNA
into
the
host
cell,
and
hijacks
its
DNA
manufacturing
resources
in
order
to
create
more
phages.
As
proteins
are
translated,
the
phages
are
assembled,
lyse
the
cell,
and
move
on
to
infect
other
cells.
In
the
lysogenic
cycle,
the
phage
DNA
is
integrated
into
the
host
chromosome,
and
is
replicated
as
the
cell
divides.₃
Phages’
ability
to
kill
bacterial
cells
provide
many
possible
uses,
ranging
from
controlling
food-borne
illness
to
treating
disease
and
infection.
Conclusion
●
Viva
and
Phicolas
are
highly
lytic
siphoviridae
mycobacteriophages,
Viva
belongs
to
the
F
cluster
and
the
F1
subcluster.
This
particular
cluster
is
very
common
in
Colorado.
●
The
clear
plaques
indicate
that
Viva
and
Phicolas
are
lytic
phages
because
there
is
a
distinct
edge
between
the
area
where
bacteria
have
been
cleared
and
the
rest
of
the
bacterial
lawn.
●
Viva
and
Phicolas
are
both
highly
infectious
phage
and
by
having
them
in
the
database
it
helps
contribute
to
the
current
advancements
being
made
in
phage
therapy
today.
●
Potential
for
environmentally-specific
uses,
as
phage
can
adapt
to
severe
environments
like
the
mountains.₂
Future
Directions
●
Determine
the
cluster
of
Phicolas
via
restriction
digestion
analysis
and
PCR
●
Confirm
Viva’s
F1
subcluster
using
PCR
●
Further
narrow
down
specific
uses:
spot
testing
to
find
host
range,
test
environmental
variables
in
the
style
of
Fister
et
al.
(2016).₂
Acknowledgments
This
research
was
made
possible
by
the
HHMI
SEA
Phages
Program,
the
University
of
Colorado
at
Boulder,
Dr.
Nancy
Guild,
Dr.
Christy
Fillman,
Megan
Greening,
David
Engelhardt,
and
Andrew
Folta.
References
1.
Doss
J,
Culbertson
K,
Hahn
D,
Camacho
J,
Barekzi
N.
A
Review
of
Phage
Therapy
against
Bacterial
Pathogens
of
Aquatic
and
Terrestrial
Organisms.
Viruses.
2017.
2.
Fister
S,
Robben
C,
Witte
AK,
Schoder
D,
Wagner
M,
Rossmanith
P.
Influence
of
Environmental
Factors
on
Phage-Bacteria
Interaction
and
on
the
Efficacy
and
Infectivity
of
Phage
P100.
Frontiers
in
Microbiology.
2016;7:1152.
3.
Fillman,
C.
Phage
Genomics
Lab
I
Laboratory
Manual.
Macmillan
Learning
Curriculum
Solutions
.
2018.
P12
Results
Figure
3:
Electron
Microscopy
Both
Viva
and
Phicolas
are
siphoviridae
with
circular
heads
and
long
tails.
Figure
4-
Agarose
Gel
using
Restriction
Enzymes:
This
agar
ose
gel
shows
the
Uncut
DNA
and
fragments
made
by
the
restriction
enzymes.
BamHI,
ClaI,
and
EcoRIl
have
clear
bands
while
HaeIII,
HindIII,
and
SalI
have
smeared
fragments
which
indicates
numerous
of
cuts
that
are
similar
in
fragment
size.
Figure
1:
Phage
Life
cycles:
Lytic
phages
replicate
within
the
cell
and
then
perform
lysis,
while
temperate
phages
integrate
into
the
host
chromosome
and
replicate
with
the
cell.₁
Phicolas
Viva
Phicolas
Viva
Figure
2:
Titer
Assay
Both
VIva
and
Phicolas
have
clear
plaques
indicating
lytic
phages.
Phicolas
produces
4mm
plaques
and
Viva
has
3mm
plaques.
The
titers
of
the
page
lysates
are
6.8
x
10
9
pfu
and
4.6
x
10
9
pfu,
respectively.
Viva
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Valeria Rodriguez
varo7480@colorado.edu
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