cluster classification of mycobacteriophages isolated from tropical soils of puerto rico...
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C luste r C lass ification of C luste r C lass ification of
Mycobacte riophage s Isolate d Mycobacte riophage s Isolate d
From Trop ical S oils of Pue rto From Trop ical S oils of Pue rto
R icoR ico
N icole C ol nóAlbe rto C intr nó
C arolina Monta e zñLuz Marie R e ye s
D r. Michae l Rub in
R IS E Program , U nive rs ity of Pue rto R ico at C aye y
http ://phage s d b .org/
Introd uctionIntrod uction• W h a t a r e M y c o b a c t e r io p h a g e s ?
- Viruse s that infe ct the Mycobacte rial host.
• W h e r e c a n t h e y b e f o u n d ?-The y can be found in the e nvironm e nt includ ing so il.
• W h a t d is e a s e s c a n M y c o b a c t e r ia c a u s e ?-The y cause d is e ase s such as tube rculos is .
• W h a t a r e M y c o b a c t e r io p h a g e s u s e d f o r ?-The y can be us e d to infe ct and kill m ycobacte ria.
-The y are use d as a m od e l to s tud y b iological
proce sse s .
(Rubin, M. 2012), (Simmons, M., Snustad, P. 2012).
• H o w a r e M y c o b a c t e r io p h a g e s c h a r a c t e r iz e d ?-The y are characte rize d base d on the ir
ge nom e .
• C la s s i f ic a t io n in C lu s t e r s-The y share characte ristics base d on
se que nce and annotate d ge nom e s .
• P r o t e o m ic s-S tud y of prote in s tructure and function.
-C an also be use d to class ify phage s .
Introd uctionIntrod uction
(Rubin, M. 2012).
Bacte riophage S tructure
D NA
H e ad
Tail
(Rubin, M. 2012).
Lytic C ycle
Is u se d by virule nt phage s .
Is characte rize d by phage
re prod uction followe d by host ce ll
lys is .
S tage s includ e :
1. Ad sorption
2. E ntry
3. R e plication
4. G e ne E xpre ss ion
5. Asse m bly
6. Lys is (Simmons, M., Snustad, P. 2012)
Lysoge nic cycle
•Is characte rize d by
thre e proce sse s :
-Inte gration of
phage D NA into
host ge nom e .
-E xcis ion of
prophage .
-E ntry into the
lytic cycle .
(Simmons, M., Snustad, P. 2012)
O bje ctive sO bje ctive s
• To analyze d iffe re nt unse que nce d
m ycobacte riophage s and class ify the m
into the ir re spe ctive cluste rs us ing PC R
and G e l E le ctrophore s is .
• To provid e ge nu ine re se arch e xpe rie nce
for und e rgrad uate s tud e nts .
Proble m and H ypothe s is
• P r o b le m : C an we class ify
Mycobacte riophage s us ing cluste r spe cific
PC R prim e rs?
• H y p o t h e s is : Mycobacte riophage s
will be class ifie d whe n cluste r spe cific
prim e rs am plify a PC R prod uct of the
e xpe cte d s ize .
P h a g e / C lu s t e r
A1
A2
B1
B2
B3
C 1
C 2
D
E
F1
H 1
H 2
I
Mycobacte riophage C luste rs
In Phage sd b
Mycobacte riophage C luste rs
With PC R Prim e rs
Mate rials and Me thod s Mate rials and Me thod s
A d d R e a g e n t s
t o P C R T u b e s
P la c e P C R T u b e s in t h e T h e r m o c y c le r
P r e p a r a t io n o f G e l
a n d B u f f e r
A d d L o a d in g D y e t o P C R R e a c t io n s
L o a d W e l l s
w it h P C R R e a c t io n
s
P r e p a r a t ion o f
A g a r o s e G e l
Mate rials and Me thod s Mate rials and Me thod s
R u n G e l a t 8 0 v o l t s
Photo provid e d
by:e cs .um ass .e d u
P h o t o g ra p h G e l
A n a ly z e R e s u l t s
Mate rials and Me thod s Mate rials and Me thod s
C lass (G e l
#4)
E xpe rim e nt
al
C ontrol
R e sultsR e sults
C ontrol G e l
M B1 C1 M E M
http://phagesdb.org/
700 bp
400 bp
800 bp
C onclus ionsC onclus ions
• Am plification of C olbe rt and Puhltonio
ge nom ic D NAs re sulte d as be longing to
C luste r B1 .
• Am plification of G host and LR R H ood
ge nom ic D NAs re sulte d as be longing to
C luste r C 1 .
• Am plification of Pum pkin ge nom ic D NA
re sulte d as be longing to C luste r E .
Phagius_ Maxim us G e l
C onclus ionC onclus ion
• Am plification of Phagius_ Maxim us
ge nom ic D NA re sulte d in a PC R prod uct
us ing B2 cluste r spe cific prim e rs .
S uave G e l
C onclus ionsC onclus ions
• Am plification of S uave ge nom ic D NA d id
not re sult in a PC R prod uct us ing any
cluste r spe cific prim e rs .
• We conclud e that S uave d oe s not be long
to any of the cluste rs we te ste d with our
colle ction of cluste r spe cific prim e rs .
• Furthe r e xpe rim e nts are ne e d e d with
PC R prim e rs for othe r cluste rs .
Bloo G e l
C onclus ionsC onclus ions
• Am plification of Bloo ge nom ic D N A d id
not re sult in a PC R prod uct us ing any
cluste r spe cific prim e rs .
• We conclud e that Bloo d oe s not be long
to any of the cluste rs we te ste d with our
colle ction of cluste r spe cific prim e rs .
• Furthe r e xpe rim e nts are ne e d e d with
PC R prim e rs for othe r cluste rs .
Wilie G e l
C onclus ionsC onclus ions
• Am plification of Wilie ge nom ic D NA d id
not re sult in a PC R prod uct us ing any
cluste r spe cific prim e rs .
• We re com m e nd pre paring phage s from
Wilie with gre ate r am ount of ge nom ic
D N A.
• Furthe r e xpe rim e nts are ne e d e d with
PC R prim e rs for othe r cluste rs .
S um m ary of C onclus ionsS um m ary of C onclus ions
M y c o b a c t e r io p h a g e s
C lu s t e r
Phagius_ Maxim us B2
S uave n/d
Bloo n/d
Wilie n/d
N/d = Not Determined
C o n t r o l M y c o b a c t e r iop h a g e s
S iz e in B a s e P a ir s
Puhltonio (C luste r B1 ) 700
C olbe rt (C lus te r B1 ) 700
G host (C luste r C 1 ) 400
LR R H ood (C luste r
C 1 )
400
Pum pkin (C luste r E ) 800
Future D ire ctions
• U se cluste r spe cific prim e rs to class ify the
m ycobacte riophage s isolate d from Pue rto R ico.
• D e s ign ad d itional cluste r spe cific prim e rs for
cluste rs J - Q .
• Pre pare ad d itional D NA from
m ycobacte riophage Wilie and re pe at the PC R
e xpe rim e nts u s ing the cluste r spe cific prim e rs .
• C alculate the e xpe cte d s ize s of all cluste r
spe cific am plifie d PC R prod ucts .
R e fe re nce s
• H atfull, G raham F ., C re sawn, S te ve n E ., H e nd rix, Roge r,
W. 2008. C om parative G e nom ics of the
Mycobacte riophage s : Ins ights into Bacte riophage
E volution. R e se arch in Microb iology Volum e 1 59, Is sue 5.
P . 332-339.
• Ross , Robe rt. 201 2. G e ne ral Botany S tud y G u id e .
D e partm e nt of Biology U PR C aye y. Pue rto R ico pp xxvii,
xxviii, xxix.
• Rub in. M, 201 2. E xpe rim e ntal C lass ification of
Mycobacte riophage s : The ore tical Background on Im portant
C once pts and Te chnique s .
• S im m ons , Michae l J., S nustad , D . Pe te r. 201 2. Principle s of
G e ne tics . John Wile y & S ons , Inc. N e w Je rse y pp . 1 65,
1 67, 1 68.
Acknowle d gm e nts
• D r. Michae l R ub in
• Yad ira O rtiz
• R IS E Program
-D ra. E ne id a D azí-D ra. E le na
G onz le zá-D r. R obe rt R oss
-Me lisa Me d ina
-Vale ria R ive ra
C luste r C lass ification of C luste r C lass ification of
Mycobacte riophage s Mycobacte riophage s
Isolate d From Trop ical Isolate d From Trop ical
S oils of Pue rto R icoS oils of Pue rto R icoN icole C ol nó
Albe rto C intr nóC arolina Monta e zñLuz Marie R e ye s
Michae l Rub in
RISE Program, University of Puerto Rico at Cayeyhttp ://phage s d b .org/