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/