This document describes a study that aimed to classify mycobacteriophages isolated from soils in Puerto Rico into clusters based on their genomes. Several mycobacteriophages were tested using PCR with cluster-specific primers and gel electrophoresis. Phagius_Maximus was classified into Cluster B2, while Colbert and Puhltonio were classified into Cluster B1. Ghost and LRR Hood were classified into Cluster C1, and Pumpkin was classified into Cluster E. Further experiments are needed to classify Suave, Bloo, and Wilie using additional cluster-specific primers. The results provide insights into mycobacteriophage evolution and classification.

1. C lu s te r C las s ification of
M ycob acte riop h age s Is olate d
F rom Trop ical S oils of P u e rto
R ico
N icole C olón
Alb e rto C intrón
C arolina Montañe z
Lu z Marie R e ye s
D r. Mich ae l R u b in
R IS E P rogram , U nive rs ity of P u e rto R ico at C aye y
h ttp :/ p h age s d b .org/
/

2. Introd u ction
• W h a t a r e M y c o b a c t e r io p h a g e s ?
- Viru s e s th at infe ct th e M ycob acte rial h os t.
• Wh e re c a n th e y b e fo u n d ?
-Th e y can b e fou nd in th e e nvironm e nt inclu d ing s oil.
• 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 ?
-Th e y cau s e d is e as e s s u ch as tu b e rcu los 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 fo r?
-Th e y can b e u s e d to infe ct and kill m ycob acte ria.
(Rubin, M. 2012), (Simmons, M., Snustad, P. 2012).

3. Introd u ction
• How a re
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 ?
-Th e y are ch aracte rize d b as e d on th e ir
ge nom e .
• C la s s if ic a t io n in C lu s t e r s
-Th e y s h are ch aracte ris tics b as e d on
s e qu e nce and annotate d ge nom e s .
• P r o t e o m ic s
-S tu d y of p rote in s tru ctu re and fu nction.
-C an als o b e u s e d to clas s ify p h age s .
(Rubin, M. 2012).

4. Bacte riop h age S tru ctu re
D NA
H e ad
Tail
(Rubin, M. 2012).

5. Lytic C ycle
Is u s e d b y viru le nt p h age s .
Is ch aracte rize d b y p h age
re p rod u ction followe d b y h os t ce ll
lys is .
S tage s inclu d e :
1. Ad s orp tion
2. E ntry
3. R e p lication
4. G e ne E xp re s s ion
5. As s e m b ly
(Simmons, M., Snustad, P. 2012)
6. Lys is

6. Lys oge nic cycle
•Is ch aracte rize d b y
th re e p roce s s e s :
-Inte gration of
p h age D N A into
h os t ge nom e .
-E xcis ion of
p rop h age .
-E ntry into th e
lytic cycle .
(Simmons, M., Snustad, P. 2012)

7. O b j ctive s
e
• To analyze d iffe re nt u ns e qu e nce d
m ycob acte riop h age s and clas s ify th e m
into th e ir re s p e ctive clu s te rs u s ing P C R
and G e l E le ctrop h ore s is .
• To p rovid e ge nu ine re s e arch e xp e rie nce
for u nd e rgrad u ate s tu d e nts .

8. P rob le m and H yp oth e s is
• P r o b l e m : C an we clas s ify
M ycob acte riop h age s u s ing clu s te r s p e cific
P C R p rim e rs ?
• H y p o t h e s i s : M ycob acte riop h age s
will b e clas s ifie d wh e n clu s te r s p e cific
p rim e rs am p lify a P C R p rod u ct of th e
e xp e cte d s ize .

9. M ycob acte riop h age C lu s te rs M ycob acte riop h age C lu s te rs
In P h age s d b With PC R P rim e rs
P ha g e /
C l uA1 t e r
s
A2
B1
B2
B3
C1
C2
D
E
F1
H1
H2
I

10. M ate rials and M e th od s
Add P la c e P C R P re p a ra ti
R e a g e nts T u b e s in t h e on of Gel
to P C R T h e r m o c y c le r a nd

11. M ate rials and M e th od s
A d d L o a d in g Load
P r e p a r a t io D ye to P C R W e lls
n of R e a c t io n s w it h P C R
Ag a ros e R e a c t io n
Gel s

12. M ate rials and M e th od s
Run G e l a t 8 0 P ho to g r A n a ly z e
v o lt s aph Gel R e s u lt s
P h o to p ro vid e d
b y:e cs .u m as s .e d u

13. R e s u lts
C las s (G e l E xp e rim e nt C ontrol
#4) al

14. C ontrol G e l
M B1 C1 M E M
800 bp
700 bp
400 bp
http://phagesdb.org/

15. C onclu s ions
• Am p lification of C olb e rt and P u h ltonio
ge nom ic D N As re s u lte d as b e longing to
C lu s te r B1 .
• Am p lification of G h os t and LR R H ood
ge nom ic D N As re s u lte d as b e longing to
C lu s te r C 1 .
• Am p lification of P u m p kin ge nom ic D N A
re s u lte d as b e longing to C lu s te r E .

16. P h agiu s _ M axim u s G e l

17. C onclu s ion
• Am p lification of P h agiu s _ M axim u s
ge nom ic D N A re s u lte d in a P C R p rod u ct
u s ing B2 clu s te r s p e cific p rim e rs .

18. S u ave G e l

19. C onclu s ions
• Am p lification of S u ave ge nom ic D N A d id
not re s u lt in a P C R p rod u ct u s ing any
clu s te r s p e cific p rim e rs .
• We conclu d e th at S u ave d oe s not b e long
to any of th e clu s te rs we te s te d with ou r
colle ction of clu s te r s p e cific p rim e rs .
• F u rth e r e xp e rim e nts are ne e d e d with
P C R p rim e rs for oth e r clu s te rs .

20. Bloo G e l

21. C onclu s ions
• Am p lification of Bloo ge nom ic D N A d id
not re s u lt in a P C R p rod u ct u s ing any
clu s te r s p e cific p rim e rs .
• We conclu d e th at Bloo d oe s not b e long
to any of th e clu s te rs we te s te d with ou r
colle ction of clu s te r s p e cific p rim e rs .
• F u rth e r e xp e rim e nts are ne e d e d with
P C R p rim e rs for oth e r clu s te rs .

22. Wilie G e l

23. C onclu s ions
• Am p lification of Wilie ge nom ic D N A d id
not re s u lt in a P C R p rod u ct u s ing any
clu s te r s p e cific p rim e rs .
• We re com m e nd p re p aring p h age s from
Wilie with gre ate r am ou nt of ge nom ic
D N A.
• F u rth e r e xp e rim e nts are ne e d e d with
P C R p rim e rs for oth e r clu s te rs .

24. S u m m ary of C onclu s ions
C o ntro l S iz e in B a s e M yc o b a c te r i C lu s t e r
M y c o b a c t e r io P a ir s opha g e s
pha g e s
P u h ltonio (C lu s te r B1 ) 700 P h agiu s _ M axim u s B2
C olb e rt (C lu s te r B1 ) 700
S u ave n/d
G h os t (C lu s te r C 1 ) 400
Bloo n/d
LR R H ood (C lu s te r 400
C1 )
Wilie n/d
P u m p kin (C lu s te r E ) 800
N/d = Not Determined

25. F u tu re D ire ctions
• U s e clu s te r s p e cific p rim e rs to clas s ify th e
m ycob acte riop h age s is olate d from P u e rto R ico.
• D e s ign ad d itional clu s te r s p e cific p rim e rs for
clu s te rs J - Q .
• P re p are ad d itional D N A from
m ycob acte riop h age Wilie and re p e at th e P C R
e xp e rim e nts u s ing th e clu s te r s p e cific p rim e rs .
• C alcu late th e e xp e cte d s ize s of all clu s te r
s p e cific am p lifie d P C R p rod u cts .

26. R e fe re nce s
• H atfu ll, G rah am F ., C re s awn, S te ve n E ., H e nd rix, R oge r,
W. 2008. C om p arative G e nom ics of th e
M ycob acte riop h age s : Ins igh ts into Bacte riop h age
E volu tion. R e s e arch in M icrob iology Volu m e 1 59, Is s u e 5.
P . 332-339.
• R os s , R ob e rt. 201 2. G e ne ral Botany S tu d y G u id e .
D e p artm e nt of Biology U P R C aye y. P u e rto R ico p p xxvii,
xxviii, xxix.
• R u b in. M , 201 2. E xp e rim e ntal C las s ification of
M ycob acte riop h age s : Th e ore tical Backgrou nd on Im p ortant
C once p ts and Te ch niqu e s .
• S im m ons , M ich ae l J., S nu s tad , D . P e te r. 201 2. P rincip le s of
G e ne tics . Joh n Wile y & S ons , Inc. N e w Je rs e y p p . 1 65,
1 67, 1 68.

27. Acknowle d gm e nts
• D r. M ich ae l R u b in
• Yad ira O rtiz
• R IS E P rogram
-D ra. E ne id a D íaz
-D ra. E le na
G onz ále z
-D r. R ob e rt R os s
-M e lis a M e d ina
-Vale ria R ive ra

29. C lu s te r C las s ification of
M ycob acte riop h age s
Is olate d F rom Trop ical
S oils of P u e rto R ico
N icole C olón
Alb e rto C intrón
C arolina M ontañe z
Lu z M arie R e ye s
M ich ae l R u b in
RISE Program, University of Puerto Rico at Cayey
h ttp :/ p h age s d b .org/
/