Raspberry Pi 5: Challenges and Solutions in Bringing up an OpenGL/Vulkan Driv...
Cluster classification mycobacteriophage
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 .
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 .
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 .
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 .
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
28.
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/
/
Editor's Notes
Are viruses that infect the micobacterial host Since they are highly diverse, they can be found in the environment including soil. Can cause diseases such as tuberculosis Are used as a model to study biological process such as…
-These are characterized based in their morphology and genome ; using proteomic techniques they can be classified in clusters using genomic sequence comparisons. -Proteome is the complete set of proteins encoded in the genome that is present in all organism and specify their genome. They can be compared to better understand the similarities and differences between phages. -To be classified in clusters they have to share similar characteristics based on their sequence and annotated genomes
Head- DNA Tail- is used to inject the genetic material into the host
Infect the host by attaching to the outer membrane of the bacterial cell. 2) the genetic material is injected into the bacteria Degradation of the host bacterial chromosomal DNA ( eliminate competing instructions from the bacterial genome) Phage replicates 5) phage assembly: the phage genomic DNA is packaged within the mature protein coat 6) Bacterial cell lysis
Used by temperate phage 2) characterized by phage integration into the bacterial chromosome. Steps: 1) the genetic is injected into the bacteria 2) the entering phage genome is confronted with a decision to determine which of two pathways to follow. 3) If the environment conditions are favorable integration occurs. ( the phage inserts its genome into the host chromosome). After this genome is replicated and each dividing cell receive a copy. 4) Unfavorable conditions cause excision ( phage genome exit the host chromosome) followed by the lytic cycle.
Buffer:
Dr. Rubin--- We want to thank Dr. Rubin for his mentoring and for his support during this project Yadira- We want to thank Yadira for being so helpful while doing the project. Rise Program– And also the Rise Program because thanks to opportunities like this we are forming ourselves to become great scientists in the future.
