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Vectors cont..
Dr. Dinithi Peiris
Dept. of Zoology
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Pattern of Infection
Lytic cycle
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Pattern of Infection
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Question
•  What is the unique feature in this life
cycle
– Phages causes lysis & cell death of
the host cell
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Lysogenic cycle
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Question
•  What is the unique feature in this life
cycle
– Prophage DNA incorporated in host
DNA
– Phage conversion
– Specialized transduction
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Types of Bacteriophages
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Lambda Bacteriophage
•  Lambda genome is approximately 49 kb
in length.
•  Many types but 2 are used
•  λ (Lambda)
•  Only 30 kb is required for lytic growth.
•  Thus, one could clone 19 kb of foreign
DNA.
•  M13
•  Packaging efficiency 78%-100% of the
lambda genome.
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Cosmid cloning vectors
Lambda cloning vectors
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COS site: Cohesive
sticky ends
Protein capsule result in
Lysis
tight constraint of
Replication
amount of DNA fit inside ori
it (~ 55kb)
It is known that a good
portion of lambda wasLysogeny
not required= Junk
DNA
Head
Tail
Circularized
lambda
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Lambda cloning vectors
the nonessential parts of
lambda
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• Filamentous
¡  Eliminate
Lysis
Replication
ori
COS
Head
• ss-circular DNA (size 6407 bp)
• DNA enter in to cell converted to double
Tail
stranded molecule known as replicative form or
¡  Can
now insert large
pieces of DNA (~ 20 kb)
RF.
• Replicates until there are about 100 copies in the
cell.
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How M13 infects and reproduces
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Uses
infects through “Pillus”
Protein coat is stripped and ss DNA is
converted to double stranded replicative form
DNA replicated by “rolling circle method”
New particles assembled
200 particles per infected cell per generation
M13 released without lysis
No lysis on bacterial lawn, generally do in
liquid culture.
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Cloning
Ss DNA for probes, sequencing
Phage display technology, M13 will
produce foreign protein on surface as part
of its protein coat, can use to generate
specific antibodies
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Advantages of using
Bacteriophages
Question…
• As a cloning vector used for in vitro recombination
• Its molecular genetics is well known
•  Do you remember how bacteria protect
themselves from bacteriophages (foreign
DNA)?
• DNA is efficiently packed into phage particles
• They have restriction enzymes to cut
them up!
• Larger insert sites
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Cosmid cloning vectors
l 
Fragments from 30 to
46 kb can be
accommodated by a
cosmid vector.
Hybrid vector: plasmid
vector and
bacteriophage lambda
vector.
l  cos ends.
Cosmid Cloning Vectors
l  DNA
that can insert in to cosmid vectors
are limited by the amount that fit into the
capsule.
ori
TetR
21.5 kb
cos
l  Somewhat
l 
EcoRI
usable but difficult to maintain
Cos site is the only
requirement for
packaging into
phage particle
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Cosmid cloning vectors
Other vectors
l 
Cosmids can infect bacteria & replicate in it.
l 
Infected cells grow in to normal cells.
l 
Cosmids are extracted from bacteria and mixed
with restriction endonucleases.
¡  BACs
(Bacterial artificial chromosomes)
l  Large
low copy number plasmids (have ori
and selectable marker)
l  Can be electroporated into E. coli
l  Useful for sequencing genomes, because
insert size 100 - 300kb
Cleaved cosmids are mixed with foreign DNA that
has been cleaved with the same endonucleases.
l  .
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l 
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Other vectors
Vectors
THERE IS NO PERFECT VECTOR
¡ 
¡ 
YAC (Yeast Artificial Chromosome)
l  Can be grown in E.coli and Yeast
l  Miniature chromosome (contains ori, selectable
markers, two telomeres, and a centromere
l  Can accept 200 kb -1000 kb; useful for sequencing
Ti plasmids; to introduce genes into plants
As much as we try to combine the properties
of several vector systems, e.g. λ, there is
now no vector that can combine all
properties in one molecule. Thus a fine
tuning process is necessary to optimize
parameters.
THE HISTORY OF VECTOR DESIGN IS A BALANCE
BETWEEN COMPETING OBJECTIVES!!
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How is Foreign DNA Inserted
in to Cells
n 
n 
n 
To open up the DNA a restriction
enzyme is used.
How is Foreign DNA Inserted
in to Cells
n 
Cut the DNA at a specific place called a
restriction site
n 
The result is a set of double-stranded
DNA pieces with single-stranded ends
n 
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These ends that jut out are not only "sticky" but
they have gaps that can be now be filled with a
piece of foreign DNA
For DNA from an outside source to bond with an
original fragment, one more enzyme is needed
DNA ligase seals any breaks in the DNA
molecule
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