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Plasmids and Vectors

DNA analysis single stranded DNA vector Plasmids Phage

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Askarinazz
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12 views

Plasmids and Vectors

DNA analysis single stranded DNA vector Plasmids Phage

Uploaded by

Askarinazz
Copyright
© © All Rights Reserved
Available Formats
Download as PPT, PDF, TXT or read online on Scribd
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Lecture-2

Plasmids and Vectors

MBBT–26605 4(3-1)
Main contents

1. Plasmid

2. λ Phage

3. Single strand DNA vectors


1. Plasmid
Introduction to vector

A self-replicating DNA molecule that


transfers a DNA segment between host
cells.

Vectors are ships for


carrying the target DNA
into a host cell.
Types of plasmids

1. F plasmid: (confer conjugative property to host


bacterium)
2. R plasmid: (confer resistant against particular
antibiotic to host bacterium)
3. Col plasmid: (produce colicine protein, which kill
the other strain of bacteria)
4. Vir plasmid: (converts bacteria into a pathogen)
5. Degradative plasmid: (provide bacteria a
capability to degrade certain chemicals like toluene
and salicylic acid)
Essential parts of all vectors

 A cloning site (multi-clone site)


 A replicator (origin of replication)
 A selectable marker
Plasmid

SacⅠ SmaⅠ XbaⅠ PstⅠ HindⅢ


agtgaattCGAGCTCGGTACCCGGGGATCCTCTAGAGTCGACCTGCAGGCATGCAAGCTTGGcgtaatcatggtcat

EcoRⅠ KpnⅠ BamHⅠ SalⅠ SphⅠ

LacZ

pUC19
2686 bp Ori
AmpR
lacZ
Plasmid properties occur in
bacteria

(a) Be double-stranded, circular DNA molecules


Distinct from the bacterial chromosome
(b)
Be extrachromosomal structures
Replicate autonomously (independently).

(c) Size range: less than 1 to more than 500 kb.


(d) Has a sequence that functions as an origin of
replication (ori).
(e) Has at least one selectable genetic maker
(a gene for antibiotic resistance).

(f) Has a polylinker site (multiple cloning site)


(a cluster of restriction endonuclease recognition sites).

(g) E. coli lacZ gene: α-galactosidase


target DNA inserted without insertion

inactivates the lacZ gene lacZ gene function normally

white colonies blue colonies


Keep in mind
Many different The distribution of any
types of plasmids have plasmid is natural.
been found in bacteria.
 Vectors carry genes the drug-resistant phenotype
for drug resistance. can be used to select for
transformed cells
Plasmids are an
efficient means of there are many copies per cell,
amplifying DNA (as many as several hundred
for some plasmids.)
4.1.3 Host range of plasmids

is determined by its ori region.


e.g

Plasmids whose ori region is derived


from plasmid Col E1 have a restricted
host range:
they only replicate in enteric bacteria,
such as E. coli, Salmonella, etc
Partitioning and segregative stability of plasmids

Due to defective partitioning, plasmids may loss,


which is called segregative instability
may happened for recombinant vector

Naturally occurring plasmids are stably maintained


contain a par gene

of partitioning function


ensures the plasmids are
stably maintained at each cell
division.
What are these par genes?
• HOK/SOK genes are called par genes.
HOK proteins when expressed kill the bacterium.
SOK is an unstable antisense mRNA and is not
translated into protein.
When HOK and SOK genes both present in the
plasmid, HOK proteins will not be formed and
bacteria will survive.
B/C mRNA of both HOK and SOK genes will pair
each other to form double strained mRNA which
will be chopped down by the RNAase enzyme.
• But if SOK gene not present (defective
partitioning) the HOK proteins will be translated
at ease and kill the bacteria.

• So these par genes ensure the plasmids are


stably maintained at each cell division.
Desirable properties of cloning plasmid
An ideal cloning vehicle has following properties:
Low molecular weight
facilitates
is much easier to handle their
usually present as multiple copies isolation
Ability to confer readily selectable
phenotypic traits on host cells

Has single site for a large number of


restriction endonucleases
Choosing a cloning vector
Ideal one is a small one, facilitating manipulation.
Capable of prolific replication in a living cell, to
enable the amplification of the inserted fragment.

Have convenient restriction sites to make the


inserted DNA be cloned.
Unique sites target DNA to one site in the
vector.
Have a method to identify and recover the
recombinant molecule easily.
NEXT LECTURE

• λ Phage

• Single strand DNA vectors

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