description of plasmids and types and importance of plasmids and artificial plasmids(PBR322,cosmids,phagemids) and selection of the recombinants and uses and advantages and disadvantages of the plasmids
2. What is?
• A plasmid is a small, circular, double-stranded DNA molecule that is distinct
from a cell's chromosomal DNA It replicates independently of
chromosomal DNA Plasmids naturally exist in bacterial cells, and they also
occur in some eukaryotes.
• plasmids carry genes that are beneficial to the host organisms. Often, the
genes carried in plasmids provide bacteria with genetic advantages, such as
antibiotic resistance. Plasmids have a wide range of lengths, from roughly one
thousand DNA base pairs to hundreds of thousands of base pairs.
• When a bacterium divides, all of the plasmids contained within the cell are
copied such that each daughter cell receives a copy of each plasmid. Bacteria
can also transfer plasmids to one another through a process called conjugation
5. ADVANTAGE
• Scientists have taken advantage of plasmids to use them as tools to
clone, transfer, and manipulate genes. Plasmids that are used
experimentally for these purposes are called vectors. Researchers
can insert DNA fragments or genes into a plasmid vector, creating a
so-called recombinant plasmid. Then, because bacteria divide
rapidly, they can be used as factories to copy DNA fragments in
large quantities. Artificial plasmids are widely used
as vectors in molecular cloning, serving to drive the replication
of recombinant DNA sequences within host organisms. In the
laboratory, plasmids may be introduced into a cell
via transformation.
• Characteristics such as flexibility, versatility, safety, and cost-
effectiveness enable molecular biologists to broadly utilize
plasmids across a wide range of applications. Some common
plasmid types include cloning plasmids, expression plasmids, gene
knock-down plasmids, reporter plasmids, viral plasmids,
and genome engineering plasmids.
6.
7. Types of Plasmids
There are five main types of plasmids:
•fertility F-plasmids
• resistance plasmid
• virulence plasmids
• degradative plasmids
•Col plasmids.
8.
9.
10.
11. ARTIFICIAL PLASMID
• pBR322 is a purpose built plasmid vector and was one of the first
widely used cloning vector it has a relatively small size of 4361
bp. This is important because transformation efficiency is inversly
proportional to size.
Nomenclature of pBR322
• Created in 1977 in the laboratory of Herbert Boyer at the
university of california, san Francisco
‘p’ plasmid
‘BR’ Boliver and Rodriguez (researchers who developed
it)
‘322’ distinguishes those plasmids from others (like
pBR325,pBR327 etc) developed in the same laboratory
Features of pBR322
• pBR322 has 4361 base pairs in length
• It has two antibiotic resistance genes they are:
a)Ampicillin resistance gene(AmpR)
R
12. • Origin of replication(ori)
• It has unique restriction sites for more than 40 restriction enzymes.11
of these 40 sites lie with in the TetR gene. There are 2 sites for
restriction enzymes HindIII and claI with in the promoter of TetR
gene. They are 6 restriction sites inside the AmpR gene.
Construction of pBR322
• The ampR gene originally resided on the plasmid RSF2124(a
naturally occurring antibiotic resistance plasmid in E.coli)
• The tetR is derived from Psc101 (a second antibiotic resistant
13. • The origin of replication is derived from pMB1, which is closely
related to the colicin producing plasmid colE1
INSERTIONAL INACTIVATION
• It is a technique used in recombinant DNA technology to select
recombinant colonies
• It involves the inactivation of a gene up on insertion of another
gene inside in its place or with in its coding sequence
For example-pBR322 is opened by using a restriction enzyme
BamHI where restriction site lies with in the tetracycline resistant
gene. The foreign DNA also isolated with the same type of
restriction enzyme and DNA ligase is added to the mixture
containing linearized pBR322 and the foreign gene two types of
vectors are formed
Recombinant vector foreign gene
Un altered vector foreign gene
15. ampR tetR
Foreign gene
ampR ampRtetR
Insertion
inactivation
Unaltered vector Altered vector
E.coli
Non transformed cell Transformed cells with and with
Out recombinant vector
INTERPRETATION
Non transformed ampS,tetS
T.With nonrecombinant ampR,tetR
T,with recombinant ampR,tetS
Transformation
16. Non transformed cell Transformed cells with and with
Out recombinant vector
Amp
Non transformed cells dead
Transformed cells live
Amp
Transformed cells are transferred
Master plate
tet
Replica plate
Transformed with recombinant dead
Transformed with unaltered live
Compared with the master plate and recombinants are picked
INTERPRETATION
Non transformed ampS,tetS
T.With nonrecombinant ampR,tetR
T,with recombinant ampR,tetS
17. USES
• It is widely used as a cloning vector
• It is used as a model system for study of prokaryotic transcription
and translation
ADVANTAGES
• Due to small size enables easy purification and manipulation
• As due to the presence of 2 selectable markers (amp,tet) allow
easy selection of recombinant DNA
• It can be amplified up to 1000-3000 copies
DISADVANTAGES
• It has very high mobility that is it can move to another cell in the
presence of conjugative plasmid like F-cell.due to this the vector
may lost in the mixed host of cells
• There is a limitation in the size of gene of interest that it can
accommodate
• Screening process is time consuming and laborious
• No high copy number
19. • These are artificially constructed
• Phagemids are the vectors that are made up of a plasmid and phage DNA
Nomenclature of phagemid
Phage part of DNA obtained from bacteriophage
Mid part of DNA obtained from plasmid
Features of phagemid
• It contains 2961bp
• It has ampicillin resistant containing gene
• It has 2 ori genes
• It has Mcs
• It has lacZ gene
• Example – most commonly used phagemids are PBluescript series
Construction of PBluescript
• marker genes plasmid
• Ori plasmid produce dsDNA
F1/M13 phage produce ss DNA
• Mcs plasmid (21 unique restriction sites) inserted in lacZ
• T7 &T3 promoters produce RNA polymerase
20. Some imp characters
• Similarly to a plasmid, a phagemid can be used to clone DNA
fragments and be introduced into a bacterial host by a range of
techniques (transformation, electroporation).
Production of dsDNA
• When a phagemid is introduced in to a host it normally replicates
and produce dsDNA
E.coli
pBluescript II SK (+/-) phagemids
21. Production of ssDNA
• However, infection of a bacterial host containing a phagemid with a
'helper' phage. e.g. VCSM13 or M13K07, provides the necessary viral
components to enable single stranded DNA replication and packaging
of the phagemid DNA into phage particles.
•These are secreted through the cell wall and released into the medium.
Filamentous phage (M13)retard bacterial growth but, in contrast to
lambda and T7 phage, are not generally lytic.
•Helper phage are usually engineered to package less efficiently than the
phagemid so that the resultant phage particles contain predominantly
phagemid DNA. F1 Filamentous phage infection requires the presence of
a pilus so only bacterial hosts containing the F-plasmid or its derivatives
can be used to generate phage particles.
22. Production of 2 diff series (depend on sense + & anti sense –
strand)
• The pBluescript II SK and KS vector series represent two
orientations of the MCS within the lacZ gene encoding the N-
terminal fragment of beta-galactosidase (KS represents the
orientation of the MCS in which lacZ transcription proceeds from
KpnI to SacI, while SK - from SacI to KpnI).
Production of RNA transcripts
• In vitro transcription of T3 and T7 RNA polymerase
pBluescript II SK (+/-) phagemids
23. Screening of recombinants
Interpretation
• The recombinants are screened by growing the cells in a medium
containing IPTG(iso propyl β D1 thio galactopyranoside)and x-gal
• When a foreign gene is inserted in the LacZ the E.coli cells fails to
produce β galactosidase so no hydrolysis of x-gal so the
bacterial colony remains white
LacZ gene produces β galactosidase it hydrolysis the x-gal and IPTG containing medium
and produce blue colour
White recombinants
Blue Non recombinants
24. Uses
pBluescript II phagemids are designed for DNA cloning, dideoxy
DNA sequencing, in vitro mutagenesis and in vitro transcription
in a single system.
Advantages
• It can be used to provide single strand or double stranded material
with out recloning
• Carrying capacity is higher than phage vectors
• Higher efficiency in transformation than phage vectors
• Phagemids are generally more stable than the recombinant phage
vectors
• Screening and storage is easier
26. • These are artificially constructed (hybrid)
• Cosmids are the vectors that are made up of a plasmid and phage DNA
Nomenclature of cosmid
Cos part of DNA containing cohesive sites
Mid part of DNA obtained from plasmid
Construction
Cosmids were first developed by collins and
Hohn in 1978
Features of cosmid
• It can carry about 44 kbp
• It has antibiotic resistant containing gene(selectable marker)
• It has ori gene
• It has Mcs(poly linker)
• One or two cos sites (require for packaging in to phage particle)
• The cos sites allow the vector to be packaged and transmitted to bacteria
like a phage vector
27. • Cloning Steps :-
• Preparation . - Target DNA: the genomic DNA to be cloned has to be cut into the
appropriate size range of restriction fragments , By using Restriction Enzymes , And
Cut the Cosmid Cloning site ( poly linker ) by the same Restriction enzyme , Then
Ligation of physically unlinked fragments.it results in the formation of long
concatamers and these are used efficiently for packing
• Packaging - the total DNA is transferred into an appropriate E. coli host via a
technique called in vitro packaging (recombinant plasmids contained in phage shells
) . - they can also be packaged in phage capsids, which allows the foreign genes to
be transferred into or between cells by transduction (a process resembling viral
infection )
28. • In general when a phage infects a bacteria it infects with high efficiency
and when it enters it gets circularized due to the presence of cos sites and
undergo recombination with the bacterial genome with site directed
integrated manner
• So during cloning of cosmid vector when the difference between cos sites is
about at a distance of 50kb away from each other they could be efficiently
packaged in to the head if the distance is less than <50kb or <30kbp then no
packaging in to the head takes place
29. More distance between cos sites Less distance between cos sites
Transduction : The phage particle with cosmid vector is now inserted in to
the host bacteria that process similar to the infection of viral particle and
when the DNA enters in to the host it gets circularized
30. • Plating & Growing & Selection & Isolation - Colonies are formed in
selective media , just as with a plasmid vector . - The media is : Liquid
media . - So that the transformed cells can be identified by plating on a
medium containing the antibiotic ( Ampicillin Agar plate ). - Those cells
which did not take up the cosmid would be unable to grow.
Uses of Cosmid Vectors:
• Cosmids are used for construction of genomic libraries of eukaryotes since
these can be used for cloning large fragments of DNA.
31. Advantages of cosmids
• Can be used as cloning vectors
• Cosmids are high insert capacity vectors
• Cosmids are used for the analysis of complex genomes
• Large genes can be studied intact and genetic linkage studies can be carried
out at molecular level
• Background molecules which do not have the insert DNA or have the
smaller inserts are eliminated during packaging
• Transformation efficiency of cosmids is high when compared to plasmids
• Easy screening method is found
Disadvantages of cosmids
• Not easy to handle these are large plasmids
• Not stable can acquire mutations
Examples of cosmid vectors
• Commonly used vectors include "SuperCos 1"
