Bacterialplasmids

2.  Plasmids are extrachromosomal and self
replicating close circular DNA molecule present
the Bacterial cell.
 Plasmids are physically separated from a
chromosomal DNA and can replicate
independently.
in
 Plasmids are some times present in archaea and
eukaryotic organism.

3. Bacterial Plasmids
The term plasmid was first
introduced by
theAmerican molecular
biologist
1952.
Joshua Lederberg in
Aplasmid is a short, usually
circular, and double- stranded
segment of DNA that is found in the
cytoplasm separate from the main
bacterial chromosome.

4. The bacterial chromosome and bacterial plasmids, as shown
in the electron microscope. The plasmids(arrow) are the
circular structures, much smaller than the main
chromosomal DNA.

5. It can be really isolated from the Bacterial
cell.
It possess single restriction site for one or
more restriction enzymes.
Insertion of linear molecule at one of these
site does not alter it’s replication process.
It can be reintroduce into bacterial cells and
cells carry the plasmid.
Not all plasmid exist as circular molecule.
Linear plasmid have been found in a variety
of bacteria. For ex., Streptomyces species
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6. Plasmid features
Plasmid sizes vary from 1 to over 1,000 kbp.
They usually contain 5 to 100 genes
usually carry genes that are useful but
essential to survival: e.g. genes which
bacteria resistant to antibiotics.
and
not
make
As long as the bacterium is thriving in a low-stress
environment, removing all the plasmids would not
affect the ability of the bacterium to survive.
Specifically, plasmids are nonessential,
extrachromosomal pieces of DNA.

7.  Plasmid Replication
Plasmids carry genes that direct their own replication and
additional factors that ensure that the copies get separated
into the new daughter cells. This ensures that the plasmids
are not lost from the cells during binary fission.

8. Classification Of Plasmids.
I.Based on their ability to transfer
bacteria.
to other
a) Conjugative plasmids - contain tra genes,
which perform the complex process of sexual
conjugation, the transfer of plasmids to another
bacterium or different cells.
These are high molecular weight plasmid and
present at one to 3 copies per chromosome.
e.g., F plasmid, many R plasmid & some Col
plasmid.

9. b) Non-conjugative plasmids - incapable of
initiating conjugation, hence they can be
transferred only with the assistance of
conjugative plasmids. These are low molecular
weight plasmid and present in multiple copies
per chromosome.
e. g., many R plasmid & most Col plsmid.
c) Mobilisable plasmid - An intermediate
class of plasmid. They carry only a subset of
the genes required for transfer. They can
parasitize another plasmid, transferring at
high frequency in the presence of a
conjugative plasmid.

10. II.Based on
function.
a) Degradative plasmids – They are able to
digest unusual substances like toluene and
salicylic acid.
e.g.,TOL plasmid of Psedomonas putida.
b) Virulence plasmids – contains vir genes
which turn the bacterium into a pathogen.
e. g., Ti & Ri plasmids
c) Fertility (F)-plasmids - contain tra genes.
They are capable of conjugation and result
in the expression of sex pilli.
Example: F plasmid of E. coli.

11. d) Resistance (R)plasmids – contain
genes that provide resistance
against antibiotics or poisons.
Historically known as R-factors, before
the nature of plasmids was understood.
e. g., pRP4 of Pseudomonas sp.
e) Col plasmids - contain genes that code
for bacteriocins & toxins that can kill other
bacteria.
e. g., ColE1

12. III. Based on copy number.
a) Stringent Plasmid – It replicates only
along with the main bacterial
chromosome & is present as a single
copy, or at most several copies, per cell.
a) Relaxed Plasmid – It replicates within a
cell independently of the chromosomal
DNAreplication. Thus multiple copies of
plasmids are present.

13. IV.Based on compatibility
It is possible for plasmids of different types to
coexist in a single cell. Several different
plasmids have been found in E. coli. However,
related plasmids are often incompatible, in the
sense that only one of them survives in the
cell line, due to the regulation of vital plasmid
functions. Thus, plasmids can be assigned
into incompatibility groups.

14.  ColE1 is a col plasmid found in bacteria.
 These plasmids belongs to family of
Bacteriocins.

15.  The
and
 The
mass of the plasmid is 4,200 kilo daltons
it’s length is 6,646 bp
cell will contain 10 to 15 copies of this
plasmid and therefore is considered to be
present in high copy number.
 Copy number is a function of the rate at
which DNA synthesis is initiated.
 The plasmid are replicate unidirectionally.

16.  It’s not self transmissible meaning that it
cannot initiate and complete the transmission
into a recipient cell.
 Because, it lacks transfer apparatus, such as
mob gene for mobility and bom sequence
(basis of mobility) and physical requirements
like pilli.
 This plasmid produce colicin E1.
 It’s non conjugative plasmid

17.  The plasmids containing the trp operon
(ColEl-trp) and the kanamycin resistance
gene were maintained under logarithmic
growth conditions at a level of 25-30 copies
per cell and accumulate to the extent of
several hundred copies per cell in the
presence of chloramphenicol.

18. ColE1 plasmid as a vehicle for the molecular
cloning of the tryptophan operon.
Cells carrying the colE1 trp plasmid determined
the production of highly elevated levels of trp
operon – specific mRNA and tryptophan
biosynthetic enzymes.
In normal condition produce 24 copies of colE1
in cell
After addition of chloramphenicol the
chromosome stops the replication and after a
short period of time. Whereas colE1 DNA
synthesis continues for 12-16hr and form
1000-3000 copies of plasmid per cell.
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19.  Sv 40 is a simian vacuolating 40 or simian
virus 40.
 It is a polyomavirus that is found in both
monkeys and humans.
 It’s non enveloped small DNA virus whose
capsid is composed of major capsid protein
Vp1 and minor capsid protein Vp2 and Vp3

20.  Sv 40 can not replicate autonomously if the
replication origin is defective.
 It can integrate into chromosomal locations
of green monkey cells and can then be
replicated along the chromosomal DNA
 Sv 40 plasmids can be packaged only if their
DNA is within the range of 3900 to 5300bp.

21.  Sv 40 was used to transduce gene expression
in inside a living
culture(in vivo)
 The Sv 40 genome
fragment into PBR
 Ex., pBSV -8His –
organism (in vitro) and in cell
had been cloned as a Bam HI
322 make pBSV plasmid
baculovirus expression vector used
for secretion of Histidine – tagged protein

22.  Viruses have been used as cloning vectors for
higher organism for specialized applications.
 Human adeno viruses are used in gene
therapy.
 Baculoviruses are used to synthesize
important pharmaceutical proteins in insect
cells.
 Caulimo viruses and gemini viruses have
been used for cloning in plants.

23. PMB 9 is a derived plasmid created by
Moore, July 1995.
 It is circular DNA, and it’s length is 5295 bp.
 PMB 9 plasmid is similar to col E 1 plasmid in
it’s replication. But it’s not produce colicin E1
protein`
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24.  EcoRI-HindIII fragment of pMB9 provides for
efficient transcription of the uvrB gene.
 The plasmid pMB 9, derived from pMB 1
contains the kil gene which responsilble
colicin E1 release by cell lysis.
 pMB 9 is important role in excretion of
penicillinase.
for

25. Uses of plasmids
Plasmids serve as important tools in genetics and
biotechnology labs, where they are commonly used
to multiply (make many copies of) or express
particular genes.
Disease Models - Plasmids were historically used
to genetically engineer the embryonic stem cells of
rats in order to create rat genetic disease models.

26. Gene therapy- plasmid vectors are used for
the insertion of therapeutic genes at pre-
selected chromosomal target sites within the
human genome.
Another major use of plasmids is to make
large amounts of proteins. In this case,
researchers grow bacteria containing a plasmid
harboring the gene of interest.
eg: insulin & antibiotics.