plasmids history properties classification ColE1 plasmid characteristics replication regulation application Sv 40 plasmid history properties applications PMB 9 Plasmid construction application

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

3.  1952 – Joshua Lederberg suggests name
‘Plasmid ’
 1962 – plasmids are circular DNA molecule.

4.  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

5.  Plasmids can be broadly classified into two
groups depending upon the whether or not they
carry the set of transfer genes called tra genes.
 Conjugative plasmids
 Non-conjugative plasmids
 Conjugative plasmids contain a set of transfer
or tra genes which promote sexual conjugation
between different cells.
 It’s high molecular weight plasmid and present
at one to 3 copies per chromosome.

6.  Non-conjugative plasmids are incapable of
initiating conjugation. Hence they can be
transferred only with the assistance of
conjugative plasmids.
 These plasmids are low molecular weight
and present at multiple copies per
chromosome.
 Based on copy number:
 Stringent plasmid – 1 to 3 copies of plasmid
per cell
 Relaxed plasmid – several copies of plasmids
per cell

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

8.  The mass of the plasmid is 4,200 kilo daltons
and it’s length is 6,646 bp
 The 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.

9.  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

10.  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.

11.  RNA I, RNA II, and Rom (Rop protein)
 RNA I is a small inhibitory RNA. That bind to
RNA II will act as a primer for DNA replication.
 Rom plasmid encoded protein. Which
stabilizes the RNA I - RNA II complex

13.  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.

14.  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

15.  It was first idendified by Ben Sweet and
Maurice Hilleman in 1960.
 In 1962 Bernice E. Eddy described the sv 40
oncogenic function including sarcoma
 The complete viral genome was sequenced by
walter and fiers in 1978

17.  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.

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

19.  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.

20.  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`

21.  The EcoRI-HindIII fragment of plasmid pMB9
originally derived from plasmid pSC1O1.
 This fragment consist of 354 basepair and
it’s contains a promoter for transcription.
 This plasmid containing an ampicillin
resistant gene constructed from P2124.
 The plasmid has three restriction site for the
EcoR1, HindIII, Bam HI, Sal I restriction
endonuclease.

22.  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 for
colicin E1 release by cell lysis.
 pMB 9 is important role in excretion of
penicillinase.