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Bacterial plasmids
1.
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
12.
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
16.
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.
Notas do Editor
Conjucative plasmids carry the tra and mob region
It’s non conjucative plasmid
Chloramphenicol stops the protein synthesis. but replication is those on.
An origin of replication is a sequence of DNA at which replication is initiated on a chromosome. The origin of replication determines the vector copy number. The origin of replication also determines the plasmid's compatibility
since the early genic region is normal
baculovirus expression vector (pBSV-8His) directs secretion of recombinant proteins into the culture medium of infected insect cells.