Bacterial plasmids

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

1. Plasmids

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. Plasmids

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

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 Properties of Plasmids

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. Classifications of plasmids

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. ColE1 plasmid

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 . Characteristics of ColE1

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 Replication regulation in colE1

12. 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 . Applications of colE1

13. 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 Sv 40 plasmid

14. 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 History

15. 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. Properties of sv 40

16. 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 Applications of sv 40

17. 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.

18. 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` PMB 9 Plasmid

19. 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 . Construction of pMB9 plasmid

20. 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. Applications