Characteristics of bacterial Plasmid : Size : Conformation : Replication origin of replication : Replication Protein :

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1 Characteristics of bacterial Plasmid : Size : Conformation : Replication origin of replication : Replication Protein :

2 Definition of Plasmid Plasmids are extrachromosomal circular, double stranded DNA material present in cell cytoplasm. Characteristics Of Plasmid A plasmid is a small DNA molecule that is physically separate form and can replicate independently. Commonly found as small circular, double-stranded DNA molecule in bacteria. Plasmids are some time present in archae and eukaryotic organisms. Plasmids carry gene that may benefit to survival of antibiotic resistance organisms. Artificial plasmids are widely used as vectors in molecular cloning, DNA sequencing etc. Fig : Bacterial cell with plasmid

3 Plasmid size vary from 1 to over 1000 kbp. Size Generally size of plasmid ranging from 1x10 6 to 1x10 8 of bacterial chromosome. The 1x10 8 size big plasmid are observed in pseudomonas and Agrobacterium. Most of the plasmids are studied in enterobactericeae family in two groups : Small Plasmid Large Plasmid - Non self transferable - Self transferable - Non Conjugated - Conjugated

4 Conformation Plasmid DNA may appear in one of five conformation which run at different speed in gel during electrophoresis. Conformation are listed below in order of electrophoretic mobility as per given voltage from slowest to fastest. 1. Nicked open-circular DNA has one strand cut. 2. Relaxed circular DNA is fully intact with both strands, but enzymatically relaxed which can modeled for twisted cord unwind and relax & then plugging it into itself. 3. Linear DNA has free ends because both strands have been cut or because the DNA was linear in vivo 4. Supercoiled (Covalently closed-circular) DNA is fully intact with both strands and with an integral twist resulting in a compact form. 5. Supercoiled denatured DNA is like supercoiled DNA but has unpaired region that make it slightly less compact

5 The rate of migration from small linear fragment is directly proportional to the voltage ( when low voltage is applied ). At higher voltage, large fragments migrate continuously increasing yet different rate ( thus resolution of a gel decreases with increase voltage ). At a specific low voltage the migration rate of small DNA fragments is function of their length. Large linear fragments (over 20kb or so) migrate at certain fixed length. Restriction digests are frequently used to analyse purified plasmid it results in the formation of bands after gel electrophoresis. Fig : Bacterial cell with plasmid

6 Replication Origin Of Replication Many bacteria, including E. coli contain plasmid that each contain an origin of replication. These are separate from the origins of replication that are used by the bacteria to copy their genome and often function very differently. The E. coli plasmid pbr322 used a protein called Rop/Rom to regulate the number of plasmid. Fig : Replication of plasmid Fig : pbr322 plasmid

7 The most commonly origin of replication that is used in plasmid for genetic engineering is puc. Due to puc origin on plasmid allows genetic engineering to produce large quantity of DNA for research purpose. Other origins of replication include psc101 (derived from Salmonella, around 5 copies/cell) 15A origin (derived from p15a, copies per cell) and Bacterial artificial chromosome. Fig :- puc19 plasmid origin

8 Replication Of Protein(RP) Replication protein is the major protein that binds to single stranded DNA (ssdna) in eukaryotics. During DNA replication, replication of protein prevents single-stranded DNA from winding back on itself or from forming secondary structure. This keeps DNA unwound for the polymerase to replicate it. RP also binds to ssdna during the initial phase of homologous recombination, it is important process in DNA repair.

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