Plasmids and bacteriophages
- 1. Plasmids and Bacteriophages BY Dr. Sonia Goel SGT University, Gurugram.
- 2. Plasmids A plasmid is a small, extrachromosomal DNA molecule within a cell that is physically separated from chromosomal DNA and can replicate independently. They are most commonly found as small circular, double-stranded DNA molecules in bacteria; however, plasmids are sometimes present in archaea and eukaryotic organisms.
- 3. Types of plasmids Depending on their transmissibility property are of three types: 1. Transmissible plasmid: They can be transferred from cell to cell by the process of genetic transfer like conjugation, hence also known as conjugative plasmid. Large plasmids with molecular weight of 40-100 million.
- 4. Continued... 2. Non – transmissible: These cannot be transferred from cell to cell during conjugation as they lack transferase gene. They are small plasmids of molecular weight 3-20 million and are non-conjugative. 3. Episome: This plasmid lies either freely in circular from or gets integrated into the host chromosomes.
- 5. Continued... Depending on the nature of factors or function, plasmids are of following types; 1. The F factor: Also called fertility- factor or sex-factor. It contains genetic information essentail for controlling the matting process of bacteria during conjugation. These genes determine: Expression of pilli and transfer of DNA during mating.
- 6. Continued... 2. The R factor: Also known as resistance factor. Occur in 2 forms: Large plasmid and small plasmid. Large plasmids are conjugative ‘R’ factors helps in conjugation. The small plasmids only contain ‘r’ genes and are not conjugative.
- 7. Continued... 3. Colcinogenic (Col) factor: Also known as bacteriocinogenic factor. Col factor codes for the production of bacteriocins ( e.g; colicins, dipthericin, pyocyanin etc), which are antibiotic like substances that are specifically and selectively lethal to other closely related bacteria. 4. Virulence plasmid: This plasmid codes for the virulence factor in some bacteria that increases it pathogenicity.
- 8. Continued... 5. Metabolic plasmids: This plasmid helps in various metabolic activities in bacteria. E.g : Root modulation and N2 fixation genes of rhizobium are present in its plasmid.
- 9. Purification of plasmids In biochemical aspects, to purify plasmid DNA from bacteria is to isolate only plasmid DNA from the mixture of biopolymers such as protein, ribonucleic acid (RNA), chromosomal DNA and plasmid DNA, by which bacteria cell is composed
- 11. Continued... Multiple methods of nucleic acid purification exist. All work on the principle of generating conditions where either only the nucleic acid precipitates, or only other biomolecules precipitate, allowing the nucleic acid to be separated. Ethanol precipitation Ethanol precipitation works by using ethanol as an antisolvent of DNA, causing it to precipitate out of solution. The soluble fraction is discarded to remove other biomolecules.
- 12. Continued... Spin column Spin column-based nucleic acid purification precipitates nucleic acid such that it binds a solid matrix and other components flow through. The conditions are then changed to elute the purified nucleic acid. Phenol–chloroform extraction In a phenol–chloroform extraction, addition of a phenol/chloroform mixture will dissolve protein and lipid contaminants, leaving the nucleic acids in the aqueous phase.
- 13. Continued... It also denatures proteins, like DNase, which is especially important if the plasmids are to be used for enzyme digestion. Otherwise, smearing may occur in enzyme restricted form of plasmid DNA.
- 14. Applications of plasmid Plasmids are used in genetic engineering to amplify, or produce many copies of certain genes. They are used in different techniques and are involved in research of genetic engineering and gene therapy by gene transfer to bacterial cells or to cells of superior organisms, whether other plants, animals or other living organisms, to improve their resistance to diseases, growth rates, or any other required traits.
- 15. Continued... In molecular cloning, plasmids are types of vectors that are useful in cloning short segments of DNA. For example, the artificial and cost-effective bulk production of antibiotics can be achieved by incorporating an expression vector for that antibiotic in microbial cells. Similarly, other biomolecules can also be produced. In addition, plasmids are used to administer gene therapy, which is a technique used to correct defective genes responsible for disease development. They can also be used to replicate proteins, such as the protein that codes for insulin, in large amounts
- 16. Phage genetics The most intensively studied bacteriophage is the phage called lambda. It is an important model system for the latent infection of mammalian cells by retroviruses , and it has been widely used for cloning purposes. Lambda is the prototype of a group of phages that are able to infect a cell and redirect the cell to become a factory for the production of new virus particles
- 17. Continued... Lambda and other phages, which can establish lytic or lysogenic cycles, are called temperate phages. Other examples of temperate phages are bacteriophage mu and P1. Mu inserts randomly into the host chromosome causing insertional mutations where intergrations take place. The P1 genome exists in the host cell as an autonomous, self-replicating plasmid.
- 18. Continued... Phage gene expression during the lytic and lysogenic cycles uses the host RNA polymerase, as do other viruses. However, lambda is unique in using a type of regulation called antitermination.
- 19. Phage genetic organization Phages are simple organisms that consist of a core of genetic material (nucleic acid) surrounded by a protein capsid. The nucleic acid may be either DNA or RNA and may be double-stranded or single-stranded. There are three basic structural forms of phage: an icosahedral (20-sided) head with a tail, an icosahedral head without a tail, and a filamentous form.
- 20. Gene Mapping in plasmids Gene mapping describes the methods used to identify the locus of a gene and the distances between genes. The essence of all genome mapping is to place a collection of molecular markers onto their respective positions on the genome. Molecular markers come in all forms. Genes can be viewed as one special type of genetic markers in the construction of genome maps, and mapped the same way as any other markers.
- 23. Continued... The black arrows show the direction of transcription, which is essential for cloning. If you clone your gene of interest in a middle of another gene, make sure that both of them are transcribed in the same direction. Otherwise, the native promoter can interfere with your gene expression. plasmid sequences are diagrammed as linear sequences starting from the ori. “Ori” means the origin of plasmid replication.
- 24. Continued... pBR322 has two antibiotic resistance genes: tet (tetracycline resistance) and amp (ampicillin resistance). These genes encode an efflux pump (tetR) and beta-lactamase (ampR) to excrete tetracycline and ampicillin from the cell, respectively. Tet and amp are read in different directions.
- 25. Thank You