Plasmid: Introduction, Structure, Types, Isolation and Its Uses

Introduction of plasmid

A plasmid is an extrachromosomal DNA that is naturally present in some bacteria but also present in some eukaryotes e.g. Dictyostelium purpureum. It can replicate independently of the host chromosome. The plasmid is circular and double-stranded and carries few genes and their size ranges from 1 to over 200-kilo base pairs.

Plasmid structure

It contains three components:

1. an origin of replication,
2. a polylinker to clone the gene of interest (called multiple cloning site where the restriction enzymes cleave), and
3. an antibiotic resistance gene (selectable marker).

Types of plasmids

Classification on the basis of its ability to transfer to additional bacteria-

Conjugative type: It retains tra-genes, which carry out the intricate process of conjugation, the transfer of a plasmid to another bacterium.

Transitional classes: It is considered to be mobilizable, contains only a subset of the genes necessary for a successful transfer. It has the ability to parasitize a conjugative plasmid by transferring at a high frequency exclusively in the presence of this. Currently, it is used to manipulate DNA and could potentially be used as a device for curing disease. It is possible for various plasmids to coexist in a single cell.

Functional classification of plasmids

1. Fertility
2. R-plasmid
3. Ti plasmid tumor-inducing in plant
4. Degradative
5. Col-Plasmid 

    

    

   Fertility Plasmid: F-Plasmid carries the fertility genes (tra-genes) for conjugation, the transfer of genetic information between two cells. It is also known as episome because it integrates into the host chromosome and promotes the transfer of chromosomal DNA bacterial cells.

   Antibiotic-resistant plasmid: R-Plasmid contains genes that encode resistance to antibiotics or poisons. Examples of R- pBR322 Plasmid. It contains genes for resistance to tetracycline and ampicillin.

   Ti plasmid tumor-inducing in plant:  It contains A. tumefaciens, which carry instructions for bacteria to become a pathogen by switching to the tumor state synthesize opines, toxins, and other virulence factors. This effectively transfers foreign genes into certain plant cells.

   Degradative plasmids: Also called catabolic plasmids that allow the host bacterium to metabolize normally difficult or unusual compounds such as pesticides.

   Col-Plasmids: They contain genes that encode for the antibacterial polypeptides called bacteriocins, a protein that kills other strains of bacteria. The col proteins of E. coli are encoded by proteins such as Col E1.

    

Plasmid Isolation

1. 1.5 ml of overnight growth culture was taken into an Eppendorf tube.
2. The cells were then centrifuged at 12000 rpm for 2 minutes at 4°C.
3. After harvesting, the supernatant was discarded and the pellets were resuspended in 100  ml of solution 1.
4. Then, 200 ml of solution 2 was added and tubes were mixed by inversion
5. 150 ml of solution 3 was added to the mixture and mixed by rotating the tubes.
6. The tubes were then incubated in ice for 5 minutes.
7. Following this, the tubes were centrifuged at 12000 rpm for 5 minutes.
8. Then, the supernatant was taken and transferred to the new tube.
9. 0.6 times the volume of isopropanol of twice the volume of ethanol was added to the and tubes and they were vortexes.
10. Following vortexing, the tubes were centrifuged at 12000 rpm for 10 minutes.
11. The supernatant was discarded and the pellet was washed with 70% ethanol.
12. The tubes were centrifuged at 12000 rpm for 5 minutes and the supernatant was discarded.
13. Then, the tubes were left for drying after which the pellet was dissolved in TE buffer.

Keys: Solution 1 (resuspension buffer)  contains tris hydrochloride- buffer

EDTA – chelating agent

Glucose-osmotic agent -cell to cell contact loss

Solution 2 ( lysis buffer) freshly prepared, contains 0.2% sodium hydroxide and 1% SDS

Solution 3 (neutralization buffer) contains

potassium acetate : 60 ml (5.0 M )

Glacial acetic acid: 28.5 ml

Water: 11.5 ml

Uses of plasmid

• It provides a versatile tool in genetic engineering because of its unique
  properties as a vector.
• It is utilized to create transgenic organisms by introducing new genes into recipient cells. For example, the Ti plasmids from the soil bacterium Agrobacterium tumefaciens are very valuable in plant pathology in developing plants with resistance to diseases such as Holcus spot on leaves and crown gall tumors.
• It also carries medical significance because of its role in antibiotic synthesis. Streptomyces coelicolor plasmid can give rise to thousands of antibiotics, as well as that of S. lividans or S. reticuli. In another example, E. coli plasmids are used to clone the gene of penicillin G acylase, the enzyme that turns penicillin G into the antibacterial 6-amino-penicillanic acid. Once again, these cloning processes are carried out with the assistance of a type II restriction enzyme to put the gene of interest into the plasmid vector.
• In DNA recombinant technology, the plasmid-based reporter gene is crucial as they allow observation of organisms in real-time. The gene for Green Fluorescent Protein can be integrated into a plasmid of the organism under investigation. The encoded protein is small and does not alter the function of the host protein. This feature of GFP makes it very easy to observe cell dynamics.

Bibliography

1. Shakibaie, MR., Dhakephalker, PA., Kapadnis, BP., Chopade, BA:
   Conjugational transfer and survival of plasmid encoding silver and antibiotic
   resistance genes of Acinetobacter baumannii BL54, E.coli K12 J53.2
   transconjugants and pseudomonas transformants in different soil microcosms.Journal bacteriology research. (2009) (Accepted for publication).
2. Sinkovics J, Horvath J, Horak A (1998). “The origin and evolution of viruses (a review)”. Acta Microbiologica et Immunologica Hungarica. 45 (3–4): 349–90. PMID 9873943.
3. Thomas CM, Summers D (2008). Bacterial Plasmids. Encyclopedia of Life Sciences. doi:10.1002/9780470015902.a0000468.pub2. ISBN 978-0470016176.
4. Finbarr Hayes (2003). “Chapter 1 – The Function and Organization of Plasmids”. In Nicola Casali, Andrew Presto (eds.). E. Coli Plasmid Vectors: Methods and Applications. Methods in Molecular Biology. 235. Humana Press. pp. 1–5. ISBN 978-1-58829-151-6.
5. T. A. Brown (2010). “Chapter 2 – Vectors for Gene Cloning: Plasmids and Bacteriophages”. Gene Cloning and DNA Analysis: An Introduction (6th ed.). Wiley-Blackwell. ISBN 978-1405181730.
6. Kandavelou K, Chandrasegaran S (2008). “Plasmids for Gene Therapy”. Plasmids: Current Research and Future Trends. Caister Academic Press. ISBN 978-1-904455-35-6.
7. Andrew Preston (2003). “Chapter 2 – Choosing a Cloning Vector”. In Nicola Casali, Andrew Preston (eds.). E. Coli Plasmid Vectors: Methods and Applications. Methods in Molecular Biology, Vol. 235. Humana Press. pp. 19–26. ISBN 978-1-58829-151-6.
8. https://www.mybiosource.com/learn/testing-procedures/plasmid-isolation