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  • 1. PLASMIDS Dr.T.V.Rao MD
  • 2. Joshua LederbergThe term plasmid was first introduced by the American molecular biologist Joshua Lederberg in 1952• Joshua Lederberg was an American molecular biologist known for his work in genetics, artificial intelligence, and space exploration. He was just 33 years old when he won the 1958 Nobel Prize in Physiology or Medicine for discovering that bacteria can mate and exchange genes. He shared the prize with Edward L. Tatum and George Beadle who won for their work with genetics.
  • 3. Joshua LederbergWins Nobel Prize
  • 4. What is a Plasmid• A plasmid is a DNA molecule that is separate from, and can replicate independently of, the chromosomal DNA. They are double stranded and, in many cases, circular. Plasmids usually occur naturally in bacteria, but are sometimes found in eukaryotic organisms (e.g., the 2- micrometre-ring in Saccharomyces cerevisiae).
  • 5. Basic understanding of Plasmid• A plasmid is a vehicle that can carry artificially inserted DNA. It will replicate in E. coli, and with its own replication it will also replicate the inserted DNA, independent of its origin. In a way one can see a plasmid as a minute DNA factory. The main criteria for a good plasmid is that it takes up the insert you want to put in, and that it replicates in sufficient amounts,and that it does not destroy your insert during the process.
  • 6. What is a plasmid?• A circular piece of autonomously replicating DNA• Originally evolved by bacteria• May express antibiotic resistance gene or be modified to express proteins of interest
  • 7. Bacterial plasmid
  • 8. Plasmids• Plasmids are molecules of DNA that are found in bacteria separate from the bacterial chromosome.• They: are small (a few thousand base pairs) usually carry only one or a few genes are circular have a single origin of replication
  • 9. Multiplication of Plasmids• Plasmids are replicated by the same machinery that replicates the bacterial chromosome. Some plasmids are copied at about the same rate as the chromosome, so a single cell is apt to have only a single copy of the plasmid. Other plasmids are copied at a high rate and a single cell may have 50 or more of them.
  • 10. Plasmids enters the Bacteria with ease. • Plasmids enter the bacterial cell with relative ease. This occurs in nature and may account for the rapid spread of antibiotic resistance in hospitals and elsewhere. Plasmids can be deliberately introduced into bacteria in the laboratory transforming the cell with the incoming genes.
  • 11. INTRODUCTION• Plasmids are self replicative extrachromosomal DNA molecules of finite size that are stably inherited and exchanged promiscuously between a broad spectrum of bacteria and other domains (Perlin, 2002).• Plasmids allow bacterial (including other domains) populations to ‘sample’ the horizontal gene pool for adaptive traits that might be advantageous for survival under local selective pressure (Sorensen et al., 2005).• Plasmids also provide genetic variation, acts as sources of recombination and can allow faster gene fixation leading to greater likelihood that the ‘new’ trait will persist (Sorensen et al., 2005).
  • 12. INTRODUCTION• Plasmid classification is generally based on incompatibility group (determined by their replication/partitioning functions) or the genetic information specified by their DNA (Perlin, 2002). Incompatibility grouping had been used to group plasmid of Pseudomonas species (Jacoby, 1977) and the Enterobacteriaceae into 26 incompatibility group (Couturier et al., 1988).• Most plasmids have a narrow host range allowing only intra-species transfer and replication. However, a small group of plasmids called the broad host range (BHR) plasmids (Inc P, Q, W, N and C) can be transferred and replicated in a wide range of bacteria (Hill and Top, 1998; Dale and Park, 2004). BHR plasmids may either be self-transmissible (Tra+, Mob+) or mobilizable but not self-transmissible (Tra-, Mob+) (Perlin , 2002).• pr
  • 13. Plasmids are Complex • Prokaryotes were initially thought to harbour only circular plasmids. However with the discovery of double stranded linear plasmids in the spirochaete that cause lyme disease, Borrelia borgdorferi (Barbour and Garon, 1987), linear plasmids have also been detected in the genera Streptomyces (Kinashi et al., 1994), Mycobacterium (Le Dantec et al., 2001), Rhodococcus (Larkin et al., 2005) and Arthrobacter (Overhage et al., 2005).
  • 14. Understanding a Plasmid• Many bacteria have accessory DNA molecules in addition to their larger chromosome. These molecules, called plasmids, are extensively used in genetic engineering. In order to be useful in labs, these plasmids need to have an origin of replication (ori), which enables them to replicate within a bacterial cell. They also need to have multiple restriction enzyme sites to enable cutting and pasting of DNA into a plasmid.
  • 15. Role in Antibiotic Resistance • Most plasmids have one or two identifiable markers that give a distinct phenotype to the bacterial cell. Examples of such markers include antibiotic resistance (ampR) or expression of an enzyme that catalyzes a reaction that produces a color change (lacZ).
  • 16. Plasmids and Ampicillin Resistance• Some plasmids have the ampR gene, which confers resistance to the antibiotic ampicillin. E. coli cells containing this plasmid, termed "+ampR" cells, can survive and form colonies on LB agar that has been supplemented with ampicillin. In contrast, cells lacking the ampR plasmid, termed "–ampR" cells, are sensitive to the antibiotic, which kills them. An ampicillin-sensitive cell (– ampR) can be transformed to an ampicillin-resistant (+ampR) cell by its uptake of a foreign plasmid containing the ampR gene
  • 17. Plasmids and Microbes• In microbiology, an extra chromosomal genetic element that occurs in many bacterial strains. Plasmids are circular deoxyribonucleic acid (DNA) molecules that replicate independently of the bacterial chromosome. They are not essential for the bacterium but may confer a selective advantage. One class of plasmids, colicinogenic (or Col ) factors, determines the production of proteins called colicins, which have antibiotic activity and can kill other bacteria. Another class of plasmids, R factors, confers upon bacteria resistance to antibiotics. Some Col factors and R factors can transfer themselves from one cell to another and thus are capable of spreading rapidly through a bacterial population. A plasmid that is attached to the cell membrane or integrated into the bacterial chromosome is called an episome.
  • 18. Plasmids in Genetic Engineering • Plasmids are extremely valuable tools in the fields of molecular biology and genetics, specifically in the area of genetic engineering. They play a critical role in such procedures as gene cloning, recombinant protein production (e.g., of human insulin), and gene therapy research. In such procedures, a plasmid is cut at a specific site (or sites) using enzymes called restriction endonucleases
  • 19. Structure of Plasmids• Plasmid size varies from 1 to over 1,000 kilo base pairs (kbp). The number of identical plasmids within a single cell can range anywhere from one to even thousands under some circumstances. Plasmids can be considered to be part of the mobilome, since they are often associated with conjugation, a mechanism of horizontal gene transfer
  • 20. Plasmids used as Vectors • Plasmids • small (1-1000 kb) • circular • extrachromosomal DNA • Growth is independent of the host’s cell cycle; amplification of gene product • A type of cloning vector used to carry a gene not found in the bacterial host’s chromosome
  • 21. VARIETIES OF PLASMIDS BASED ON STRUCTUREGenetic map of ColE1circular plasmid: colE1,imm: genes for productionof, and immunity to colicinE1; mob codes for nucleaserequired for mobilization;rom codes for proteinrequired for effectivecontrol of copy number;oriT: origin of conjugaltransfer; oriV: origin ofreplication (Dale and Park,2004).
  • 22. Plasmids can …• Plasmids are considered transferable genetic elements, or "replicons", capable of autonomous replication within a suitable host. Plasmids can be found in all three major domains, Archea, Bacteria and Eukarya. Similar to viruses, plasmids are not considered a form of "life" as it is currently defined. Unlike viruses, plasmids are "naked" DNA and do not encode genes necessary to encase the genetic material for transfer to a new host, though some classes of plasmids encode the sex pilus necessary for their own transfer
  • 23. Plasmids act as Vectors• Plasmids are key vectors of horizontal gene transfer and essential genetic engineering tools. They code for genes involved in many aspects of microbial biology, including detoxication, virulence, ecological interactions, and antibiotic resistance.
  • 24. Plasmids originated several Drug resistance Mechanisms• The role of plasmids in evolution of bacterial genome and adaptation to specific environmental changes has contributed immensely to the emergence of antibiotic and heavy metal resistance plasmids.• This feat is aided by the activities of transposons, which promote the movement of resistance genes between plasmids or from the chromosome of naturally resistant organisms onto a plasmid ( Dale and Park, 2004; Kapil, 2005).• Bacteria can be resistant to various antibiotics either by acquisition of several independent plasmids or through acquiring a single plasmid with many resistance determinant on it (Hill and Top, 1998).
  • 25. VARIETIES OF PLASMIDS BASED ON FUNCTIONS Pumping of the toxic metal out of the bacterial cell, bioaccumulation in physiologically inaccessible compound and redox chemistry in which a more toxic ion species is converted to a less toxic ion (Endo et al., 2002)• Plasmid-mediated mercury (mer) (Silver and Walderhaug, 1994), arsenic (ars) (Wu and Rosen, 1993), cadmium (Cad) (Tsai et al., 1993), and chromate (Chr) (Pimentel et al., 2002) resistance have been reported for Gram positive and Gram negative with various mechanisms of resistance.
  • 26. VARIETIES OF PLASMIDS BASED ON FUNCTIONS • Genetic map of Escherichia coli F factor showing the four major regions: The inc, rep region determines replication and plasmid incompatibility properties; the tra region provides conjugative DNA mobilization functions; the region containing the four transposable elements that facilitates interaction between F factor and other DNA molecules; and the silent region (Porter, 2002).
  • 27. Escherichia coli Cloning and Expression Vectors • Gene technology methods together with the DNA sequencing and PCR methodologies have revoluzionarized basic and applied research in molecular biology involving all kinds of organisms from eubacteria up to humans. Since almost all of the cloning experiments start by insertion of DNA fragments into Escherichia coli plasmid
  • 28. VARIETIES OF PLASMIDS BASED ON FUNCTIONS Table 1: some antibiotic resistance plasmids, their incompatibility groups and phenotypes Abbreviations used: Km, kanamycin; Tc, tetracycline; Ap, ampicillin; Sm, streptomycin; Sp, spectinomycin; Cm, chloramphenicol, Tp, trimethoprim; BHR, broad host range (Hill and Top, 1998)
  • 29. VARIETIES OF PLASMIDS BASED ON FUNCTIONS Sex pheromone plasmids• Pheromones are secreted chemicals used for signalling between two or more individuals. Pheromone conjugative plasmids are confined to the Enterococci and encode antibiotic resistance, bacteriocins and hemolysins (Grohmann et al., 2003).• In this novel transfer system, recipient cells secrete a family of heat stable peptide pheromones with specificities for donor carrying various conjugative plasmid that trigger response from donor bacteria harbouring a particular plasmid, which synthesize an adhesin that facilitates the formation of mating aggregate with nearby recipients (Dale and Park, 2004).• Examples of sex pheromone plasmids are pAD1 (59.3-kb, hemolysin/bacteriocin plasmid), and pCF10 (65-kb, tetracycline resistance) (Grohmann et al., 2003).
  • 30. VARIETIES OF PLASMIDS BASED ON FUNCTIONSSenescence plasmids in fungi• Senescence is an inherent degenerative program in multicellular organisms that is manifested by a progressive decline in cellular energy production culminating in the death of a part or the whole organism (D’souza and Maheshwari, 2002).• This phenomenon was found to be associated with the accumulation of high copy number circular plasmids (sen DNAs) in the mitochondrial respiration resulting in the death of the fungus (D’souza and Maheshwari, 2002).• Mitochondrial-based linear plasmids from Neurospora sp. were also found to be responsible for the death of the strains harbouring them by insertion of their DNA into the mitochondrial genome resulting in disruption of several genes leading to senescence and death due to defective respiration (Bertrand, 2000; Griffiths, 1998).
  • 31. 1973 Genetic Engineering Invented
  • 32. Cloning: Digestion TARGET GENESOURCE DNA
  • 33. Cloning: Digested Fragments TARGET GENE
  • 34. Cloning: LigationDNA LIGASE TARGET GENE
  • 35. Cloning: Recombinant Plasmid TARGET GENE
  • 36. Cloning: Transformation E. coli
  • 37. Cloning: Competence Ca2+ Ca2+ Ca2+ Ca2+ Ca2+ Ca2+ Ca2+ Ca2+ Ca2+ Ca2+ Ca2+ Ca2+ Ca2+ Ca2+ Ca2+ Ca2+ Ca2+ E. coli Ca2+ Ca2+ Ca2+ Ca2+ Ca2+ Ca2+ Ca2+ Ca2+ 2+ Ca 2+ Ca2+ Ca Ca2+ Ca2+
  • 38. Cloning: Competence E. coli
  • 39. Cloning: Transformation E. coli
  • 40. Cloning: Transformation E. coli
  • 41. Plasmids in Antibiotic Resistance• In addition plasmids carry antibiotic resistance genes and their spread in pathogenic bacteria is of great medical importance. Plasmids are used in molecular studies of various organisms with ramifications in synthetic biology, medicine, ecology
  • 42. Antibiotic resistance • Plasmid often contain genes or gene-cassettes that confer a selective advantage to the bacterium harboring them, e.g., the ability to build an antibiotic resistance. Every plasmid contains at least one DNA sequence that serves as an origin of replication or ori (a starting point for DNA replication), which enables the plasmid DNA to be duplicated independently from the chromosomal DNA
  • 43. Plasmids can be Engineered for Selective Needs• While there are only a couple essentials that a plasmid must have, DNA workers have engineered a number of features into many plasmids that make them easier to use. First the essentials: 1) it must have the _ori_ gene, which enables the plasmid to be replicated in the host cells, 2) it must have a "selectable marker" so that only cells that have the plasmid will grow in culture. This is almost always a gene that confers resistance to a particular antibiotic, so that cells grown in the presence of the drug will all contain the plasmid.
  • 44. Plasmid-based Expression Systems for Mammalian Cells• In contrast to bacteria and yeast no natural plasmids are found in mammalian cells. Therefore many attempts to construct different expression vector systems for mammalian cells have been made in recent years. These vector systems can be categorized in terms of vector administration, mechanisms of vector replication and mechanisms to achieve nuclear persistence of the vectors
  • 45. Limitations of Plasmids• There are limitations to each of these steps: large inserts require specialized plasmids (cosmids or YACs for megabase sized- inserts), the larger a plasmid+insert, the lower its replication rate, but there are ways to improve the yield, and certain plasmids result in frequent deletions of (parts of) the insert, although this is sometimes due to the host (the E. coli or another host cell) or due to the nature of the insert as well.
  • 46. Plasmids in Gene therapy• The success of gene therapy depends on the efficient insertion of therapeutic genes at the appropriate chromosomal target sites within the human genome, without causing cell injury, oncogenic mutations or an immune response. Although viral vectors offer excellent vehicles for highly efficient transduction of human cells, the associated safety concerns make non-viral delivery of therapeutic genes by using plasmid DNA into cells more attractive.
  • 47. Dual Expression Cassette Plasmids for the Expression of One Gene of Interest pSELECT• pSELECT plasmids offer all the features necessary to express a gene of interest at high levels in a large number of cell types. pSELECT plasmids contain two transcription units, the first drives the expression of the gene of interest and the second drives the expression of a large choice of dominant selectable markers for both E. coli and mammalian cells.
  • 48. Horizontal Gene Transfer Mediated by Plasmids• Among the mobile elements and mechanisms of HGT, plasmids are undoubtedly critical players because of their ability to transfer by conjugation among both closely and very distantly related bacterial hosts. This feature allows them to broadly distribute genes or gene clusters that code for various host-beneficial phenotypes. The event, generally recognized as horizontal gene transfer (HGT), is now considered as a strong driving force for the evolution of bacterial genome organization and for rapid adaptation to the surrounding environments
  • 49. Plasmid DNA as Prophylactic and Therapeutic vaccines for Cancer and Infectious Diseases• Concept of plasmid DNA as a protective vaccine strategy for cancer and infectious diseases. However, the crossover application into human studies has been met with poor results based on the DNA vector¹s inability to provide clinically relevant prophylactic and therapeutic benefit
  • 50. Plasmid profiles in epidemiologic surveillance of disease outbreaks and Drug Resistance• Plasmids can also serve as markers of various bacterial strains when a typing system referred to as plasmid profiling, or plasmid fingerprinting is used. Because many species of bacteria contain plasmids, plasmid profile typing has been used to investigate outbreaks of many bacterial diseases and to trace inter- and intra-species spread of antibiotic resistance.
  • 51. Programme created by Dr.T.V.Rao MD for ‘e’ learning for Medical and paramedical students in Developing World Email