Plasmids & Nucleoid
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Joshua Lederberg was an American molecular biologist who first introduced the term "plasmid" in 1952 and won the 1958 Nobel Prize in Physiology at age 33 for his work in genetics. Plasmids are small, circular DNA molecules that naturally exist in bacterial cells and some eukaryotes. They often carry genes that provide advantages to bacteria like antibiotic resistance. Plasmids are useful in research because they are easy to isolate, manipulate, and replicate in bacteria. The nucleoid refers to the region in a prokaryotic cell that contains its chromosomal DNA and is more compact than the nucleus in eukaryotic cells.
Plasmids & Nucleoid
- 1. PLASMIDS & NUCLEOID Vivek kumar MSC MICROBIOLOGY Bangalore University
- 2. JOSHUA LEDERBERG WINS NOBEL PRIZE The 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
- 3. WHAT IS A PLASMID * A plasmid is a small, circular, double-stranded DNA * Plasmids naturally exist in bacterial cells. * They also occur in some eukaryotes. Often, the genes carried in plasmids provide bacteria with genetic advantages, such as antibiotic resistance.
- 5. IMPORTANCE OF PLASMID Easy to work with - Plasmids are a convenient size (generally 1,000-20,000 basepairs) for physical isolation (purification) and manipulation. Self-replicatclonin - you can easily make an endless number of copies of the plasmid using bacteria, which can uptake plasmids and amplify them during cell division.
- 6. Stable Plasmids are stable long-term either as purified DNA or within bacterial cells that have been preserved as glycerol stocks.
- 7. PLASMID ELEMENT 1- origin of replication (ORI) 2-Antibiotic-resistance gene 3- At least one unique restriction enzyme recognition site. 4-Additionally, the restriction enzyme site(s)
- 8. DESCRIPTIONS
- 9. Origin of Replication (ORI) DNA sequence which directs initiation of plasmid replication (by bacteria) by recruiting bacterial transcriptional machinery. The ORI is critical for the ability of the plasmid to be copied (amplified) by bacteria, which is an important characteristic of why plasmids are convenient and easy to use.
- 10. 1- Allows for selection of plasmid-containing bacteria by providing a survival advantage to the bacterial host. 2- Each bacterium can contain multiple copies of an individual plasmid, and ideally would replicate these plasmids upon cell division in addition to their own genomic DNA. 3- Bacteria without plasmids can replicate faster and out- populate bacteria with plasmids, thus selecting against the propagation of these plasmids through cell division Antibiotic Resistance Gene
- 11. Multiple Cloning Site (MCS) Short segment of DNA which contains several restriction enzyme sites, enabling easy insertion of DNA by restriction enzyme digestion and ligation. In expression plasmids, the MCS is often located downstream from a promoter, such that when a gene is inserted within the MCS, its expression will be driven by the promoter. As a general rule, the restriction sites in the MCS are unique and not located elsewhere in the plasmid backbone.
- 12. Insert The insert is the gene, promoter, or other DNA fragment cloned into the MCS. The insert is typically the genetic element one wishes to study using a particular plasmid.
- 13. Promoter Region Drives transcription of the insert. The promoter is designed to recruit transcriptional machinery from a particular organism or group of organisms. The promoter can also direct cell-specific expression.
- 14. Selectable Marker The selectable marker is used to select for cells that have successfully taken up the plasmid for the purpose of expressing the insert. This is different than selecting for bacterial cells that have taken up the plasmid for the purpose of replication. The selectable marker enables selection of a population of cells that have taken up the plasmid and that can be used to study the insert. The selectable marker is typically in the form of another antibiotic resistance gene (this time, under the control of a non-bacterial promoter) or a fluorescent protein (that can be used to select or sort the cells by visualization or FACS).
- 15. Primer Binding Site A short single-stranded DNA sequence used as an initiation point for PCR amplification or DNA sequencing of the plasmid. Primers can be utilized to verify the sequence of the insert or other regions of the plasmid
- 16. GENERAL TYPE OF PLASMID Conjugative and Non-Conjugative There are many ways to classify plasmids from general to specific. One way is by grouping them as either conjugative or non-conjugative. Bacteria reproduce by sexual conjugation, which is the transfer of genetic material from one bacterial cell to another, either through direct contact or a bridge between the two cells. Some plasmids contain genes called transfer genes that facilitate the beginning of conjugation. Non- conjugative plasmids cannot start the conjugation process, and they can only be transferred through sexual conjugation with the help of conjugative plasmids.
- 18. SPECIAL TYPES OF PLASMID Fertility F-plasmids Resistance plasmids Virulence plasmids, Degradative plasmids Col plasmids
- 19. FERTILITY F-PLASMIDS Fertility plasmids, also known as F-plasmids, contain transfer genes that allow genes to be transferred from one bacteria to another through conjugation. These make up the broad category of conjugative plasmids. F-plasmids are episomes , which are plasmids that can be inserted into chromosomal DNA. Bacteria that have the F- plasmid are known as F positive (F+), and bacteria without it are F negative (F–). When an F+ bacterium conjugates with an F– bacterium, two F+ bacterium result. There can only be one F-plasmid in each bacterium.
- 21. RESISTANCE PLASMIDS Resistance or R plasmids contain genes that help a bacterial cell defend against environmental factors such as poisons or antibiotics. Some resistance plasmids can transfer themselves through conjugation. When this happens, a strain of bacteria can become resistant to antibiotics. Recently, the type bacterium that causes the sexually transmitted infection gonorrhea has become so resistant to a class of antibiotics called quinolones that a new class of antibiotics, called cephalosporins, has started to be recommended by the World Health Organization instead. The bacteria may even become resistant to these antibiotics within five years. According to NPR, overuse of antibiotics to treat other infections, like urinary tract infections, may lead to the proliferation of drug-resistant strains.
- 23. VIRULENCE PLASMID When a virulence plasmid is inside a bacterium, it turns that bacterium into a pathogen, which is an agent of disease. Bacteria that cause disease can be easily spread and replicated among affected individuals. The bacterium Escherichia coli (E. coli) has several virulence plasmids. E. coli is found naturally in the human gut and in other animals, but certain strains of E. coli can cause severe diarrhea and vomiting. Salmonella enterica is another bacterium that contains virulence plasmids.
- 25. DEGRADITIVE PLASMID & COL PLASMID Degradative plasmids help the host bacterium to digest compounds that are not commonly found in nature, such as camphor, xylene, toluene, and salicylic acid. These plasmids contain genes for special enzymes that break down specific compounds. Degradative plasmids are conjugative. Col plasmids contain genes that make bacteriocins (also known as colicins), which are proteins that kill other bacteria and thus defend the host bacterium. Bacteriocins are found in many types of bacteria including E. coli, which gets them from the plasmid ColE1.
- 28. NUCLEIOD
- 29. . The nucleoid is mostly composed of multiple compacted copies of DNA in a continuous thread, with the addition of some RNA and proteins. The DNA in prokaryotes is double- stranded and generally takes a circular shape. That DNA can sometimes also be found in other regions outside the nucleoid. Eukaryotes, such as plants and animals, have a nucleus that houses their genetic material, with a surrounding double membrane, or what we call the nuclear envelope. This membrane separates the contents of the nucleus from the cytoplasm. As in prokaryotes, the DNA of eukaryotes is also double-stranded.
- 30. Bacterial chromosome Chromosomes carry most prokaryotic genes. Genes essential for bacterial growth are carried on chromosome. It is present in all bacteria. Only one chromo-some is present in a bacterial nucleoid. Bacterial chromosome is a single circle containing about 4000 kbp (kilobase pairs) of DNA. DNA is a double stranded helical structure. A molecule of DNA consists of many mononucleotides. A mononucleotide consists of a molecule of sugar, a molecule of phosphate and a molecule of base. In each strand of DNA, molecules of phosphate and sugar alternate, and one of the four bases present in DNA is attached with the sugar. The four bases are adenine, thymine, guanine and cytosine. A base of a strand joins with its complementary base of the other strand. Adenine is always complementary to thymine, and guanine to cytosine. During cell division, duplication of chromosome occurs so that each daughter cell receives an identical set.
- 31. . The bacterial chromosomal DNA is folded into a compact structure called nucleoid. The shape and size of this ‘body’ is determined by a number of factors. Major players are DNA supercoiling, macromolecular crowding and architectural proteins, associated with the nucleoid The early preparations of Varshavsky et al. revealed the presence of two abundant proteins, H-NS (histone-like nucleoid structuring protein) and HU (heat unstable protein)
- 34. REFERENCES Hiraga, S. (1992). Chromosome and plasmid partition in Escherichia coli. Annual Review of Biocheristry 61: 283–306. Ippen-Ihier, K.A., Skurray, R.A. (1993). Genetic organization of transfer- related determinants on the sex factor F and related plasmids, pp. 23–52. In: Clewell, D.B. (ed.), Bacterial Conjugation. New York: Plenum Press.