Plasmids and their features for genetic engineering
- 1. Plasmids
- 2. Plasmids • A plasmid is a small DNA molecule within a cell that is physically separated from a chromosomal DNA and can replicate independently. • The term was coined by Lederberg and Hays and shortly discovered by Tatum. • They are most commonly found in bacteria as small circular, double-stranded DNA molecules; however, plasmids are sometimes present in archaea and eukaryotic organisms. • Plasmids carry genes that may benefit the survival of the organism, for example antibiotic resistance. • While the chromosomes are big and contain all the essential genetic information for living under normal conditions, plasmids usually are very small and contain only additional genes that may be useful to the organism under certain situations or particular conditions. • Artificial plasmids are widely used as vectors in molecular cloning, serving to drive the replication of recombinant DNA sequences within host organisms.
- 3. • Plasmids are considered replicons, a unit of DNA capable of replicating autonomously within a suitable host. • Plasmids can be transmitted from one bacterium to another via three main mechanisms: transformation, transduction, and conjugation. • This host-to-host transfer of genetic material is called horizontal gene transfer, and plasmids can be considered part of the mobilome. • Unlike viruses (which encase their genetic material in a protective protein coat called a capsid), plasmids are "naked" DNA and do not encode genes necessary to encase the genetic material for transfer to a new host. • However, some classes of plasmids encode the conjugative "sex" pilus necessary for their own transfer. • The size of the plasmid varies from 1 to over 200 kbp, and the number of identical plasmids in a single cell can range anywhere from one to thousands under some circumstances.
- 4. • The relationship between microbes and plasmid DNA is neither parasitic nor mutualistic, because each implies the presence of an independent species living in a detrimental or commensal state with the host organism. • Plasmids may carry genes that provide resistance to naturally occurring antibiotics in a competitive environmental niche, or the proteins produced may act as toxins under similar circumstances, or allow the organism to utilize particular organic compounds that would be advantageous when nutrients are scarce.
- 5. Properties and characteristics • In order for plasmids to replicate independently within a cell, they must possess a stretch of DNA that can act as an origin of replication. • The self-replicating unit, in this case the plasmid, is called a replicon. • A typical bacterial replicon may consist of a number of elements, such as the gene for plasmid-specific replication initiation protein (Rep), repeating units called iterons, DnaA boxes, and an adjacent AT-rich region. • Smaller plasmids make use of the host replicative enzymes to make copies of themselves, while larger plasmids may carry genes specific for the replication of those plasmids. • A few types of plasmids can also insert into the host chromosome, and these integrative plasmids are sometimes referred to as episomes in prokaryotes.
- 6. • Plasmids almost always carry at least one gene. • Many of the genes carried by a plasmid are beneficial for the host cells, for example: enabling the host cell to survive in an environment that would otherwise be lethal or restrictive for growth. • Some of these genes encode traits for antibiotic resistance or resistance to heavy metal, while others may produce virulence factors that enable a bacterium to colonize a host and overcome its defences, or have specific metabolic functions that allow the bacterium to utilize a particular nutrient, including the ability to degrade recalcitrant or toxic organic compounds. • Plasmids can also provide bacteria with the ability to fix nitrogen. • Some plasmids, have no observable effect on the phenotype of the host cell or its benefit to the host cells cannot be determined, and these plasmids are called cryptic plasmids.
- 7. • Naturally occurring plasmids vary greatly in their physical properties. • Their size can range from very small mini-plasmids of less than a 1 kilobase pairs (Kbp), to very large megaplasmids of several megabase pairs (Mbp). • Plasmids are generally circular, however examples of linear plasmids are also known. • Plasmids may be present in an individual cell in varying number, ranging from one to several hundreds. • The normal number of copies of plasmid that may be found in a single cell is called the copy number, and is determined by how the replication initiation is regulated and the size of the molecule. • Larger plasmids tend to have lower copy numbers.
- 8. Classification • Plasmids may be classified in a number of ways. • Plasmids can be broadly classified into conjugative plasmids and non- conjugative plasmids. Conjugative plasmids • Conjugative plasmids contain a set of transfer or tra genes which promote sexual conjugation between different cells. • In the complex process of conjugation, plasmid may be transferred from one bacterium to another via sex pili encoded by some of the tra genes. Non-conjugative plasmids • Non-conjugative plasmids are incapable of initiating conjugation, hence they can be transferred only with the assistance of conjugative plasmids. • An intermediate class of plasmids are mobilizable, and carry only a subset of the genes required for transfer. • They can parasitize a conjugative plasmid, transferring at high frequency only in its presence.
- 10. Incompatibility groups • Plasmids can also be classified into incompatibility groups. • A microbe can harbor different types of plasmids, however, different plasmids can only exist in a single bacterial cell if they are compatible. • If two plasmids are not compatible, one or the other will be rapidly lost from the cell. • Different plasmids may therefore be assigned to different incompatibility groups depending on whether they can coexist together. • Incompatible plasmids normally share the same replication or partition mechanisms and can thus not be kept together in a single cell.
- 11. Based on function • There are five main classes: • Fertility F-plasmids • Resistance (R) plasmids • Col plasmids • Degradative plasmids • Virulence plasmids
- 12. • F-plasmid which contain tra genes. • They are capable of conjugation and result in the expression of sex pili. • The Fertility factor allows genes to be transferred from one bacterium carrying the factor to another bacterium lacking the factor by conjugation. • The F plasmid belongs to a class of conjugative plasmids that control sexual functions of bacteria with a fertility inhibition (Fin) system.
- 13. • OriT (Origin of Transfer): The sequence which marks the starting point of conjugative transfer. • OriC (Origin of Replication): The sequence starting with which the plasmid-DNA will be replicated in the recipient cell. • tra-region (transfer genes): Genes coding the F-Pilus and DNA transfer process. • IS (Insertion Elements) composed of one copy of IS2, two copies of IS3, so-called "selfish genes" (sequence fragments which can integrate copies of themselves at different locations). • Some F plasmid genes and their Function Genes Function traA Pilin, Major subunit of the pilus.
- 14. R-Plasmids • Resistance (R) plasmids, which contain genes that provide resistance against antibiotics or poisons. • Historically known as R-factors, before the nature of plasmids was understood. R-factor was first demonstrated in Shigella in 1959 by Japanese scientists. • Often, R-factors code for more than one antibiotic resistance factor: genes that encode resistance to unrelated antibiotics may be carried on a single R- factor, sometimes up to 8 different resistances. • Many R-factors can pass from one bacterium to another through bacterial conjugation and are a common means by which antibiotic resistance spreads between bacterial species, genera and even families. • For example, RP1, a plasmid that encodes resistance to ampicillin, tetracycline and kanamycin originated in a species of Pseudomonas, from the Family Pseudomonadaceae, but can also be maintained in bacteria belonging to the family Enterobacteriaceae, such as Escherichia coli.
- 16. • Col plasmids, which contain genes that code for bacteriocins, proteins that can kill other bacteria. • Degradative plasmids, which enable the digestion of unusual substances, e.g. toluene and salicylic acid. • Virulence plasmids, which turn the bacterium into a pathogen.
- 17. Plasmid purification from bacteria relies on their unique physical properties Bacterial cell with plasmids contains MANY different, well-folded proteins 1-2 copies of large (>Mbp) , circular bacterial DNA complexed with proteins Multiple copies of small (5-15 kbp) plasmids Purification involves sequential denaturation and renaturation steps
- 18. Cells are first treated with base and a detergent breaks open membrane and denatures both DNA and proteins Proteins denature irreversibly Chromosomal DNA denatures—will have difficulty renaturing because of its length and many proteins complexed to it Plasmids denature, but strands stay together because of supercoiling
- 19. Extract is neutralized to allow DNA molecules to renature Plasmids renature and are suspended in the SUPERNATANT following centrifugation Proteins and chromosomal DNA form aggregate irreversibly, forming a PRECIPITATE that can be collected by centrifugation When purifying plasmids, use a micropipette to remove the supernatant for further processing steps
- 20. Zyppy purification kit use multiple steps to purify plasmids Alkaline lysis Neutralization Purification of plasmid DNA on a silica resin Elution of purified DNA from he silica resin Let's look at the individual steps……………..
- 21. 1 Transformed E. coli cultures are concentrated by centrifugation 2. The cell pellet is resuspended in 600 µL TE buffer by vortexing 3. 100 µL of 7X Blue Zyppy lysis buffer is added 0.1 N NaOH in buffer lyses the cells GENTLY mix the contents by inverting the tube 4-6 times Solution changes from cloudy to clear when cells are lysed Warning: too much mechanical agitation can shear chromosomal DNA Alkaline lysis
- 22. Neutralization 4. Add 350 µL yellow Zyppy Neutralization buffer Mix by inverting several times A heavy precipitate will begin to form immediately! The initial “glop” will become more granular when neutralization is complete—but don’t overdo it! The precipitate contains denatured proteins and the denatured chromosomal DNA. 5. Spin down the denatured molecules for 3 minutes at top speed. CAREFULLY remove the supernatant containing the plasmid – Don't be greedy! Purity is preferred to yield!
- 23. Purification on Zyppy silica resin 6. Apply the supernatant to the spin column. Place the column in the collection tube. Centrifuge the column for ~15 seconds at top speed. 7. Discard the flow through in the collection tube. Add 200 µL Zyppy EndoWash. Centrifuge ~15 sec. EndoWash contains guanidine hydrochloride and isopropanol. It removes contaminating proteins that are bound to the resin. 8. Discard the flow through in the collection tube. Add 400 µL Column Wash. Centrifuge 1 min.
- 24. Plasmid Elution 9. Transfer the column to a clean, LABELED microcentrifuge tube 10. Add 50 µL TE buffer directly on top of the column. Allow the column to stand upright in the test tube for ~10 min. (Plasmid is being eluted.) 11. Spin the column for 30 seconds. Plasmid DNA will be collected in the microcentrifuge tube. Pure plasmid DNA collects here!
- 25. Applications - Vectors • Artificially constructed plasmids may be used as vectors in genetic engineering. • These plasmids serve as important tools in genetics and biotechnology labs, where they are commonly used to clone and amplify (make many copies of) or express particular genes. • A wide variety of plasmids are commercially available for such uses. • The gene to be replicated is normally inserted into a plasmid that typically contains a number of features for their use. • These include a gene that confers resistance to particular antibiotics (ampicillin is most frequently used for bacterial strains), an origin of replication to allow the bacterial cells to replicate the plasmid DNA, and a suitable site for cloning.
- 26. Cloning • Plasmids are the most-commonly used bacterial cloning vectors. • These cloning vectors contain a site that allows DNA fragments to be inserted, for example a multiple cloning site or polylinker which has several commonly used restriction sites to which DNA fragments may be ligated. • After the gene of interest is inserted, the plasmids are introduced into bacteria by a process called transformation. • These plasmids contain a selectable marker, usually an antibiotic resistance gene, which confer on the bacteria an ability to survive and proliferate in a selective growth medium containing the particular antibiotics. • • The cells after transformation are exposed to the selective media, and only cells containing the plasmid may survive. • In this way, the antibiotics act as a filter to select only the bacteria containing the plasmid DNA.
- 28. • The vector may also contain other marker genes or reporter genes to facilitate selection of plasmid with cloned insert. • A plasmid cloning vector is typically used to clone DNA fragments of up to 15 kbp. To clone longer lengths of DNA, lambda phage with lysogeny genes deleted, cosmids, bacterial artificial chromosomes, or yeast artificial chromosomes are used.
- 29. Protein production • Another major use of plasmids is to make large amounts of proteins. • In this case, researchers grow bacteria containing a plasmid harboring the gene of interest. • Just as the bacterium produces proteins to confer its antibiotic resistance, it can also be induced to produce large amounts of proteins from the inserted gene. • This is a cheap and easy way of mass-producing the protein the gene codes for, for example, insulin.
- 30. Gene therapy • Plasmid may also be used for gene transfer into human cells as potential treatment in gene therapy so that it may express the protein that is lacking in the cells. • Some strategies of gene therapy require the insertion of therapeutic genes at pre-selected chromosomal target sites within the human genome. • Plasmid vectors are one of many approaches that could be used for this purpose.
- 31. Plasmid genes 1) Essential genes for keeping the plasmid within the cell Replication: • uses the replication system of the host cell • have its own initiation, elongation and termination • occurs during the entire cell cycle Copy number: • a certain amount of copies present per cell • controlled by the initiation frequency • low (1-4) to high (10-100)
- 32. Partitioning: • only a problem for low and medium copy number • Host specificity/range: • low to broad
- 33. 2) Non-essential –important for transfer • Are spread horizontally (among bacteria) • Important genes - pili-genes - oriT - tra/ mob genes
- 34. 3) Non-essential –with surviving value • Resistance against antibiotics • Production of antibacterial substances (colicins) • genes for pathogenesis/virulence • genes to be able to use special energy/carbon sources, e.g. phenol