- Many bacteria possess additional DNA molecules that are in addition to their bigger genome. These molecules, referred to as plasmids, are widely used in the field of genetic engineering.
- In order to function in laboratories, these plasmids must have an origin (ori) that allows them to reproduce within bacteria. They should also include several restriction enzymes that allow cutting and pasting of DNA into the genome.
- A plasmid can be described as a vehicle that is able to carry artificially implanted DNA. It is able to duplicate within E. coli, and by its own process, it can also reproduce the DNA inserted, independent of the source.
- It is possible to consider a plasmid to be an extremely small DNA manufacturing facility.
- The most important requirement for a “good” the plasmid is to take up the insert that you would like to insert and reproduces in sufficient quantities, and that it doesn’t destroy the insert in the process.
What is plasmid?
- The plasmid can be described as a DNA-based molecule that is distinct from and is able to replicate independently of the chromosomal DNA.
- They are double-stranded and, in a lot of instances circular.
- Plasmids are typically found in bacteria, however they are occasionally found in eukaryotic organisms (e.g. the two micrometre-sized ring found in Saccharomyces cerevisiae).
- The term”plasmid” was first used by American molecular biologist Joshua Lederberg in 1952.
- Joshua Lederberg was an American molecular biologist who was famous for his research in genetics, artificial intelligence, as well as space exploration.
- He was just 33 of age when he received the 1958 Nobel Prize in Physiology or Medicine because he discovered bacteria are able to mate with each other and share genes.
- He was awarded the prize along with Edward L. Tatum and George Beadle who won for their work on genetics.
Characteristics of Plasmids
- Plasmids are DNA-containing molecules that are present in bacteria that are distinct from the bacteria’s the chromosome.
- Plasmids are present in archae, bacteria, and other eukaryotes, including yeast as well as plants.
- They are very small (a couple of thousands of base pair) usually carrying only one or two genes. Genes that are circular, and they share a single source of replication.
- Plasmids replicate using the same mechanism that reproduces the bacteria’s genome. Certain plasmids are copied approximately the same speed as the chromosome. Therefore, it is possible for a single cell to only have one replica of the plasmid. Other plasmids are copied at an large rate, and one cell could have 50 or more copies.
- Plasmids are able to enter the bacterial cells easily. It happens naturally and is a possible reason for the rapid development of resistance to antibiotics in hospitals and other places. Plasmids are introduced into bacteria during laboratory experiments to alter the cell using the new genes.
- Plasmids are self-replicating extrachromosomal DNA molecules that have a small size, which are transferred and exchanged in a sporadic manner among a variety of bacteria and different domains.
- Plasmid classification is generally based on incompatibility group (determined by their replication/partitioning functions) or the genetic information specified by their DNA. Incompatibility grouping has been utilized to classify plasmids from Pseudomonas species and Enterobacteriaceae into 26 groups of incompatibility.
- The majority of plasmids operate within a narrow host range, which allows only intraspecies transmutation and reproduction. However, a select collection of plasmids known as”the broad host range (BHR) the plasmids (Inc the plasmids P, Q. W. N., and C) are transferable and replicated in a vast variety of bacteria. BHR plasmids can have self-transmissibility (Tra+ Mob+, Tra+) or able to be mobilized however they are not self-transmissible (TraMob+, Tra).
- The circular DNA fragment autonomously reproducing DNA
- Could express the antibiotic resistance gene and be altered to produce proteins that are of interest.
Shapes of plasmids
There are 5 shapes of plasmids;
- Nicked Open-Circular: DNA has one strand cut.
- Relaxed Circular: DNA is fully intact.
- Linear: DNA has free ends.
- Supercoiled: DNA is completely intact, but it has a twist to it which makes it smaller.
- Supercoiled Denatured: A little smaller in size than supercoiled.
Size of Plasmid
Smaller plasmids are few in number and only a single larger plasmid is present in each bacterium.
- The size of the plasmid varies from several Kelo basepair to several megabase pairs, depending on the type of plasmid.
- One of the important properties of the plasmid DNA is the horizontal gene transfer through the process of conjugation.
- The plasmid is a self-replicating element that is inherited in each bacterium during cell division.
- The size of the plasmid DNA is generally 1kb to 2 kb (used in genetic research), the smaller size of it makes it easy to create and modify for genetic engineering.
- Also, the stability of the plasmid DNA is very high. It can remain stable for a longer period of time as in a purified state or in the bacterial cell.
- The plasmid can be used in many different types of species for gene transfer and gene therapy experiments.
Structure of plasmids
- Regarding the structure of plasmids, they are composed by circular double chain DNA.
- The circular shape of plasmids is created through the two ends of double chains becoming joined through covalent bonds.
- The molecules are also very small in size, specifically in comparison to DNA of living organisms.
- They range between a few thousand kilobases to several hundred kilobases.
- While a majority of plasmids exhibit the covalently closed circular shape however, some plasmids possess an elongated structure and don’t have the circular shape.
- In general, plasmids are made up of three main components which comprise:
1. Origin of replication (replicon)
- The place of origin for replication (ori) is an exact location within the strand from which replication starts.
- For plasmids this area is mostly composed of A-T base pair pairs that are simpler to divide when undergoing replication.
- In comparison to the organisms’ DNA, which has many types in replication.
- Moreover, the plasmids are one of the few sources of replication since their size is smaller.
- The origin of the process, they contain several regulators that are involved in replication (e.g. Rep proteins).
2. Polylinker (multiple cloning sites)
- In the plasmid the polylinker (MCS) is among the most crucial components of the molecular. It is due to its ability to allow students to gain knowledge about the process of cloning.
- A polylinker is a brief sequence of DNA composed of several locations for cleavage through restriction enzymes. In this way, MCS allows for the simple insertion of DNA by the ligation process or digestion with restriction enzymes.
- On the site of cleavage different polylinkers are able to slice the DNA strand.
- So any of these restriction enzymes may cut the plasmid in specific places on the sire, allowing for DNA to be inserted.
3. Antibiotic resistance gene
- The gene for resistance to antibiotics is one of the primary elements of plasmids. These genes play a significant part in the development of resistance to drugs (to any or all of the antibiotics) and make treatment for certain diseases more difficult.
- Plasmids are renowned today for their capacity to transfer genes from one species or bacterium to another in the process of conjugation (contact between cells followed by the transfer of the DNA contents).
- Through this process, they can confer antimicrobial resistance to different species of bacteria. Although plasmid replication offers an advantage for bacteria (resistance to specific antibiotics) however, it can also impact the division of bacteria’s cells due to the increased burden of replication.
- This is why bacteria that have plasmids tends to be fewer populated than the ones without plasmids because of the lower rate of cell division.
4. Some of the other components of plasmids include
- A promoter region: A promoter region is the part of plasmids responsible for recruiting the transcriptional machinery.
- Primer binding site: A primer binding site is a small sequence of DNA that is on one strand, which is usually used to aid in the process of PCR Amplification as well as DNA sequencing.
- Selection Marker: The antibiotic resistance gene (ARG) plays a significant function in the development of resistance to drugs, that allows the selection of bacteria that have plasmids. Apart from ARGs some plasmids contain other selection markers, either in nature , or via artificial introduction.
While plasmids have a variety of general features, there are several kinds of plasmids that exist.
Types of Plasmids
Classification of Plasmids based on Function
1. Fertility Plasmids (F PLASMID)
- The F-plasmid is also known as the fertility or sex-factor determines the sexuality of E. Coli bacteria. Cells that contain this plasmid are identified as F+ and those lacking it are referred to as F-.
- F+ bacteria may be regarded to be male due to the fact that they may act as a donor of not just the plasmid but also chromosomal genes that are transferred to the Fcells that act as recipients, and are consequently, considered female.
- Transfer is accomplished by the conjugation of F+ cell to the F-cell. The F-plasmid can be described as a conjugative plasmid.
- A distinctive aspect of the F-plasmid is that it is able to exist as an individual entity that replicates in isolation from the chromosomal DNA. Or, it could be integrated into the chromosome as an integral component.
- When an F-plasmid gets integrated in the E. the chromosome of coli, the bacterial cell shifts from a P-cell to an Hfr-strain (high frequency of Recombination). There are a variety of locations within the E. Coli chromosome that the F-plasmid is able to be integrated.
- The site of integration is dependent on the one each integration leads to a new Hfr-strain. For F+ x F conjugation, only the plasmid is transmitted.
- In Hfr x F conjugation, chromosomal genes will be transferred, and sometimes also the F-plasmid. The F-plasmid can be described as a self-transmitting plasmid with the double-stranded circular DNA molecules.
- Its molecular weight is 63x the number of Daltons, and it has several genes that control how the plasmid is transferred its donor into the receiver.
- Any mutation to any of the genes that are essential result in the demise of transmission of the plasmid.
- As an F-plasmid is able to be embedded into the chromosome that is found in the cells of its host this can, in occasions be isolated or removed of the E. Coli chromosome in its unbound state, forming an elongated plasmid.
- It has been found that the process of excision is not always perfect in the sense that portions from the E. cocci chromosome that connect to the F-plasmid which has been linearly integrated are contained in the excised F DNA as well certain parts of the plasmid DNA remain within the E. the chromosome of coli.
- The F-plasmids that contain a portion that are chromosomal DNA have been designated as F’-plasmids.
- If an F-plasmid sheds certain vital genes in the process of elimination the plasmid becomes in a state of non-existence and, in the end, removed when cells divide.
- If an F-plasmid is transferred through conjugation with an F-recipient, it is able to transfer the genes that it carries on its chromosomes.
- In this way, the recipient is diploid in relation to the transferred genes (because it now has one copy of its own , and another version of that gene that is transmitted through its F the plasmid). Therefore, the exchange of chromosomal genes can be carried out via F’-plasmids. This is known as sexual-duction.
- Fertility Plasmids (F PLASMID) carry the fertility genes (tra-genes) to facilitate conjugation, which is the transmission of information genetic between two cells.
2. Resistance Plasmids (R PLASMID)
- R-plasmids conferring resistance to various drugs individually or multiple resistance to several antibacterial agents were first discovered in Japan in the 1950s in the gastroenteritis-causing Shigella dysenteriae.
- Since then, these plasmids were discovered inside E. coli and other enteric bacteria. The plasmids are an extremely dangerous threat to medical sciences.
- The massive R-plasmids that have molecular weights that range from 30 to x the size of 106 Daltons are self transmittable through the conjugation process to other bacteria.
- They are therefore conjugative plasmids like the F-plasmid. smaller R-plasmids that have molecular masses of 5-6 x 106 Daltons are not transmissible.
- The majority of self-transmissible large plasmids such as R100 of Shigella that confer multiple drug resistance are composed comprised of 2 DNA fragments linked to one another by covalent linkage , forming an unidirectional circular molecule.
- One DNA segment is known as”the factor that transfers resistance (RTF) in the other one contains drug resistance genes. The RTF plays a major role with the function for transfer of R-plasmid.
- It has a range in the form of gene (the transfers genes) along with other ones that regulate the replication of the plasmid within cells of the hosts.
- The resistance genes in the second segment produce enzymes to destroy antibiotics, such as penicillins streptomycin and chloramphenicol. the sulfonamides, kanamycin, tetracycl and others.
- In some bacteria that are resistant to drugs for example, Salmonella Typhimurium strain 29, The resistance gene is not on the same plasmid however, in separate plasmids with different sizes. This is often referred to as the aggregation of plasmids.
- The transposable elements in complex transposons could contain genes for resistance to drugs. They can be incorporated into plasmids resulting in a plasmid for resistance to drugs.
- So, R plasmids can consist of transposons each carrying at least one gene that confer resistance to antibiotics. For instance, Tn 5 carrying a resistance gene to kanamycin might be added to the plasmid R100 of Shigella giving the plasmid the ability to fight the antibiotic.
- In addition to resistance to drugs, plasmids could also render bacterial hosts immune to the harmful negative effects caused by heavy metals.
- Plasmid-coded resistance to nickel mercury, cobalt, arsenic, and cadmium has been documented in various species that belong to the genera Pseudomonas, Escherichia, Salmonella and Staphylococcus.
- Include genes that create resistance to antibiotics or poisons.
3. Col Plasmids
- The Col-plasmids reside in a variety of varieties from E. coli and they contain genes that control the production of a family of proteins known as colicines.
- Colicines can inhibit the growth of the related bacteria that do not have the colon-plasmid (Cor). A variety of different Col-plasmids have been identified and each one produce colicines with a distinct mechanism of inhibiting susceptible bacteria.
- For instance Col B is responsible for causing damage to the cytoplasmic membranes of the bacteria in question, while the Col E2 or Col E3 are responsible for the degrading of the nucleic acids. Similar to R-plasmids and Col-plasmids they could be self-transmitting or not.
- Large Col plasmids like Col I or Col V-K94, with molecular weights of 60x more than 106 Daltons are self-transmissible. They possess a tiny copy number, typically one to three replicas per cell. Small Col-plasmids like Col-El, possess a molecular weight that is between 4 and 5 x the number of Daltons.
- They possess a large number of copies, typically between 10 and 30 copies for each cell. They are self-non-transmissible, but may be mobilized with the help of F-plasmid. This means that if an F+-cell also has the Col El plasmid and conjugates with an F-cell and is able to mat with an F-cell, it is possible that the Col El plasmid can be transferred to the recipient via the mating bridge that is created through the F-plasmid.
- It is evident that F-ColEl+ cells are incapable of transferring the Col-plasmid into another cell because it is not able to construct the mating bridge. Contrary to this, the large Col-plasmids are self-transmitting, as they possess the genes to build the conjugation apparatus on their own and don’t rely on the F-plasmid’s the transfer of their genes to cells.
- As with F and the large R-plasmids the large Col-plasmids also function as conjugative plasmids. Colicins are part of a broad group of proteins known as Bacteriocins.
- A variety of bacteria have been observed to make bacteriocins, which can kill others that are related to or unrelated to bacteria. The proteins that are created are coded by genes found in Bacteriocinogenic (plasmids).
- Bacteriocins made by different bacteria can be identified by different names, for example, the pyocine created by Pseudomonas aeruginosa, megasine developed through Bacillus megaterium and nisin produced by lactobacilli, and so on.
- Bacteriocins generally exert their antibacterial effects by adhering to the cell wall of the target cells .
- They also work by blocking one of the essential metabolic processes like replication of nucleic acid, the transcription process, protein synthesizing, or the metabolism of energy.
- Bacteriocins that are produced by bacteria in the enteric environment aid in maintaining the balance of nature within the human colon.
- Other bacteriocins created by the bacteria in the natural environment may work by removing competition. Nisin that is made by the lactic acid bacteria is commercially employed to preserve dairy products and foods.
- Col Plasmids contain genes that encode antibacterial polypeptides known as bacteriocins. which kills different strains of bacteria. The proteins that make up the col of E. Coli contain proteins, such as Col E1.
4. Degradative Plasmids
- Dissimilation or degradation of organic compounds as part of mineralization is usually controlled by plasmid-borne genes within numerous microorganisms.
- The plasmids that contain genes that code to catabolize organic compounds are commonly referred to as dissimilation or degradative. For instance, in the species like Pseudomonas both chromosomal and plasmid genes generate enzymes that aid in the degrading complex compounds.
- A few of the plasmid gene encode enzymes that degrade these unusual compounds such as camphor, toluene and naphthalene salicylate, as well as complex hydrocarbons from crude petroleum.
- By utilizing these enzymes, bacteria can make use of these compounds to produce carbon and energy sources. This means that bacteria with degradative plasmids have a greater chance of survival in conditions in which these unique substances are in the market.
- Normal bacteria lacking such enzymes coding for plasmids would be destroyed in similar conditions. The ability of organisms that carry degradative plasmids that can metabolize various complex compounds raises that they could be employed to help in the bioremediation process of the environment that is polluted.
- The advent of techniques for genetic engineering has prompted scientists to create strains of bacteria that are genetically enhanced which contain plasmids capable of the degradation of a variety of complex chemicals like those found naturally in petroleum crude.
- A Pseudomonas strain that is synthetic was created through Anandamohan Chakraborty of the university of Illinois, USA offering prospects that can be used in the future to eliminate spills of oil in the oceans resulted from leaks in tankers of petroleum crude. Oil spills pose a serious threat for marine life, including plants as well as animals.
- Degradative Plasmids allow digestion of unusual substances.
5. Virulence Plasmids
- Virulence-plasmids can confer pathogenesity to the host bacteria.
- They make the bacterium pathogenic because the bacterium becomes better equipped to fight the host’s defense or produce toxic substances.
- For instance, Ti-plasmids belonging to Agrobacterium tumefaciens can cause the disease known as crown gall in angiospermic plants.
- Entertoxigenic species from E. coli cause traveller’s diarrhoea as a result of the plasmid which encodes for an enterotoxin, which triggers the discharge of salts and water in the bowel.
- Virulence Plasmids transform a bacterium into a pathogen.
- Episomes are an abacus-like plasmid or viral DNA that has the ability to incorporate itself in the DNA chromosomal an organism that hosts it .
- This is why it is able to remain in place for a lengthy period and be replicated in every cell division in its host. It can also eventually become an essential part of its genetic composition.
Classification of Plasmids based on their Ability To Transfer To Other Bacteria
- Conjugative plasmids: Conjugative plasmids contain the ‘tra’ ( sexual transmission to genetic material) that carry out the complicated process of conjugation. It is the transfer of plasmids into another bacteria.
- Non-conjugative plasmids: Non-conjugative Plas cannot initiate conjugation, so they are only transferable with the help from conjugative plasmids.
- Intermediate classes of plasmids: The intermediate classes have the ability to be mobilized and carry only one subset of genes necessary to transfer. They can also parasitize an elongative plasmid, and transmit at a high rate only when it is present. Plasmids are currently being used to manipulate DNA , and could be a method of treating a variety of diseases.
Replication of Plasmids
- Plasmids replicate independently because they are able to replicate independently due to their own sources. The enzymes that are involved with plasmid reproduction are common cell enzymes, particularly in the instances smaller plasmids. However, some plasmids have genes that encode for specific enzymes for replication of plasmids.
- Plasmids have a small number of genes, usually under 30, and these genes are involved predominantly with controlling the replication initiation process as well as distribution of replicated plasmids to daughter cells. The genetic information contained in the plasmid genes are not vital to the host since the bacteria without them typically function normally.
- Because the plasmid DNA itself is very small and is very small, its replication happens very fast, possibly in one tenth or less than the duration of the cell division.
- The majority of plasmids found in bacteria that are gram-negative replicate in a similar manner to the replication process of the bacterial genome, with the chromosome beginning at the origin of replication in bidirectional replication of the DNA circular resulting in the Theta (O) middle.
- But, some plasmids from bacteria that are gram-negative replicate using a unidirectional methods. The majority of gram-positive plasmids replicate using a circular process similar to that employed by phages phx174. The majority of linear plasmids reproduce by using a mechanism that involves a protein bonded to the 5′-ends of each DNA strand used to initiate DNA synthesizing.
Functions of Plasmids
- Plasmids permit bacteria (including different realms) populations to “sample” the horizontal gene pool to identify adaptive traits that could be advantageous to survival under pressures of local selection.
- Plasmids also allow genetic variation, and act as a source of recombination, and allow for quicker gene fixation, leading to a greater chance that the “new” characteristic will last.
- A key role in the process of forming Play a crucial role in. Many plasmids possess at least two distinct markers that confer distinct characteristics to the bacteria. Some of these markers are antimicrobial resistance (ampR) or the expression in an enzyme which catalyzes the reaction that causes a change in color (lacZ).
Plasmids and Ampicillin Resistance: Certain plasmids contain ampR genes, which confers resistance to ampicillin, an antibiotic. E. bacteria cells that have this plasmid, referred to as “+ampR” cells are able to thrive and grow into colonies on LB Agar which is added with ampicillin. However, cells that do not have ampR plasmids, also known as “-ampR” cells are tolerant to the antibiotic that eliminates them. The ampicillin-sensitive cells ( ampR) is able to transform into the ampicillin-resistant (+ampR) cell through the acceptance of an external plasmid that contains ampR genes.
- Plasmids play a crucial role also in Genetic Engineering. Plasmids are highly valuable instruments in the field of genetics and molecular biology particularly in the field in genetic engineering. They play an essential part in procedures such as gene creation, cloning, and recombinant protein production (e.g. the insulin from humans) and research into gene therapy. In these processes the plasmid is cut at a specific location (or locations) with enzymes known as restriction endonucleases.
- Plasmids are vectors. Plasmids are essential vectors of horizontal gene transfer, and are crucial tools for genetic engineering. They encode for genes that are involved in a variety of aspects of the microbial biochemistry, including detoxicationand virulence, ecological interactions, as well as antibiotic resistance.
- Plasmids created a variety of Drug Resistance mechanisms. The role played by plasmids in the evolution of the bacterial genome as well as adapting to changes in the environment has been a major factor in the rise of antibiotic as well as heavy resistance plasmids.
- Plasmids are used in Gene Therapy. The effectiveness of gene therapy is dependent on the successful introduction of therapeutic genes to the correct chromosomal targets within the human genome without causing cell damage as well as oncogenic mutations that trigger the triggering of an immune reaction. While viral vectors are an excellent vehicle for the highly efficient transmission of human cells the security concerns associated with them makes non-viral delivery for therapeutic genes using the plasmid DNA of cells more appealing.
- A vector is a component of a molecule which contains genetic material that could be duplicated and expressed when transferred to another cell.
- In light of this definition, it’s easy to see the reason why “vector” or “plasmids” are often used interchangeably. But, this isn’t to suggest that all plasmids can be considered vectors.
- One of the main characteristic of plasmid vectors is their small in size. In addition to their size they also have an origin for replication, a specific marker and multiple Cloning sites.
- The most effective plasmid vectors are those that are those that have high copy numbers within the cell. Therefore, they ensure that there are plenty of copies of the gene targeted to be cloned for purposes.
- It also assures that the gene of importance is enhanced during the process of genomic division. Additionally the plasmid may contain an indicator gene that acts as a visual marker that helps determine whether the cloning process was successful.
- Because of their many sites for cloning, plasmids are among the best sources to clone. Because of this it is feasible for restriction enzymes that cleave several parts of the plasmid in order to allow Cloning.
- In the past, the use of these vectors has made it possible for the introduction of recombinant DNA into host cells for research for research purposes.
- In particular, with this method of cloning it has been possible for scientists to sequence the genomes of various species, examine how genes are expressed, and even examine various cellular processes.
- Although smaller plasmids can be capable of carrying lengthy DNA segments, their smaller size may also allow for the removal of non-essential genes which aren’t needed to clone.
Isolation of Plasmid
In order to get pure plasmid DNA for processes as cloning and transfection and PCR the process of plasmid isolation must be carried out. This involves using various methods to collect the plasmid DNA of host cells to utilize it for molecular biology. Plasmid extraction involves several steps.
- Cell development (growth of the bacterial cells) – The process involves the growth of the plasmid-containing bacteria in the specific shaken culture. The antibiotics could be used to inhibit the growth of unwelcome bacteria.
- Centrifugation – Bacterial growth will be followed by centrifugation to remove the cells. After the supernatant has been eliminated, the it is possible to isolate plasmids.
The most well-known methods for isolation is the classic method, which is often called alkaline lysis. It includes the steps below:
- Suspension of the pellets in an isotonic solution bacteria’s pellet that is obtained by centrifugation can be re-suspended within the isotonic liquid (ethylene diamine Tetraacetate) which blocks nuclease activity.
- The cells are lysed by alkaline is the process of removing cells with sodium dodecyl sulfur to degrade the lipid structure of the cell membrane
- Precipitation of proteins that are dissolved using the solution of acidic potassium acetate
- The process of sedimentation, centrifugation, is employed to help in sedimentation
- Purification A mixture of chloroform and phenol is used to purify the DNA plasmid. This procedure removes protein from the DNA.
- Include ethanol to help precipitate (to be sifted through centrifugation)
- Clean the solution using 70% of ethanol (to get rid of salt)
- Centrifuge to remove the plasmid DNA
- In TE solutions, dissolve, and keep in a storage container.
Example of Plasmids
1. pBR322 Plasmid
- PBR322 is a plasmid that was among the first widely utilized E. coli cloning vectors.
- It was created in 1977 in the lab by Herbert Boyer at the University of California, San Francisco It was named in honor of Francisco Bolivar Zapata, the postdoctoral researcher, and Raymond L. Rodriguez. The p symbolises “plasmid,” and BR stands for “Bolivar” as well as “Rodriguez.” The pBR322 genome is 4361 base pair in length.
- It also contains two resistance genes to antibiotics – the gene bla that encodes the ampicillin resistance (AmpR) protein as well as the gene tetA that encodes the tetracycline resistance (TetR) Protein.
- It is the source of replication of pMB1 and the rop gene that encodes a restriction factor for copy number of the plasmid.
- The plasmid is unique in its restriction sites that are used by more than forty restriction enzymes. Eleven of the forty sites are located inside the TetR gene.
- There are two the restriction enzyme HindIII and ClaI in the promoter of TetR gene. There are six important restriction sites within the AmpR gene.
- The origin for these resistance genes comes from pSC101 for Tetracycline as well as RSF2124 to make Ampicillin.
- Its circular structure is named in such a way that 0 represents in the middle distinctive EcoRI site, and the number rises with the TetR gene.
- If you need to get rid of ampicillin, for instance you must employ restriction endonuclease, or molecular scissors to remove PstI.
- Then pBR322 becomes anti-resistant to ampicillin .The identical procedure of Insertional Inactivation could also be used to Tetracycline.
- This gene is known as the AmpR gene encodes penicillin beta-lactamase. Promoters P1 as well as P3 are the beta-lactamase genes.
- P3 is the primary promoter and P1 is made by the ligation of two DNA fragments in order to make pBR322.
- P2 is located in the exact area as P1, however it’s situated on the opposite strand, and starts transcription that is directed towards the TCR gene.
2. Ti Plasmid
The Tumour-inducing plasmid is found in the bacteria Agrobacterium Tumifaciens.
It is extensively used to clone vectors in order to transfer desired genes to host plants for the purpose of creating transgenic plants. The most important traits of Ti plasmid include:
- The size of the plasmid about 250kbp
- There are a variety of Ti plasmids, based on the diverse genes they carry and that code for different opioids, e.g. leucinopine, nopaline, octopine, etc.
- The pathogen is species that affects numerous dicotyledonous plants. It is responsible for the development of the disease known as crown gall in plants.
- It includes one or more T-DNA regions.
- Agrobacterium tumifaciens is able to convert normal cells into tumour cells through the introduction of the DNA fragment known as T DNA . it begins producing chemicals which are required by the bacterium.
- Once the gene is inserted into Ti plasmid the plasmid is no longer pathogenic, but can still introduce the gene desired into the plant cells
- It has vir or virulence gene, which transfers the T-DNA region onto plants cells, and is incorporated into the genome of plants.
- Ti plasmid is able to be altered according to the need to insert desired genes
- Agrobacterium tubifaciens is known as “nature’s Genetic Engineer”