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Study of Cloning Vectors.pptx unit-2 Biotechnology 6th Sem

Study of cloning vectors.. plasmids,cosmids,bacteriophage,yeast,In biotechnology, a cloning vector is a DNA molecule used to carry foreign DNA into a host cell, where it can be replicated and/or expressed. Essentially, it's a vehicle for transferring genetic material for cloning, which is the process of creating multiple copies of a specific DNA sequence. Common types of cloning vectors include plasmids, bacteriophages, cosmids, and artificial chromosomes. Origin of Replication (ori): A specific DNA sequence that allows the vector to replicate independently within the host cell. Multiple Cloning Site (MCS): A region with several unique restriction enzyme recognition sites where the foreign DNA fragment can be inserted. Selectable Marker: A gene that provides a way to identify host cells that have successfully taken up the vector, often conferring antibiotic resistance. Types of Cloning Vectors: Plasmids: Small, circular DNA molecules found in bacteria that can replicate independently. They are widely used as cloning vectors due to their ease of manipulation and ability to carry relatively small DNA inserts. Bacteriophages: Viruses that infect bacteria. Some bacteriophages can be engineered to carry foreign DNA and are used as vectors. Cosmids: Hybrid vectors combining features of plasmids and bacteriophages, allowing them to carry larger DNA fragments than typical plasmids. Artificial Chromosomes: Engineered chromosomes that can carry very large DNA fragments. Examples include Yeast Artificial Chromosomes (YACs) and Bacterial Artificial Chromosomes (BACs). How Cloning Vectors are Used: 1. Insert DNA: The gene of interest is cut out of a source DNA using restriction enzymes and then ligated (joined) into the cloning vector. 2. Transformation: The recombinant vector (vector with the inserted DNA) is introduced into a host cell (often bacteria). 3. Selection: Cells that have taken up the vector are identified using the selectable marker (e.g., antibiotic resistance). 4. Replication and Expression: The vector replicates within the host cell, producing many copies of the inserted DNA. If needed, the gene can also be expressed, producing the corresponding protein. A cloning vector is a small piece of DNA that can be stably maintained in an organism, and into which a foreign DNA fragment can be inserted for cloning purposes. The cloning vector may be DNA taken from a virus, the cell of a higher organism, or it may be the plasmid of a bacterium.

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Study of Cloning Vectors By G.Raghava Ravali M.Pharm Pharmaceutical Chemistry
G.RAGHAVA RAVALI Terms Gene Manipulation(deliberate modification-change of nucleic acid) DNA cloning(separating and copying of genetic sequence) Foreign DNA or Passenger DNA or DNA insert(DNA fragments)
G.RAGHAVA RAVALI Cloning Vectors • The Vectors are the DNA molecules that carry foreign DNA segment and replicate inside the host cell. • Also Called Vehicle DNA • Any extrachromosomal small genome can be used as a vector.
G.RAGHAVA RAVALI Characteristics of Cloning Vectors • Autonomous Replication • Easy Isolation, purification and insertion into the host • Easy selection of transformed cells through marker genes • Integration with the DNA inserts • Specific control systems like promoters, terminators, operators.
G.RAGHAVA RAVALI
G.RAGHAVA RAVALI Types of Vectors • Plasmid Vectors • Bacteriophage Vectors • Cosmid Vectors • Shuttle Vectors • Yeast Vectors • Expression Vectors
G.RAGHAVA RAVALI Plasmid Vectors A plasmid vector is a DNA molecule, typically circular and double- stranded, that is used to introduce foreign genetic material (DNA) into a host cell. It's a type of cloning vector, essentially a vehicle for carrying and replicating genes. Plasmids naturally exist in bacteria and some other microorganisms, but they can also be engineered for use in genetic engineering and biotechnology.
G.RAGHAVA RAVALI Characteristics of plasmid Vectors Circular and Extrachromosomal: Plasmid vectors are typically circular DNA molecules, separate from the host cell's chromosomal DNA. Self-Replication: They contain an origin of replication (ori) that allows them to replicate independently within the host cell. Multiple Cloning Site (MCS): Many plasmid vectors include a multiple cloning site, which is a region with several unique restriction enzyme recognition sites, making it easy to insert foreign DNA fragments. Selectable Markers: They often carry genes that provide a selectable advantage to the host cell, such as antibiotic resistance, allowing researchers to easily identify cells that have taken up the plasmid. Promoters and Other Regulatory Elements: Some plasmid vectors include promoters and other regulatory elements to control the expression of the inserted gene.
G.RAGHAVA RAVALI How Plasmid Vectors Work: 1. Gene Insertion: Foreign DNA is inserted into the MCS of the plasmid. 2. Transformation: The recombinant plasmid (containing the foreign DNA) is introduced into a host cell (e.g., bacteria, yeast, or mammalian cells). 3. Replication: The plasmid replicates inside the host cell, producing multiple copies of the foreign DNA. 4. Gene Expression: If the plasmid is designed as an expression vector, the inserted gene can be transcribed and translated into protein.
G.RAGHAVA RAVALI Working of Plasmids 1. Introduction of the Foreign DNA:A gene of interest (the foreign DNA) is isolated and cut using restriction enzymes. A plasmid vector is also cut with the same restriction enzyme, creating compatible "sticky ends". The foreign DNA is then inserted into the plasmid vector using DNA ligase, forming a recombinant plasmid. 2. Replication and Propagation:The recombinant plasmid is introduced into a suitable host cell (often bacteria) through a process called transformation. The plasmid carries an origin of replication (ori) sequence, which allows it to replicate independently of the host cell's chromosomal DNA. As the host cell replicates, the plasmid and its cargo of foreign DNA are also replicated, resulting in multiple copies of the recombinant DNA. 3. Expression and Applications:If the plasmid contains the necessary regulatory elements (like promoters), the foreign gene can be transcribed and translated into a protein within the host cell.This principle is used to produce proteins of interest, such as insulin, in large quantities using bacteria as factories.Plasmids can also be used for gene therapy, where a functional gene is introduced into a patient's cells to correct a genetic defect.They are also essential tools in research for studying gene function and manipulating DNA.
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G.RAGHAVA RAVALI
G.RAGHAVA RAVALI
G.RAGHAVA RAVALI pBR322 pBR322 is a widely used cloning vector, specifically a plasmid, in molecular biology. It is a circular, double-stranded DNA molecule derived from the E. coli plasmid ColE1. pBR322 is known for its usefulness in recombinant DNA technology, particularly for cloning and gene expression studies.
G.RAGHAVA RAVALI 1. Origin of Replication (ori): The ori site allows the plasmid to replicate independently within the host cell, ensuring that the inserted gene is also replicated.This feature is crucial for amplifying the cloned gene along with the plasmid DNA. 2. Selectable Markers (Antibiotic Resistance Genes): pBR322 contains two antibiotic resistance genes: one for ampicillin resistance (AmpR) and another for tetracycline resistance (TetR).These genes act as selectable markers, allowing researchers to differentiate between cells that have taken up the plasmid (transformed cells) and those that haven't.For example, if a gene of interest is inserted into the TetR gene, it disrupts the TetR gene, and the transformed cells will only be resistant to ampicillin, not tetracycline. 3. Multiple Cloning Site (MCS): pBR322 has several unique restriction enzyme sites within its MCS.These sites allow for the insertion of foreign DNA fragments into the plasmid using enzymes that cut at specific DNA sequences.The MCS is typically located within one of the antibiotic resistance genes, allowing for the use of insertional inactivation for selecting recombinant clones. 4. Other important features: Size: pBR322 is relatively small (4361 base pairs), making it easier to handle and manipulate. Sequenced: It has been completely sequenced, meaning its entire DNA sequence is known, which is helpful for designing experiments. Mobility: pBR322 is known to be relatively mobile between cells, which can be a limitation for some applications. In summary: pBR322 functions as a stable and reliable cloning vector due to its origin of replication, selectable markers, and multiple cloning sites. It has been extensively used for gene cloning, recombinant DNA research, and protein expression studies in various applications. Working Of Pbr322
G.RAGHAVA RAVALI pUC19 pUC19 is a widely used, small, circular, double-stranded DNA molecule that serves as a cloning vector in bacteria like E. coli. It's a modified version of the pBR322 plasmid, known for its high copy number and multiple cloning sites. The plasmid is 2,686 base pairs long and contains an ampicillin resistance gene, allowing for selection of cells carrying the plasmid.
G.RAGHAVA RAVALI Working of pUC19 High Copy Number: pUC19 replicates frequently in host cells, resulting in many copies of the plasmid, which is useful for producing many copies of inserted DNA. Multiple Cloning Site (MCS):A region containing several restriction enzyme recognition sites, facilitating the insertion of foreign DNA fragments. Ampicillin Resistance Gene:Enables selection of bacteria that have taken up the plasmid by growing them in the presence of ampicillin. LacZ Gene Fragment:A portion of the lacZ gene, encoding β-galactosidase, is present and allows for blue-white screening of recombinant colonies.
G.RAGHAVA RAVALI Uses of pUC19: Cloning:pUC19 is commonly used for cloning genes and other DNA fragments in E. coli. Blue-White Screening:The presence of the MCS within the lacZ gene allows for easy identification of bacterial colonies that contain inserted DNA fragments. Teaching Aid:Its well-defined characteristics and ease of use make it a popular tool for teaching molecular biology techniques. In essence, pUC19 is a versatile and widely used tool for genetic engineering and molecular biology research.
G.RAGHAVA RAVALI Bacteriophage vectors Bacteriophage vectors are viruses that infect bacteria and are used in genetic engineering to carry foreign DNA into bacterial cells. They are particularly useful because they can accommodate larger DNA fragments than some other cloning vectors like plasmids. Common bacteriophage vectors include lambda phage, M13 phage, and P1 phage.
G.RAGHAVA RAVALI Characteristics of Bacteriophage Large DNA capacity: Bacteriophages can carry larger DNA fragments than plasmids, allowing for the cloning of larger genes or gene libraries. Efficient delivery: Bacteriophages are highly efficient at infecting and delivering their DNA into bacterial cells. Versatile: They can be used in various applications, including gene cloning, DNA sequencing, and creating genomic libraries.
G.RAGHAVA RAVALI Working of Bacteriophage • Bacteriophage vectors are viruses that only infect bacteria and transform them efficiently while carrying large inserts. • Bacteriophages or phages have higher transformation efficiencies which increase the chances of recovering a clone containing the recombinant DNA segments. • The most important feature of a phage is the packaging system which enables the incorporation of large eukaryotic genes and their regulatory elements. • The use of phages also facilitates the isolation of larger quantities of DNA that can be used for the analysis of the insert. • Even though there are a number of phages that can and have been used as vectors, phage λ is the most convenient cloning vector. • It can selectively package a chromosome about 50 kb in length, and the size of the phage can be adjusted by removing the central part of the genome as it is not necessary for replication or the packaging of the donor DNA. • The use of a bacteriophage vector that can incorporate larger DNA segments decreases the number of clones required to obtain a particular DNA library with the entire genome of the organism. • Phage vectors are also effective as cloning vectors as the recombinant molecules formed after the cloning process are packaged into infective particles that can then be stored or handle efficiently. • Some of the common phages used as vectors include M13 phages, λ phages, and P1 phages.
G.RAGHAVA RAVALI λ phage λ-phage is an example of a bacteriophage that infects the bacterial species, Escherichia coli (E. coli). This vector is more effective than other plasmid vectors as it has a higher efficiency in entering bacterial cells so as to incorporate the recombinant DNA within the host genome. It is a double-stranded DNA bacteriophage that contains an ori sequence requires for replication and a number of DNA sequences encoding regulatory and replicative proteins. The phage DNA replicates by the combination of theta and rolling circle replication process to produce a linear dsDNA. It is then followed by the cos sequence, which enables the circularization of the genome after infection. The DNA sequences between the two arms of the vector are not essential which are then replaced with the recombinant DNA during cloning.
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G.RAGHAVA RAVALI Uses of Bacteriophage 1. Gene Cloning: Cloning larger DNA fragments:Bacteriophages can accommodate larger DNA inserts compared to plasmids, making them suitable for cloning larger genes or genomic DNA fragments. Efficient screening:Screening for recombinant phage clones (those containing the desired DNA insert) is often easier than screening bacterial colonies, particularly for large libraries. Examples of phage vectors:Lambda phage: Used for cloning larger fragments of DNA, with both insertion and replacement vectors available. M13 phage: Used for generating single-stranded DNA copies, which is useful for sequencing and mutagenesis. 2. Molecular Biology Research: Studying gene structure and function:Bacteriophage vectors can be used to introduce specific genes into bacterial cells, allowing researchers to study gene expression and protein production. DNA sequencing:M13 vectors are particularly useful for generating single-stranded DNA templates for sequencing.
G.RAGHAVA RAVALI 3. Biotechnology Applications: Phage display:Bacteriophages can be engineered to display foreign peptides or proteins on their surface, allowing for the selection and identification of binding partners (e.g., for drug discovery). Phage therapy:Bacteriophages are being explored as a potential alternative to antibiotics for treating bacterial infections, especially those caused by antibiotic-resistant bacteria. Vaccine development:Bacteriophages can be used to deliver vaccine antigens to the immune system. Biosensors:Bacteriophages or their components can be incorporated into biosensors for detecting bacteria, toxins, or other molecules. Food preservation:Bacteriophages can be used to control the growth of spoilage or pathogenic bacteria in food products. 4. Other Uses: Bacterial typing:Bacteriophages can be used to identify specific strains of bacteria (phage typing). Delivery of therapeutic genes:Bacteriophages can be modified to deliver therapeutic genes into specific cells.
G.RAGHAVA RAVALI Cosmid Vectors Cosmid vectors are hybrid cloning vectors that combine features of plasmids and bacteriophages, specifically utilizing cos sites from lambda phage. They are used to clone and analyze large DNA fragments, particularly for constructing genomic libraries. Cosmids can accommodate DNA inserts up to 45kb, which is larger than typical plasmids, and can be packaged into phage particles for efficient delivery into bacteria.
G.RAGHAVA RAVALI Features Large DNA Insert Capacity: Cosmids can carry DNA fragments up to 45kb, making them suitable for cloning larger genes and constructing genomic libraries. Hybrid Vector: They combine plasmid replication origins for stable maintenance in bacteria with cos sites from bacteriophage lambda for efficient packaging and transduction. Genomic Libraries: Cosmids are frequently used to create genomic libraries, which are collections of cloned DNA fragments representing the entire genome of an organism
G.RAGHAVA RAVALI
G.RAGHAVA RAVALI Working of Cosmid Vectors 1. Hybrid Nature: Cosmids are essentially plasmids that have been engineered to include the cos sites from the bacteriophage lambda. The cos sites are crucial for packaging the recombinant DNA into phage particles. These vectors can accommodate large DNA fragments, typically up to 45 kb, making them suitable for genomic library construction. 2. Packaging and Delivery: The cos sites allow the recombinant DNA molecule (cosmid + insert) to be packaged into lambda phage particles in vitro. This process utilizes the bacteriophage lambda's packaging machinery. The packaged phage particles can then infect bacterial cells, delivering the cosmid vector and its DNA insert into the host. 3. Replication and Maintenance: Once inside the bacterial cell, the cosmid vector can replicate as a plasmid due to its plasmid origin of replication. This allows for stable maintenance and amplification of the recombinant DNA within the bacterial host.
G.RAGHAVA RAVALI Applications of Cosmid Vectors: Cosmid vectors are commonly used to construct genomic libraries, which are collections of cloned DNA fragments representing the entire genome of an organism. They are also used in various molecular biology experiments that require the cloning and manipulation of large DNA fragments
G.RAGHAVA RAVALI Shuttle Vector A shuttle vector is a type of plasmid vector that can replicate in two different host organisms, typically a prokaryote and a eukaryote. This dual- host capability allows researchers to clone and manipulate DNA sequences in one organism, often E. coli, and then transfer the modified DNA into another, such as yeast or mammalian cells, for further study or application.
G.RAGHAVA RAVALI Key Features of Shuttle Vectors: Dual Origins of Replication: Shuttle vectors contain at least two origins of replication, one for each host organism they are designed to shuttle between. This allows the vector to be replicated and maintained in both host cell types. Selection Markers: They also include selectable markers, such as antibiotic resistance genes or genes that complement auxotrophic mutations in the host cells, to allow for the selection of cells that have taken up the vector. Example: A common example is a vector that can replicate in both E. coli and yeast, allowing for initial cloning and manipulation in E. coli and then expression or further study in yeast.
G.RAGHAVA RAVALI Working of Shuttle Vectors 1. Cloning and Amplification in a Prokaryotic Host: The gene of interest is inserted into the shuttle vector using restriction enzymes and ligases. The recombinant shuttle vector is then introduced into a prokaryotic host, usually E. coli, for replication and amplification. E. coli is often chosen because it's easy to grow, manipulate, and produce large quantities of the plasmid. 2. Transfer to a Eukaryotic Host: Once the gene is sufficiently amplified in E. coli, the recombinant shuttle vector is extracted. This vector is then introduced into the eukaryotic host where the gene will be expressed. 3. Gene Expression and Analysis in the Eukaryotic Host: The shuttle vector replicates and expresses the target gene within the eukaryotic cell. Researchers can then analyze the gene's expression, the protein it produces, or any other phenotypic changes it causes.
G.RAGHAVA RAVALI Advantages of Shuttle Vectors: Flexibility: Shuttle vectors allow for initial cloning and manipulation in E. coli, where procedures are generally faster and easier, and then transfer to a more complex eukaryotic system for expression. Efficient Protein Production: If the goal is to produce a protein, a shuttle vector can be used to amplify the gene in E. coli and then express it in a eukaryotic cell (like yeast) that is more likely to produce the protein correctly, including any necessary post- translational modifications. Studying Gene Function: Shuttle vectors can be used to study gene function in different organisms, allowing for cross-species comparisons and a better understanding of gene roles.
G.RAGHAVA RAVALI
G.RAGHAVA RAVALI Phagemid Vector A phagemid vector is a type of DNA cloning vector that combines features of both plasmids and bacteriophages, specifically filamentous phage M13. It allows for replication as a plasmid and can be packaged into phage particles for various applications like phage display, DNA sequencing, and site-directed mutagenesis.
G.RAGHAVA RAVALI Features of Phagemid Vectors: Plasmid Replication Origin: Phagemids contain a plasmid origin of replication, enabling them to replicate as circular DNA molecules within a bacterial cell, similar to regular plasmids. Bacteriophage Origin of Replication: They also include a bacteriophage origin of replication, allowing them to be packaged into bacteriophage particles. This is typically the f1 origin from M13 phage. Phage Packaging Signal: Phagemids contain a specific DNA sequence (packaging signal) that enables them to be incorporated into phage particles. Single-stranded DNA Production: When helper phage is present, phagemids are replicated and packaged into single-stranded DNA, which is then released from the cell as part of the phage particle.
G.RAGHAVA RAVALI Phagemids Working: 1.Cloning: A DNA fragment of interest is inserted into the phagemid vector. 2.Transformation: The phagemid is introduced into a bacterial host. 3.Replication: The phagemid replicates as a plasmid within the bacteria, producing multiple copies of the inserted DNA. 4.Helper Phage Infection: If phage display is desired, the bacterial culture is infected with a helper phage. 5.Packaging: The helper phage provides the necessary proteins to package the phagemid DNA into phage particles. 6.Phage Display: These phage particles display the foreign protein (encoded by the inserted DNA) on their surface. 7.Selection: The displayed proteins can be screened and selected for desired properties.
G.RAGHAVA RAVALI Advantages of Phagemid Vectors: Large Cloning Capacity: Phagemids can accommodate larger DNA inserts compared to some other phage vectors. Stability: Plasmid-based replication ensures good genetic stability. Versatility: Phagemids can be used for various applications, including phage display, DNA sequencing, and mutagenesis. Applications: Phage Display: Selecting and studying proteins with specific binding properties. DNA Sequencing: Generating single-stranded DNA for sequencing. Site-Directed Mutagenesis: Introducing specific mutations into DNA. RNA Probe Generation: Producing RNA probes for various molecular biology experiments.
G.RAGHAVA RAVALI
G.RAGHAVA RAVALI Bacterial artificial chromosome Bacterial artificial chromosomes are engineered DNA molecules that are used to clone DNA segments in bacteria cells (usually E. coli). These consist of a bacteria-derived F-factor replication origin which enables the propagation of large DNA fragments in a supercoiled circular form. Bacterial artificial chromosomes can carry a much larger size of insert DNA as compared to plasmid or phage vectors. These vectors are considered superior over other artificial chromosomes like yeast artificial chromosomes, and mammalian artificial chromosomes as the F-factor found in the bacteria reduces insert chimerism and instability that might arise during the process. These are highly efficient as DNA segments as large as 300,000 base pairs can be inserted into bacterial artificial chromosomes, which decreases the number of clones and cycles to be performed to obtain the desired result. BAC libraries have been used to generate large genomic DNA inserts for processes like positional cloning, physical mapping, and genome sequencing. BAC cloning system has been increasingly used in genetic engineering due to its stability and ease of use as compared to other similar vectors. However, BACs have been associated with the random insertion of DNA fragments into the host genome resulting in unpredicted expression.
G.RAGHAVA RAVALI Yeast Artificial Chromosome Yeast artificial chromosomes are engineered DNA molecules that are used to clone DNA inserts within the yeast cells, particularly Saccharomyces cerevisiae. YACs have been developed in order to clone large sequences of DNA so as to increase the efficiency of the process. YACs can clone up to 500 kb of DNA, which is much higher than most traditional cloning vectors. Even though these are frequently used as cloning vectors, they are also helpful in other genetic processes like DNA sequencing and analysis. These are also unique in their ability to clone the complete sequences of larger genomes that exceed the limits of traditional techniques. Since yeast cells are eukaryotic cells, YACs can be used for unstable sequences when cloned in prokaryotic systems. These consist of a mixture of functional units from different organisms, but once the insert DNA is cloned, these can function as normally replicating yeast chromosomes. There are some limitations with using YAC as vectors as these introduce a high degree of chimerism and insert rearrangement. Since these are eukaryotic cells, these are difficult to handle and have lower efficiencies as compared to bacterial artificial chromosomes. Different yeast artificial chromosomes have been created over the years that are then used for different purposes. One of the most commonly used examples of yeast artificial chromosomes includes pYAC4, which has been extensively used as a cloning vector.
G.RAGHAVA RAVALI Human artificial chromosome Human artificial chromosomes are extrachromosomal DNA fragments that act as a new chromosome within the human cell. The use of human artificial chromosomes has increased with advances in genetic engineering as it helps overcome problems commonly associated with traditional vector systems. HACs can exist as single copy episomes without integration into the host chromosomes allowing long-term stable maintenance. Besides, there is no upper limit in the size of the DNA insert to be incorporated into a HAC as entire genomic units can be used to mimic the natural gene expression. In spite of numerous advantages, HACs have only been used for studies related to the structure and function of human kinetochores. Limitations associated with HACs are due to technical difficulties during gene loading and ill-defined structures of the vectors.
G.RAGHAVA RAVALI Expression vectors An expression vector is a specialized type of DNA molecule, usually a plasmid or virus, designed to direct the expression of a specific gene within a host cell. It acts as a vehicle to introduce a gene of interest into a cell, enabling the cell's machinery to produce the protein encoded by that gene. Expression vectors are crucial tools in molecular biology and biotechnology for protein production and functional studies.
G.RAGHAVA RAVALI Features of Expression Vectors: Origin of Replication (ori): Allows the vector to replicate independently within the host cell. Promoter: A DNA sequence that initiates transcription of the gene of interest. Strong promoters are often used to ensure high levels of gene expression. Multiple Cloning Site (MCS) or Polylinker: A region with several unique restriction enzyme recognition sites, allowing for easy insertion of the gene of interest. Selectable Marker: A gene that provides a means of selecting for cells that have taken up the vector (e.g., antibiotic resistance). Terminator Sequence: Ensures proper termination of transcription. Enhancers (optional): DNA sequences that can further boost transcription levels. Tagging Sequences (optional): Sequences that can be added to the gene to facilitate protein purification or detection (e.g., His-tag, FLAG-tag).
G.RAGHAVA RAVALI How Expression Vectors Work: 1. Gene Insertion: An expression vector is engineered to include the gene encoding the protein of interest (the "gene of interest"). 2. Promoter Sequence: A promoter sequence is added upstream of the gene. This promoter acts as a binding site for RNA polymerase, initiating the transcription of the gene into messenger RNA (mRNA). 3. Transcription and Translation: The mRNA is then translated into a protein using the host cell's machinery. 4. Inducible Promoters: Many expression vectors utilize inducible promoters, which allow for controlled expression of the gene. This means the protein is only produced when a specific inducer molecule is added to the cell culture, preventing potential toxicity from the protein. 5. Selection Markers: Expression vectors also often contain selection markers, such as antibiotic resistance genes, to help identify and select cells that have successfully taken up the vector. 6. Protein Purification (Optional): Some expression vectors include tags like 6x-His tags, which can be used to easily purify the protein of interest from the cell lysate.
G.RAGHAVA RAVALI Applications of Expression Vectors: Protein Production: Producing large quantities of specific proteins for research, therapeutic, or industrial applications. Gene Therapy: Delivering functional genes to correct genetic defects. Vaccine Development: Producing antigens for vaccine development. Protein Localization Studies: Studying where proteins are located within cells. Studying Protein Function: Investigating the function of a protein by examining its effects on cellular processes.
G.RAGHAVA RAVALI Secretion vector Secretion vectors are a type of specialized expression vector that expresses the cloned genes in order to produce proteins at locations other than the cytoplasm. The transport of protein product from the cell is achieved by the fusion of the inset DNA with a nucleotide sequence encoding the peptide of an easily secreted protein. The use of secretion vector has many advantages like higher yield, simple purification process, and improved protein stability. Secretion vectors can be designed for more than one type of prokaryotes or eukaryotes, including mammals. A commonly associated problem with the incorporation of a protein of eukaryotic origin into a prokaryotic host is the overexpression of the protein. This problem is solved by the use of secretion vectors that alleviate the formation of inclusion bodies. Secretion vectors have replaced cloning vectors in processes focusing on the production of proteins and the expression of eukaryotic DNA fragments.
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Study of Cloning Vectors.pptx unit-2 Biotechnology 6th Sem

  • 1.
    Study of CloningVectors By G.Raghava Ravali M.Pharm Pharmaceutical Chemistry
  • 2.
    G.RAGHAVA RAVALI Terms Gene Manipulation(deliberatemodification-change of nucleic acid) DNA cloning(separating and copying of genetic sequence) Foreign DNA or Passenger DNA or DNA insert(DNA fragments)
  • 3.
    G.RAGHAVA RAVALI Cloning Vectors •The Vectors are the DNA molecules that carry foreign DNA segment and replicate inside the host cell. • Also Called Vehicle DNA • Any extrachromosomal small genome can be used as a vector.
  • 4.
    G.RAGHAVA RAVALI Characteristics ofCloning Vectors • Autonomous Replication • Easy Isolation, purification and insertion into the host • Easy selection of transformed cells through marker genes • Integration with the DNA inserts • Specific control systems like promoters, terminators, operators.
  • 5.
  • 6.
    G.RAGHAVA RAVALI Types ofVectors • Plasmid Vectors • Bacteriophage Vectors • Cosmid Vectors • Shuttle Vectors • Yeast Vectors • Expression Vectors
  • 7.
    G.RAGHAVA RAVALI Plasmid Vectors Aplasmid vector is a DNA molecule, typically circular and double- stranded, that is used to introduce foreign genetic material (DNA) into a host cell. It's a type of cloning vector, essentially a vehicle for carrying and replicating genes. Plasmids naturally exist in bacteria and some other microorganisms, but they can also be engineered for use in genetic engineering and biotechnology.
  • 8.
    G.RAGHAVA RAVALI Characteristics ofplasmid Vectors Circular and Extrachromosomal: Plasmid vectors are typically circular DNA molecules, separate from the host cell's chromosomal DNA. Self-Replication: They contain an origin of replication (ori) that allows them to replicate independently within the host cell. Multiple Cloning Site (MCS): Many plasmid vectors include a multiple cloning site, which is a region with several unique restriction enzyme recognition sites, making it easy to insert foreign DNA fragments. Selectable Markers: They often carry genes that provide a selectable advantage to the host cell, such as antibiotic resistance, allowing researchers to easily identify cells that have taken up the plasmid. Promoters and Other Regulatory Elements: Some plasmid vectors include promoters and other regulatory elements to control the expression of the inserted gene.
  • 9.
    G.RAGHAVA RAVALI How PlasmidVectors Work: 1. Gene Insertion: Foreign DNA is inserted into the MCS of the plasmid. 2. Transformation: The recombinant plasmid (containing the foreign DNA) is introduced into a host cell (e.g., bacteria, yeast, or mammalian cells). 3. Replication: The plasmid replicates inside the host cell, producing multiple copies of the foreign DNA. 4. Gene Expression: If the plasmid is designed as an expression vector, the inserted gene can be transcribed and translated into protein.
  • 10.
    G.RAGHAVA RAVALI Working ofPlasmids 1. Introduction of the Foreign DNA:A gene of interest (the foreign DNA) is isolated and cut using restriction enzymes. A plasmid vector is also cut with the same restriction enzyme, creating compatible "sticky ends". The foreign DNA is then inserted into the plasmid vector using DNA ligase, forming a recombinant plasmid. 2. Replication and Propagation:The recombinant plasmid is introduced into a suitable host cell (often bacteria) through a process called transformation. The plasmid carries an origin of replication (ori) sequence, which allows it to replicate independently of the host cell's chromosomal DNA. As the host cell replicates, the plasmid and its cargo of foreign DNA are also replicated, resulting in multiple copies of the recombinant DNA. 3. Expression and Applications:If the plasmid contains the necessary regulatory elements (like promoters), the foreign gene can be transcribed and translated into a protein within the host cell.This principle is used to produce proteins of interest, such as insulin, in large quantities using bacteria as factories.Plasmids can also be used for gene therapy, where a functional gene is introduced into a patient's cells to correct a genetic defect.They are also essential tools in research for studying gene function and manipulating DNA.
  • 11.
  • 12.
  • 13.
  • 14.
    G.RAGHAVA RAVALI pBR322 pBR322 isa widely used cloning vector, specifically a plasmid, in molecular biology. It is a circular, double-stranded DNA molecule derived from the E. coli plasmid ColE1. pBR322 is known for its usefulness in recombinant DNA technology, particularly for cloning and gene expression studies.
  • 15.
    G.RAGHAVA RAVALI 1. Originof Replication (ori): The ori site allows the plasmid to replicate independently within the host cell, ensuring that the inserted gene is also replicated.This feature is crucial for amplifying the cloned gene along with the plasmid DNA. 2. Selectable Markers (Antibiotic Resistance Genes): pBR322 contains two antibiotic resistance genes: one for ampicillin resistance (AmpR) and another for tetracycline resistance (TetR).These genes act as selectable markers, allowing researchers to differentiate between cells that have taken up the plasmid (transformed cells) and those that haven't.For example, if a gene of interest is inserted into the TetR gene, it disrupts the TetR gene, and the transformed cells will only be resistant to ampicillin, not tetracycline. 3. Multiple Cloning Site (MCS): pBR322 has several unique restriction enzyme sites within its MCS.These sites allow for the insertion of foreign DNA fragments into the plasmid using enzymes that cut at specific DNA sequences.The MCS is typically located within one of the antibiotic resistance genes, allowing for the use of insertional inactivation for selecting recombinant clones. 4. Other important features: Size: pBR322 is relatively small (4361 base pairs), making it easier to handle and manipulate. Sequenced: It has been completely sequenced, meaning its entire DNA sequence is known, which is helpful for designing experiments. Mobility: pBR322 is known to be relatively mobile between cells, which can be a limitation for some applications. In summary: pBR322 functions as a stable and reliable cloning vector due to its origin of replication, selectable markers, and multiple cloning sites. It has been extensively used for gene cloning, recombinant DNA research, and protein expression studies in various applications. Working Of Pbr322
  • 16.
    G.RAGHAVA RAVALI pUC19 pUC19 isa widely used, small, circular, double-stranded DNA molecule that serves as a cloning vector in bacteria like E. coli. It's a modified version of the pBR322 plasmid, known for its high copy number and multiple cloning sites. The plasmid is 2,686 base pairs long and contains an ampicillin resistance gene, allowing for selection of cells carrying the plasmid.
  • 17.
    G.RAGHAVA RAVALI Working ofpUC19 High Copy Number: pUC19 replicates frequently in host cells, resulting in many copies of the plasmid, which is useful for producing many copies of inserted DNA. Multiple Cloning Site (MCS):A region containing several restriction enzyme recognition sites, facilitating the insertion of foreign DNA fragments. Ampicillin Resistance Gene:Enables selection of bacteria that have taken up the plasmid by growing them in the presence of ampicillin. LacZ Gene Fragment:A portion of the lacZ gene, encoding β-galactosidase, is present and allows for blue-white screening of recombinant colonies.
  • 18.
    G.RAGHAVA RAVALI Uses ofpUC19: Cloning:pUC19 is commonly used for cloning genes and other DNA fragments in E. coli. Blue-White Screening:The presence of the MCS within the lacZ gene allows for easy identification of bacterial colonies that contain inserted DNA fragments. Teaching Aid:Its well-defined characteristics and ease of use make it a popular tool for teaching molecular biology techniques. In essence, pUC19 is a versatile and widely used tool for genetic engineering and molecular biology research.
  • 19.
    G.RAGHAVA RAVALI Bacteriophage vectors Bacteriophagevectors are viruses that infect bacteria and are used in genetic engineering to carry foreign DNA into bacterial cells. They are particularly useful because they can accommodate larger DNA fragments than some other cloning vectors like plasmids. Common bacteriophage vectors include lambda phage, M13 phage, and P1 phage.
  • 20.
    G.RAGHAVA RAVALI Characteristics ofBacteriophage Large DNA capacity: Bacteriophages can carry larger DNA fragments than plasmids, allowing for the cloning of larger genes or gene libraries. Efficient delivery: Bacteriophages are highly efficient at infecting and delivering their DNA into bacterial cells. Versatile: They can be used in various applications, including gene cloning, DNA sequencing, and creating genomic libraries.
  • 21.
    G.RAGHAVA RAVALI Working ofBacteriophage • Bacteriophage vectors are viruses that only infect bacteria and transform them efficiently while carrying large inserts. • Bacteriophages or phages have higher transformation efficiencies which increase the chances of recovering a clone containing the recombinant DNA segments. • The most important feature of a phage is the packaging system which enables the incorporation of large eukaryotic genes and their regulatory elements. • The use of phages also facilitates the isolation of larger quantities of DNA that can be used for the analysis of the insert. • Even though there are a number of phages that can and have been used as vectors, phage λ is the most convenient cloning vector. • It can selectively package a chromosome about 50 kb in length, and the size of the phage can be adjusted by removing the central part of the genome as it is not necessary for replication or the packaging of the donor DNA. • The use of a bacteriophage vector that can incorporate larger DNA segments decreases the number of clones required to obtain a particular DNA library with the entire genome of the organism. • Phage vectors are also effective as cloning vectors as the recombinant molecules formed after the cloning process are packaged into infective particles that can then be stored or handle efficiently. • Some of the common phages used as vectors include M13 phages, λ phages, and P1 phages.
  • 22.
    G.RAGHAVA RAVALI λ phage λ-phageis an example of a bacteriophage that infects the bacterial species, Escherichia coli (E. coli). This vector is more effective than other plasmid vectors as it has a higher efficiency in entering bacterial cells so as to incorporate the recombinant DNA within the host genome. It is a double-stranded DNA bacteriophage that contains an ori sequence requires for replication and a number of DNA sequences encoding regulatory and replicative proteins. The phage DNA replicates by the combination of theta and rolling circle replication process to produce a linear dsDNA. It is then followed by the cos sequence, which enables the circularization of the genome after infection. The DNA sequences between the two arms of the vector are not essential which are then replaced with the recombinant DNA during cloning.
  • 23.
  • 24.
  • 25.
    G.RAGHAVA RAVALI Uses ofBacteriophage 1. Gene Cloning: Cloning larger DNA fragments:Bacteriophages can accommodate larger DNA inserts compared to plasmids, making them suitable for cloning larger genes or genomic DNA fragments. Efficient screening:Screening for recombinant phage clones (those containing the desired DNA insert) is often easier than screening bacterial colonies, particularly for large libraries. Examples of phage vectors:Lambda phage: Used for cloning larger fragments of DNA, with both insertion and replacement vectors available. M13 phage: Used for generating single-stranded DNA copies, which is useful for sequencing and mutagenesis. 2. Molecular Biology Research: Studying gene structure and function:Bacteriophage vectors can be used to introduce specific genes into bacterial cells, allowing researchers to study gene expression and protein production. DNA sequencing:M13 vectors are particularly useful for generating single-stranded DNA templates for sequencing.
  • 26.
    G.RAGHAVA RAVALI 3. BiotechnologyApplications: Phage display:Bacteriophages can be engineered to display foreign peptides or proteins on their surface, allowing for the selection and identification of binding partners (e.g., for drug discovery). Phage therapy:Bacteriophages are being explored as a potential alternative to antibiotics for treating bacterial infections, especially those caused by antibiotic-resistant bacteria. Vaccine development:Bacteriophages can be used to deliver vaccine antigens to the immune system. Biosensors:Bacteriophages or their components can be incorporated into biosensors for detecting bacteria, toxins, or other molecules. Food preservation:Bacteriophages can be used to control the growth of spoilage or pathogenic bacteria in food products. 4. Other Uses: Bacterial typing:Bacteriophages can be used to identify specific strains of bacteria (phage typing). Delivery of therapeutic genes:Bacteriophages can be modified to deliver therapeutic genes into specific cells.
  • 27.
    G.RAGHAVA RAVALI Cosmid Vectors Cosmidvectors are hybrid cloning vectors that combine features of plasmids and bacteriophages, specifically utilizing cos sites from lambda phage. They are used to clone and analyze large DNA fragments, particularly for constructing genomic libraries. Cosmids can accommodate DNA inserts up to 45kb, which is larger than typical plasmids, and can be packaged into phage particles for efficient delivery into bacteria.
  • 28.
    G.RAGHAVA RAVALI Features Large DNAInsert Capacity: Cosmids can carry DNA fragments up to 45kb, making them suitable for cloning larger genes and constructing genomic libraries. Hybrid Vector: They combine plasmid replication origins for stable maintenance in bacteria with cos sites from bacteriophage lambda for efficient packaging and transduction. Genomic Libraries: Cosmids are frequently used to create genomic libraries, which are collections of cloned DNA fragments representing the entire genome of an organism
  • 29.
  • 30.
    G.RAGHAVA RAVALI Working ofCosmid Vectors 1. Hybrid Nature: Cosmids are essentially plasmids that have been engineered to include the cos sites from the bacteriophage lambda. The cos sites are crucial for packaging the recombinant DNA into phage particles. These vectors can accommodate large DNA fragments, typically up to 45 kb, making them suitable for genomic library construction. 2. Packaging and Delivery: The cos sites allow the recombinant DNA molecule (cosmid + insert) to be packaged into lambda phage particles in vitro. This process utilizes the bacteriophage lambda's packaging machinery. The packaged phage particles can then infect bacterial cells, delivering the cosmid vector and its DNA insert into the host. 3. Replication and Maintenance: Once inside the bacterial cell, the cosmid vector can replicate as a plasmid due to its plasmid origin of replication. This allows for stable maintenance and amplification of the recombinant DNA within the bacterial host.
  • 31.
    G.RAGHAVA RAVALI Applications ofCosmid Vectors: Cosmid vectors are commonly used to construct genomic libraries, which are collections of cloned DNA fragments representing the entire genome of an organism. They are also used in various molecular biology experiments that require the cloning and manipulation of large DNA fragments
  • 32.
    G.RAGHAVA RAVALI Shuttle Vector Ashuttle vector is a type of plasmid vector that can replicate in two different host organisms, typically a prokaryote and a eukaryote. This dual- host capability allows researchers to clone and manipulate DNA sequences in one organism, often E. coli, and then transfer the modified DNA into another, such as yeast or mammalian cells, for further study or application.
  • 33.
    G.RAGHAVA RAVALI Key Featuresof Shuttle Vectors: Dual Origins of Replication: Shuttle vectors contain at least two origins of replication, one for each host organism they are designed to shuttle between. This allows the vector to be replicated and maintained in both host cell types. Selection Markers: They also include selectable markers, such as antibiotic resistance genes or genes that complement auxotrophic mutations in the host cells, to allow for the selection of cells that have taken up the vector. Example: A common example is a vector that can replicate in both E. coli and yeast, allowing for initial cloning and manipulation in E. coli and then expression or further study in yeast.
  • 34.
    G.RAGHAVA RAVALI Working ofShuttle Vectors 1. Cloning and Amplification in a Prokaryotic Host: The gene of interest is inserted into the shuttle vector using restriction enzymes and ligases. The recombinant shuttle vector is then introduced into a prokaryotic host, usually E. coli, for replication and amplification. E. coli is often chosen because it's easy to grow, manipulate, and produce large quantities of the plasmid. 2. Transfer to a Eukaryotic Host: Once the gene is sufficiently amplified in E. coli, the recombinant shuttle vector is extracted. This vector is then introduced into the eukaryotic host where the gene will be expressed. 3. Gene Expression and Analysis in the Eukaryotic Host: The shuttle vector replicates and expresses the target gene within the eukaryotic cell. Researchers can then analyze the gene's expression, the protein it produces, or any other phenotypic changes it causes.
  • 35.
    G.RAGHAVA RAVALI Advantages ofShuttle Vectors: Flexibility: Shuttle vectors allow for initial cloning and manipulation in E. coli, where procedures are generally faster and easier, and then transfer to a more complex eukaryotic system for expression. Efficient Protein Production: If the goal is to produce a protein, a shuttle vector can be used to amplify the gene in E. coli and then express it in a eukaryotic cell (like yeast) that is more likely to produce the protein correctly, including any necessary post- translational modifications. Studying Gene Function: Shuttle vectors can be used to study gene function in different organisms, allowing for cross-species comparisons and a better understanding of gene roles.
  • 36.
  • 37.
    G.RAGHAVA RAVALI Phagemid Vector Aphagemid vector is a type of DNA cloning vector that combines features of both plasmids and bacteriophages, specifically filamentous phage M13. It allows for replication as a plasmid and can be packaged into phage particles for various applications like phage display, DNA sequencing, and site-directed mutagenesis.
  • 38.
    G.RAGHAVA RAVALI Features ofPhagemid Vectors: Plasmid Replication Origin: Phagemids contain a plasmid origin of replication, enabling them to replicate as circular DNA molecules within a bacterial cell, similar to regular plasmids. Bacteriophage Origin of Replication: They also include a bacteriophage origin of replication, allowing them to be packaged into bacteriophage particles. This is typically the f1 origin from M13 phage. Phage Packaging Signal: Phagemids contain a specific DNA sequence (packaging signal) that enables them to be incorporated into phage particles. Single-stranded DNA Production: When helper phage is present, phagemids are replicated and packaged into single-stranded DNA, which is then released from the cell as part of the phage particle.
  • 39.
    G.RAGHAVA RAVALI Phagemids Working: 1.Cloning:A DNA fragment of interest is inserted into the phagemid vector. 2.Transformation: The phagemid is introduced into a bacterial host. 3.Replication: The phagemid replicates as a plasmid within the bacteria, producing multiple copies of the inserted DNA. 4.Helper Phage Infection: If phage display is desired, the bacterial culture is infected with a helper phage. 5.Packaging: The helper phage provides the necessary proteins to package the phagemid DNA into phage particles. 6.Phage Display: These phage particles display the foreign protein (encoded by the inserted DNA) on their surface. 7.Selection: The displayed proteins can be screened and selected for desired properties.
  • 40.
    G.RAGHAVA RAVALI Advantages ofPhagemid Vectors: Large Cloning Capacity: Phagemids can accommodate larger DNA inserts compared to some other phage vectors. Stability: Plasmid-based replication ensures good genetic stability. Versatility: Phagemids can be used for various applications, including phage display, DNA sequencing, and mutagenesis. Applications: Phage Display: Selecting and studying proteins with specific binding properties. DNA Sequencing: Generating single-stranded DNA for sequencing. Site-Directed Mutagenesis: Introducing specific mutations into DNA. RNA Probe Generation: Producing RNA probes for various molecular biology experiments.
  • 41.
  • 42.
    G.RAGHAVA RAVALI Bacterial artificialchromosome Bacterial artificial chromosomes are engineered DNA molecules that are used to clone DNA segments in bacteria cells (usually E. coli). These consist of a bacteria-derived F-factor replication origin which enables the propagation of large DNA fragments in a supercoiled circular form. Bacterial artificial chromosomes can carry a much larger size of insert DNA as compared to plasmid or phage vectors. These vectors are considered superior over other artificial chromosomes like yeast artificial chromosomes, and mammalian artificial chromosomes as the F-factor found in the bacteria reduces insert chimerism and instability that might arise during the process. These are highly efficient as DNA segments as large as 300,000 base pairs can be inserted into bacterial artificial chromosomes, which decreases the number of clones and cycles to be performed to obtain the desired result. BAC libraries have been used to generate large genomic DNA inserts for processes like positional cloning, physical mapping, and genome sequencing. BAC cloning system has been increasingly used in genetic engineering due to its stability and ease of use as compared to other similar vectors. However, BACs have been associated with the random insertion of DNA fragments into the host genome resulting in unpredicted expression.
  • 43.
    G.RAGHAVA RAVALI Yeast ArtificialChromosome Yeast artificial chromosomes are engineered DNA molecules that are used to clone DNA inserts within the yeast cells, particularly Saccharomyces cerevisiae. YACs have been developed in order to clone large sequences of DNA so as to increase the efficiency of the process. YACs can clone up to 500 kb of DNA, which is much higher than most traditional cloning vectors. Even though these are frequently used as cloning vectors, they are also helpful in other genetic processes like DNA sequencing and analysis. These are also unique in their ability to clone the complete sequences of larger genomes that exceed the limits of traditional techniques. Since yeast cells are eukaryotic cells, YACs can be used for unstable sequences when cloned in prokaryotic systems. These consist of a mixture of functional units from different organisms, but once the insert DNA is cloned, these can function as normally replicating yeast chromosomes. There are some limitations with using YAC as vectors as these introduce a high degree of chimerism and insert rearrangement. Since these are eukaryotic cells, these are difficult to handle and have lower efficiencies as compared to bacterial artificial chromosomes. Different yeast artificial chromosomes have been created over the years that are then used for different purposes. One of the most commonly used examples of yeast artificial chromosomes includes pYAC4, which has been extensively used as a cloning vector.
  • 44.
    G.RAGHAVA RAVALI Human artificialchromosome Human artificial chromosomes are extrachromosomal DNA fragments that act as a new chromosome within the human cell. The use of human artificial chromosomes has increased with advances in genetic engineering as it helps overcome problems commonly associated with traditional vector systems. HACs can exist as single copy episomes without integration into the host chromosomes allowing long-term stable maintenance. Besides, there is no upper limit in the size of the DNA insert to be incorporated into a HAC as entire genomic units can be used to mimic the natural gene expression. In spite of numerous advantages, HACs have only been used for studies related to the structure and function of human kinetochores. Limitations associated with HACs are due to technical difficulties during gene loading and ill-defined structures of the vectors.
  • 45.
    G.RAGHAVA RAVALI Expression vectors Anexpression vector is a specialized type of DNA molecule, usually a plasmid or virus, designed to direct the expression of a specific gene within a host cell. It acts as a vehicle to introduce a gene of interest into a cell, enabling the cell's machinery to produce the protein encoded by that gene. Expression vectors are crucial tools in molecular biology and biotechnology for protein production and functional studies.
  • 46.
    G.RAGHAVA RAVALI Features ofExpression Vectors: Origin of Replication (ori): Allows the vector to replicate independently within the host cell. Promoter: A DNA sequence that initiates transcription of the gene of interest. Strong promoters are often used to ensure high levels of gene expression. Multiple Cloning Site (MCS) or Polylinker: A region with several unique restriction enzyme recognition sites, allowing for easy insertion of the gene of interest. Selectable Marker: A gene that provides a means of selecting for cells that have taken up the vector (e.g., antibiotic resistance). Terminator Sequence: Ensures proper termination of transcription. Enhancers (optional): DNA sequences that can further boost transcription levels. Tagging Sequences (optional): Sequences that can be added to the gene to facilitate protein purification or detection (e.g., His-tag, FLAG-tag).
  • 47.
    G.RAGHAVA RAVALI How ExpressionVectors Work: 1. Gene Insertion: An expression vector is engineered to include the gene encoding the protein of interest (the "gene of interest"). 2. Promoter Sequence: A promoter sequence is added upstream of the gene. This promoter acts as a binding site for RNA polymerase, initiating the transcription of the gene into messenger RNA (mRNA). 3. Transcription and Translation: The mRNA is then translated into a protein using the host cell's machinery. 4. Inducible Promoters: Many expression vectors utilize inducible promoters, which allow for controlled expression of the gene. This means the protein is only produced when a specific inducer molecule is added to the cell culture, preventing potential toxicity from the protein. 5. Selection Markers: Expression vectors also often contain selection markers, such as antibiotic resistance genes, to help identify and select cells that have successfully taken up the vector. 6. Protein Purification (Optional): Some expression vectors include tags like 6x-His tags, which can be used to easily purify the protein of interest from the cell lysate.
  • 48.
    G.RAGHAVA RAVALI Applications ofExpression Vectors: Protein Production: Producing large quantities of specific proteins for research, therapeutic, or industrial applications. Gene Therapy: Delivering functional genes to correct genetic defects. Vaccine Development: Producing antigens for vaccine development. Protein Localization Studies: Studying where proteins are located within cells. Studying Protein Function: Investigating the function of a protein by examining its effects on cellular processes.
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    G.RAGHAVA RAVALI Secretion vector Secretionvectors are a type of specialized expression vector that expresses the cloned genes in order to produce proteins at locations other than the cytoplasm. The transport of protein product from the cell is achieved by the fusion of the inset DNA with a nucleotide sequence encoding the peptide of an easily secreted protein. The use of secretion vector has many advantages like higher yield, simple purification process, and improved protein stability. Secretion vectors can be designed for more than one type of prokaryotes or eukaryotes, including mammals. A commonly associated problem with the incorporation of a protein of eukaryotic origin into a prokaryotic host is the overexpression of the protein. This problem is solved by the use of secretion vectors that alleviate the formation of inclusion bodies. Secretion vectors have replaced cloning vectors in processes focusing on the production of proteins and the expression of eukaryotic DNA fragments.
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