Plasmids are naturally occurring extrachromosomal DNA molecules that can autonomously replicate in bacterial cells. Plasmids are commonly used as cloning vectors to replicate and express exogenous DNA in a host cell. Ideal plasmid vectors are small in size, present in multiple copies, contain origins of replication and selectable markers like antibiotic resistance genes. Commonly used plasmid vectors include pBR322, which contains genes for ampicillin and tetracycline resistance, and has been widely used for cloning in E. coli. Artificial plasmids are also engineered by combining functional modules from different natural plasmids to improve vector characteristics for cloning and expression applications.
This document summarizes the baculovirus expression system. Baculoviruses can be used as expression vectors by replacing a non-essential viral gene with a gene of interest. The recombinant baculovirus is produced through homologous recombination or using the Bac-to-Bac system. Insect cells are infected with the recombinant baculovirus, which drives high-level expression of the foreign gene. The baculovirus expression system allows safe, scalable production of recombinant proteins for research applications.
The document discusses phage display technology. It introduces phage display and describes the biology of filamentous phages like M13 that are commonly used. It explains how phagemids combine features of plasmids and phages to allow expression of recombinant proteins. Different types of phage display systems are described, including types that display antibody fragments. The process of biopanning and screening phage display libraries to isolate antibodies with high affinity for a target is summarized in three steps: production of the library, selection through biopanning, and screening candidates. Applications of phage display technology including production of monoclonal antibodies are also mentioned.
This document discusses bacteriophages and their use in phage display. Specifically, it notes that bacteriophages infect bacterial cells and use them to replicate viruses. It then explains that phage display involves fusing foreign genes or proteins to the surface of phages, creating libraries of phages that each display a single protein. These libraries can be exposed to targets, and phages that interact are selected and amplified through multiple rounds. The document outlines several applications of proteins isolated through phage display, such as epitope mapping, drug discovery, and developing new vaccines or treatments that have a specific interaction with a target antigen, protein, or disease.
Yeast artificial chromosomes (YACs) are engineered DNA molecules that can clone and replicate large DNA sequences in yeast cells. YACs contain essential yeast elements like a centromere and telomeres that allow them to behave like natural yeast chromosomes. YACs can clone very large inserts of up to 10 megabases of foreign DNA, making them useful for generating whole genome libraries.
This document discusses nucleic acid probes and their use in hybridization experiments. It notes that probes are short sequences of nucleotides that bind to specific target sequences. The degree of homology between the probe and target determines how stable the hybridization is. Probes can range in size from 10 to over 10,000 nucleotide bases, with most common probes being 14 to 40 bases. Short probes hybridize quickly but have less specificity, while longer probes hybridize more stably. The document then describes different methods for labeling probes, including nick translation, primer extension, RNA polymerase transcription, end-labeling, and direct labeling. It also discusses factors that affect probe specificity and hybridization conditions.
Bacteriophage vectors
Bacteriophage
WHY BACTERIOPHAGE AS A VECTOR?
M13 phage
Genome of m13 phage
Life cycle and dna replication of m13
CONSTRUCTION M13 AS PHAGE VECTOR
M13 MP 2 vector
M13MP7 VECTOR
Selection of recombinants
Lambda replacement vectors
LAMBDA EMBL 4 VECTOR
P1 PHAGE
GENOME OF P1 PHAGE
P1 PHAGE AS VECTOR
P1 phage vector system
1. A cosmid is a type of plasmid vector that contains sequences from bacteriophage lambda, specifically the cos sites. This allows DNA fragments up to 45kb to be packaged into phage particles and transduced into bacteria.
2. Cosmids are developed by combining features of plasmid and phage vectors. They can accept large DNA inserts through packaging and transduction while also replicating stably inside bacteria like plasmids.
3. Cloning with cosmids involves inserting DNA fragments into the cosmid, ligating to form concatemers, in vitro packaging into phage particles, and transducing the particles into bacteria where the cosmids replicate as plasmids.
This document discusses P1 vectors, which are cloning vectors derived from the P1 bacteriophage. P1 can transfer DNA between bacterial cells through transduction. The document describes:
1) The construction of P1 vectors pNS358 and pNS582, which contain P1 packaging and replication elements to clone and propagate large DNA fragments.
2) How the P1 system was used to clone DNA fragments up to 100 kb by packaging ligated vector/insert concatemers into phage heads.
3) Applications of P1 vectors including the creation of P1-derived artificial chromosomes (PACs) which can accommodate relatively large DNA fragments for cloning and mapping genomes.
This document summarizes the baculovirus expression system. Baculoviruses can be used as expression vectors by replacing a non-essential viral gene with a gene of interest. The recombinant baculovirus is produced through homologous recombination or using the Bac-to-Bac system. Insect cells are infected with the recombinant baculovirus, which drives high-level expression of the foreign gene. The baculovirus expression system allows safe, scalable production of recombinant proteins for research applications.
The document discusses phage display technology. It introduces phage display and describes the biology of filamentous phages like M13 that are commonly used. It explains how phagemids combine features of plasmids and phages to allow expression of recombinant proteins. Different types of phage display systems are described, including types that display antibody fragments. The process of biopanning and screening phage display libraries to isolate antibodies with high affinity for a target is summarized in three steps: production of the library, selection through biopanning, and screening candidates. Applications of phage display technology including production of monoclonal antibodies are also mentioned.
This document discusses bacteriophages and their use in phage display. Specifically, it notes that bacteriophages infect bacterial cells and use them to replicate viruses. It then explains that phage display involves fusing foreign genes or proteins to the surface of phages, creating libraries of phages that each display a single protein. These libraries can be exposed to targets, and phages that interact are selected and amplified through multiple rounds. The document outlines several applications of proteins isolated through phage display, such as epitope mapping, drug discovery, and developing new vaccines or treatments that have a specific interaction with a target antigen, protein, or disease.
Yeast artificial chromosomes (YACs) are engineered DNA molecules that can clone and replicate large DNA sequences in yeast cells. YACs contain essential yeast elements like a centromere and telomeres that allow them to behave like natural yeast chromosomes. YACs can clone very large inserts of up to 10 megabases of foreign DNA, making them useful for generating whole genome libraries.
This document discusses nucleic acid probes and their use in hybridization experiments. It notes that probes are short sequences of nucleotides that bind to specific target sequences. The degree of homology between the probe and target determines how stable the hybridization is. Probes can range in size from 10 to over 10,000 nucleotide bases, with most common probes being 14 to 40 bases. Short probes hybridize quickly but have less specificity, while longer probes hybridize more stably. The document then describes different methods for labeling probes, including nick translation, primer extension, RNA polymerase transcription, end-labeling, and direct labeling. It also discusses factors that affect probe specificity and hybridization conditions.
Bacteriophage vectors
Bacteriophage
WHY BACTERIOPHAGE AS A VECTOR?
M13 phage
Genome of m13 phage
Life cycle and dna replication of m13
CONSTRUCTION M13 AS PHAGE VECTOR
M13 MP 2 vector
M13MP7 VECTOR
Selection of recombinants
Lambda replacement vectors
LAMBDA EMBL 4 VECTOR
P1 PHAGE
GENOME OF P1 PHAGE
P1 PHAGE AS VECTOR
P1 phage vector system
1. A cosmid is a type of plasmid vector that contains sequences from bacteriophage lambda, specifically the cos sites. This allows DNA fragments up to 45kb to be packaged into phage particles and transduced into bacteria.
2. Cosmids are developed by combining features of plasmid and phage vectors. They can accept large DNA inserts through packaging and transduction while also replicating stably inside bacteria like plasmids.
3. Cloning with cosmids involves inserting DNA fragments into the cosmid, ligating to form concatemers, in vitro packaging into phage particles, and transducing the particles into bacteria where the cosmids replicate as plasmids.
This document discusses P1 vectors, which are cloning vectors derived from the P1 bacteriophage. P1 can transfer DNA between bacterial cells through transduction. The document describes:
1) The construction of P1 vectors pNS358 and pNS582, which contain P1 packaging and replication elements to clone and propagate large DNA fragments.
2) How the P1 system was used to clone DNA fragments up to 100 kb by packaging ligated vector/insert concatemers into phage heads.
3) Applications of P1 vectors including the creation of P1-derived artificial chromosomes (PACs) which can accommodate relatively large DNA fragments for cloning and mapping genomes.
The document summarizes phagemid and bacterial artificial chromosome (BAC) vectors. It describes that phagemid vectors are plasmids that contain both plasmid and phage origins of replication. Specifically, it discusses the features of pBluescript II phagemid vectors, including their polylinker and RNA polymerase promoter sequences. It also describes how pBluescript II phagemid vectors can produce blue or white colonies depending on insert presence. The document then explains that BAC vectors are low-copy plasmids that can hold up to 300kb DNA fragments. Examples of BAC vectors like pBAC108L and pBeloBAC11 are provided, with details about their replication origin and partitioning functions.
BAC & YAC are artificially prepared chromosomes to clone DNA sequences.yeast artificial chromosome is capable of carrying upto 1000 kbp of inserted DNA sequence
DNA footprinting is a technique used to identify protein binding regions on DNA. It involves treating DNA with nucleases like DNase I, which will degrade the DNA except for regions bound by proteins. These protected regions, called footprints, can identify transcription factor binding sites that regulate gene expression. The technique was originally developed in 1978 to study the binding specificity of the lac repressor protein, and it provides information on DNA-protein interactions and transcriptional regulation.
Selection & Screening of Recombinant cells & expression of recombinant (2) (1)SunandaArya
This document summarizes various methods for selecting and screening recombinant clones after introducing recombinant DNA into host cells. It discusses direct selection using antibiotic resistance genes, insertional inactivation by inserting DNA into antibiotic resistance genes, and blue-white screening using beta-galactosidase activity. It also covers colony hybridization using radioactive probes and immunological tests using antibodies to identify antigen-expressing colonies. Finally, it briefly discusses protein expression in different systems like bacteria, insect cells, and mammalian cells.
Gene cloning strategies depend on whether genomic or cDNA libraries are being constructed. Shotgun cloning is used to construct genomic libraries by fragmenting genomic DNA and inserting all fragments into vectors at once. cDNA libraries are constructed by reverse transcribing mRNA to cDNA, which is then cloned into vectors. Both library types are screened to identify overlapping clones that are assembled into contigs representing the entire genome.
This document discusses key components of expression vectors that are important for efficiently expressing cloned genes. It explains that expression vectors contain regulatory sequences like promoters and terminators to control transcription, as well as elements like ribosome binding sites, fusion tags, and selection markers. Specifically, it provides details on tightly regulated promoters, commonly used viral and bacterial promoters, and considerations for promoters in prokaryotic and eukaryotic expression systems. The document also reviews other important vector elements and their functions.
Cosmid Vectors, YAC and BAC Expression VectorsCharthaGaglani
1. Cosmid vectors are hybrid vectors derived from plasmids that contain the cos site from bacteriophage lambda, allowing them to clone DNA fragments up to 40 kb in size.
2. Yeast artificial chromosomes (YACs) are engineered yeast chromosomes that can clone very large DNA fragments, averaging 200-500 kb but up to 1 MB, taking advantage of yeast cell machinery.
3. Bacterial artificial chromosomes (BACs) are DNA constructs based on fertility plasmids that can clone up to 300 kb fragments and address issues with YAC stability and recombination.
This document discusses bacteriophage T4, a virus that infects E. coli bacteria. It has a complex protein coat and large double-stranded DNA genome. T4 uses the host cell's machinery to replicate and kills the host cell. T4 plays a role in cholera and diphtheria by carrying toxin genes that allow the bacteria to cause disease. Bacteriophage may be useful for treating antibiotic-resistant bacteria or infections where antibiotics cannot reach. T4 is also used in recombinant DNA technology.
The document discusses various methods of transfection in animals. Transfection is the process of introducing nucleic acids into eukaryotic cells. It describes viral transfection using bacteria like Agrobacterium tumefaciens and viruses. Non-viral methods include chemical transfection using calcium phosphate, liposomes, polyamines. Mechanical transfection employs microinjection or particle bombardment. Common chemical methods are calcium phosphate precipitation, polyplexes, and liposomes/lipoplexes. Viruses used are retroviruses, adenoviruses, adeno-associated viruses. Bacterial and viral vectors allow for integration into the host genome while chemical and mechanical are often transient.
Restriction enzymes are endonucleases found in bacteria and archaea that provide defense against viruses by selectively cutting invading viral DNA. Over 3,000 restriction enzymes have been identified, with some being commercially available. They recognize specific DNA sequences and cut the phosphodiester bonds within or near the recognition site. Restriction Fragment Length Polymorphism (RFLP) analyzes patterns from cleavage of DNA with restriction enzymes to differentiate organisms. RFLPs have forensic and medical applications such as paternity testing and disease detection.
This document discusses yeast artificial chromosomes (YACs) and bacterial artificial chromosomes (BACs). YACs are engineered chromosomes derived from yeast DNA that can clone very large DNA sequences in yeast cells of up to 1 megabase. BACs are cloning vectors derived from bacterial DNA that can clone DNA fragments of up to 300 kilobases in E. coli. Both systems allow cloning and propagation of large DNA fragments, but YACs can hold more DNA while BACs are more stable and better for functional analysis in mammalian cells.
Structural databases like PDB, CSD, and CATH contain 3D structural information of proteins, small molecules, and macromolecules determined through techniques like X-ray crystallography and NMR spectroscopy. These databases provide bibliographic data, atomic coordinates, and other details for each entry. PDB contains protein structures, CSD contains organic and metal-organic structures, and CATH classifies protein domains hierarchically. Structural databases have wide applications in structure prediction, analysis, mining, comparison, classification, structure refinement, and database annotation.
This document discusses methylases, which are enzymes that add methyl groups to DNA. Specifically:
- Methylases transfer methyl groups from S-adenosylmethionine to adenine or cytosine bases within their recognition sequence on DNA. This methylation protects the DNA from restriction endonucleases.
- The methylase and restriction enzyme of a bacterial species together form the restriction-modification system, with the methylase protecting the host DNA.
- Methylases are of interest because methylation of some restriction enzyme recognition sites protects the DNA from being cleaved by that enzyme. This allows study of DNA isolated from strains expressing common methylases like Dam or Dcm.
MBB 501 PLANT BIOTECHNOLOGY
INFORMATION ABOUT DIFFERENT DNA MODIFYING ENZYMES
WHAT IS AN ENZYME?
Alkaline Phosphatase
Polynucleotide kinase
Terminal deoxyneucleotidyl transferase
Nucleases
Exonuclease
Bal31 Exonuclease III
Endonuclease
S1 endonulease
Deoxyribonuclease 1 (Dnase 1)
RNase A
RNase H
Restriction Endonuclease
PvuI
PvuII
Different types of endonuclease enzymes
The recognition sequences for some of the most frequently used restriction endonucleases.
Categorization of enzymes
Isoschizomers
Neoschizomers
Isocaudomers
Cosmids are hybrid cloning vectors that combine features of plasmids and bacteriophages. They contain approximately 200 base pairs of DNA from the lambda phage, including the cos site sequence, which allows the vector to be packaged into phage particles and transduced into bacteria like a phage. Cosmids can accommodate large foreign DNA inserts of 35-45 kilobase pairs and are commonly used to construct genomic libraries.
Blotting techniques includes southren,northern,western and dot blottingbbmy
This document describes various blotting techniques used to detect specific DNA or RNA sequences, including Southern blotting, Northern blotting, Western blotting, and dot blotting. Southern blotting involves transferring DNA fragments separated by gel electrophoresis to a membrane and probing for specific sequences. Northern blotting is similar but uses RNA. Western blotting detects specific proteins. Dot blotting detects sequences in non-fractionated samples by directly applying samples to a membrane. These techniques allow for detection and analysis of genetic material.
Plasmids are small, circular DNA structures that can replicate independently of the host chromosome. They are commonly found in bacteria and play important roles in processes like drug resistance. Plasmid replication involves the recognition of an origin of replication sequence by plasmid-encoded initiator proteins. This leads to unwinding of the DNA and assembly of a replisome complex. The replication then proceeds bidirectionally via a rolling circle mechanism, where the growing DNA strand displaces the parental strand. Replication terminates once the circular plasmid is completely duplicated.
This document discusses subunit and peptide vaccines. Subunit vaccines contain purified antigens from pathogens rather than whole pathogens. They often require adjuvants and multiple doses to provide long-lasting immunity. Peptide vaccines use short amino acid sequences from pathogens to stimulate immune responses. While they are stable and inexpensive to produce, peptides may not stimulate T-cells on their own and require carriers or adjuvants. The document outlines advantages and disadvantages of both subunit and peptide vaccines.
Plasmid vectors like pBR322 and pUC are commonly used cloning vectors. pBR322 was one of the first vectors created and has advantages like a small size, antibiotic resistance markers, and a high copy number. pUC vectors also have a small size and high copy number, and contain a multiple cloning site within the lacZ gene allowing visual selection of recombinants. Artificial vectors combine elements from different sources to overcome limitations of natural plasmids, and are designed for efficient cloning and expression of foreign DNA in host cells.
This document discusses different types of genetic vectors used in molecular cloning. It begins by defining vectors as DNA molecules used to artificially carry foreign genetic material into host cells. Vectors can be classified as cloning or expression vectors. Key features of cloning vectors discussed include origins of replication, selectable markers, and the ability to accommodate DNA inserts of varying sizes. Common vector types described are plasmids, bacteriophages, cosmids, fosmids, and artificial chromosomes. Specific examples like pBR322, pUC, and lambda phage vectors are explained in terms of their components and applications. The document provides an overview of genetic vectors for molecular cloning.
The document summarizes phagemid and bacterial artificial chromosome (BAC) vectors. It describes that phagemid vectors are plasmids that contain both plasmid and phage origins of replication. Specifically, it discusses the features of pBluescript II phagemid vectors, including their polylinker and RNA polymerase promoter sequences. It also describes how pBluescript II phagemid vectors can produce blue or white colonies depending on insert presence. The document then explains that BAC vectors are low-copy plasmids that can hold up to 300kb DNA fragments. Examples of BAC vectors like pBAC108L and pBeloBAC11 are provided, with details about their replication origin and partitioning functions.
BAC & YAC are artificially prepared chromosomes to clone DNA sequences.yeast artificial chromosome is capable of carrying upto 1000 kbp of inserted DNA sequence
DNA footprinting is a technique used to identify protein binding regions on DNA. It involves treating DNA with nucleases like DNase I, which will degrade the DNA except for regions bound by proteins. These protected regions, called footprints, can identify transcription factor binding sites that regulate gene expression. The technique was originally developed in 1978 to study the binding specificity of the lac repressor protein, and it provides information on DNA-protein interactions and transcriptional regulation.
Selection & Screening of Recombinant cells & expression of recombinant (2) (1)SunandaArya
This document summarizes various methods for selecting and screening recombinant clones after introducing recombinant DNA into host cells. It discusses direct selection using antibiotic resistance genes, insertional inactivation by inserting DNA into antibiotic resistance genes, and blue-white screening using beta-galactosidase activity. It also covers colony hybridization using radioactive probes and immunological tests using antibodies to identify antigen-expressing colonies. Finally, it briefly discusses protein expression in different systems like bacteria, insect cells, and mammalian cells.
Gene cloning strategies depend on whether genomic or cDNA libraries are being constructed. Shotgun cloning is used to construct genomic libraries by fragmenting genomic DNA and inserting all fragments into vectors at once. cDNA libraries are constructed by reverse transcribing mRNA to cDNA, which is then cloned into vectors. Both library types are screened to identify overlapping clones that are assembled into contigs representing the entire genome.
This document discusses key components of expression vectors that are important for efficiently expressing cloned genes. It explains that expression vectors contain regulatory sequences like promoters and terminators to control transcription, as well as elements like ribosome binding sites, fusion tags, and selection markers. Specifically, it provides details on tightly regulated promoters, commonly used viral and bacterial promoters, and considerations for promoters in prokaryotic and eukaryotic expression systems. The document also reviews other important vector elements and their functions.
Cosmid Vectors, YAC and BAC Expression VectorsCharthaGaglani
1. Cosmid vectors are hybrid vectors derived from plasmids that contain the cos site from bacteriophage lambda, allowing them to clone DNA fragments up to 40 kb in size.
2. Yeast artificial chromosomes (YACs) are engineered yeast chromosomes that can clone very large DNA fragments, averaging 200-500 kb but up to 1 MB, taking advantage of yeast cell machinery.
3. Bacterial artificial chromosomes (BACs) are DNA constructs based on fertility plasmids that can clone up to 300 kb fragments and address issues with YAC stability and recombination.
This document discusses bacteriophage T4, a virus that infects E. coli bacteria. It has a complex protein coat and large double-stranded DNA genome. T4 uses the host cell's machinery to replicate and kills the host cell. T4 plays a role in cholera and diphtheria by carrying toxin genes that allow the bacteria to cause disease. Bacteriophage may be useful for treating antibiotic-resistant bacteria or infections where antibiotics cannot reach. T4 is also used in recombinant DNA technology.
The document discusses various methods of transfection in animals. Transfection is the process of introducing nucleic acids into eukaryotic cells. It describes viral transfection using bacteria like Agrobacterium tumefaciens and viruses. Non-viral methods include chemical transfection using calcium phosphate, liposomes, polyamines. Mechanical transfection employs microinjection or particle bombardment. Common chemical methods are calcium phosphate precipitation, polyplexes, and liposomes/lipoplexes. Viruses used are retroviruses, adenoviruses, adeno-associated viruses. Bacterial and viral vectors allow for integration into the host genome while chemical and mechanical are often transient.
Restriction enzymes are endonucleases found in bacteria and archaea that provide defense against viruses by selectively cutting invading viral DNA. Over 3,000 restriction enzymes have been identified, with some being commercially available. They recognize specific DNA sequences and cut the phosphodiester bonds within or near the recognition site. Restriction Fragment Length Polymorphism (RFLP) analyzes patterns from cleavage of DNA with restriction enzymes to differentiate organisms. RFLPs have forensic and medical applications such as paternity testing and disease detection.
This document discusses yeast artificial chromosomes (YACs) and bacterial artificial chromosomes (BACs). YACs are engineered chromosomes derived from yeast DNA that can clone very large DNA sequences in yeast cells of up to 1 megabase. BACs are cloning vectors derived from bacterial DNA that can clone DNA fragments of up to 300 kilobases in E. coli. Both systems allow cloning and propagation of large DNA fragments, but YACs can hold more DNA while BACs are more stable and better for functional analysis in mammalian cells.
Structural databases like PDB, CSD, and CATH contain 3D structural information of proteins, small molecules, and macromolecules determined through techniques like X-ray crystallography and NMR spectroscopy. These databases provide bibliographic data, atomic coordinates, and other details for each entry. PDB contains protein structures, CSD contains organic and metal-organic structures, and CATH classifies protein domains hierarchically. Structural databases have wide applications in structure prediction, analysis, mining, comparison, classification, structure refinement, and database annotation.
This document discusses methylases, which are enzymes that add methyl groups to DNA. Specifically:
- Methylases transfer methyl groups from S-adenosylmethionine to adenine or cytosine bases within their recognition sequence on DNA. This methylation protects the DNA from restriction endonucleases.
- The methylase and restriction enzyme of a bacterial species together form the restriction-modification system, with the methylase protecting the host DNA.
- Methylases are of interest because methylation of some restriction enzyme recognition sites protects the DNA from being cleaved by that enzyme. This allows study of DNA isolated from strains expressing common methylases like Dam or Dcm.
MBB 501 PLANT BIOTECHNOLOGY
INFORMATION ABOUT DIFFERENT DNA MODIFYING ENZYMES
WHAT IS AN ENZYME?
Alkaline Phosphatase
Polynucleotide kinase
Terminal deoxyneucleotidyl transferase
Nucleases
Exonuclease
Bal31 Exonuclease III
Endonuclease
S1 endonulease
Deoxyribonuclease 1 (Dnase 1)
RNase A
RNase H
Restriction Endonuclease
PvuI
PvuII
Different types of endonuclease enzymes
The recognition sequences for some of the most frequently used restriction endonucleases.
Categorization of enzymes
Isoschizomers
Neoschizomers
Isocaudomers
Cosmids are hybrid cloning vectors that combine features of plasmids and bacteriophages. They contain approximately 200 base pairs of DNA from the lambda phage, including the cos site sequence, which allows the vector to be packaged into phage particles and transduced into bacteria like a phage. Cosmids can accommodate large foreign DNA inserts of 35-45 kilobase pairs and are commonly used to construct genomic libraries.
Blotting techniques includes southren,northern,western and dot blottingbbmy
This document describes various blotting techniques used to detect specific DNA or RNA sequences, including Southern blotting, Northern blotting, Western blotting, and dot blotting. Southern blotting involves transferring DNA fragments separated by gel electrophoresis to a membrane and probing for specific sequences. Northern blotting is similar but uses RNA. Western blotting detects specific proteins. Dot blotting detects sequences in non-fractionated samples by directly applying samples to a membrane. These techniques allow for detection and analysis of genetic material.
Plasmids are small, circular DNA structures that can replicate independently of the host chromosome. They are commonly found in bacteria and play important roles in processes like drug resistance. Plasmid replication involves the recognition of an origin of replication sequence by plasmid-encoded initiator proteins. This leads to unwinding of the DNA and assembly of a replisome complex. The replication then proceeds bidirectionally via a rolling circle mechanism, where the growing DNA strand displaces the parental strand. Replication terminates once the circular plasmid is completely duplicated.
This document discusses subunit and peptide vaccines. Subunit vaccines contain purified antigens from pathogens rather than whole pathogens. They often require adjuvants and multiple doses to provide long-lasting immunity. Peptide vaccines use short amino acid sequences from pathogens to stimulate immune responses. While they are stable and inexpensive to produce, peptides may not stimulate T-cells on their own and require carriers or adjuvants. The document outlines advantages and disadvantages of both subunit and peptide vaccines.
Plasmid vectors like pBR322 and pUC are commonly used cloning vectors. pBR322 was one of the first vectors created and has advantages like a small size, antibiotic resistance markers, and a high copy number. pUC vectors also have a small size and high copy number, and contain a multiple cloning site within the lacZ gene allowing visual selection of recombinants. Artificial vectors combine elements from different sources to overcome limitations of natural plasmids, and are designed for efficient cloning and expression of foreign DNA in host cells.
This document discusses different types of genetic vectors used in molecular cloning. It begins by defining vectors as DNA molecules used to artificially carry foreign genetic material into host cells. Vectors can be classified as cloning or expression vectors. Key features of cloning vectors discussed include origins of replication, selectable markers, and the ability to accommodate DNA inserts of varying sizes. Common vector types described are plasmids, bacteriophages, cosmids, fosmids, and artificial chromosomes. Specific examples like pBR322, pUC, and lambda phage vectors are explained in terms of their components and applications. The document provides an overview of genetic vectors for molecular cloning.
It is the basics of vector cloning which necessary for every and each student who is intrested in biotechnology. It is only starting, if you want to more than this then please comment on it.
Cloning vectors are small DNA molecules used to replicate, amplify and express inserted DNA fragments. There are several types of cloning vectors including plasmids, bacteriophages, cosmids, and artificial chromosomes. Plasmids are the most commonly used cloning vectors as they can replicate autonomously in bacterial cells, contain selectable markers, and accept DNA insert sizes up to 10kb. Bacteriophages such as lambda can accept larger inserts up to 20kb but have a narrow host range. Cosmids combine properties of plasmids and phages to accept inserts up to 50kb.
Gene cloning vectors are DNA molecules that carry foreign DNA into host cells. They include plasmids, which are small circular DNA molecules found in bacteria, and bacteriophages, which are viruses that infect bacteria. Plasmids and bacteriophages have been genetically modified to serve as cloning vectors. Plasmids can accommodate DNA fragment inserts up to 10kb, while certain bacteriophages like lambda can accept larger inserts up to 20kb. Cloning vectors must be able to replicate in host cells and contain selectable marker genes and restriction enzyme sites for inserting foreign DNA.
This document discusses host cells and vectors used in gene cloning. It describes various prokaryotic and eukaryotic host cells, including E. coli, yeast, and mammalian cells. It also discusses the key features and types of vectors, including plasmids, bacteriophages, cosmids, and phagemids. Plasmids are the most commonly used prokaryotic vectors and come in various types including low-copy and high-copy plasmids. Common plasmid vectors discussed include pBR322, pUC18, and commercially available vectors. Bacteriophages like lambda phage and M13 phage are also described as viral vectors.
Cloning vectors are small pieces of DNA that can be stably maintained in an organism and have foreign DNA inserted into them for cloning purposes. The most commonly used cloning vectors are genetically engineered plasmids. Plasmids are taken from bacteria and can replicate within bacterial cells. Other types of cloning vectors include bacteriophages, cosmids, yeast artificial chromosomes, and bacterial artificial chromosomes, which can accommodate larger DNA fragments. Restriction enzymes and DNA ligase are used to cut and join DNA fragments for cloning into vectors.
The document discusses cloning vectors. It describes what a cloning vector is, including that it is a small piece of DNA that can stably maintain foreign DNA for cloning purposes. Common types of cloning vectors are described in detail, including plasmids, bacteriophages, cosmids, yeast artificial chromosomes, bacterial artificial chromosomes, and plant virus vectors. Key features of cloning vectors like origins of replication, antibiotic resistance genes, and cloning sites are also summarized.
This document provides an overview of plasmids. It defines plasmids as small, circular, extrachromosomal DNA molecules that can replicate independently in bacteria. Plasmids contain genes that provide benefits to bacteria like antibiotic resistance. They are transferred between bacteria through processes like transformation, transduction, and conjugation. Plasmids are classified based on their functions and are important tools in biotechnology as they allow cloning, protein production, and other applications.
This document provides information about various types of cloning vectors used in genetic engineering. It discusses plasmids like pBR322, pUC18, and pET21 that are commonly used as cloning vectors in E.coli. It also mentions bacteriophage vectors like M13 and lambda phage that can accommodate larger DNA inserts. Other vectors discussed include yeast episomal plasmids, cosmids, and mammalian virus SV40 that is used for cloning in animal cells. The document provides details on the characteristics, components, and advantages of these different cloning vectors.
The document discusses different types of cloning vectors. It describes cloning vectors as DNA fragments capable of self-replication that can transport foreign genetic material into host cells. The main types discussed are plasmids, bacteriophages, cosmids, BACs, YACs, and retroviral vectors. Each type has distinct features like replication origin and cloning capacity that determine their suitability for different applications. Plasmids were among the earliest vectors and can clone inserts up to 10kb, while BACs and YACs can accommodate much larger DNA fragments. The key factors in choosing a vector are the size of the DNA insert and cloning efficiency required.
Vectors are DNA molecules that can carry foreign genes into host cells. There are several types of vectors including plasmids, bacteriophages, cosmids, bacterial artificial chromosomes (BACs), and yeast artificial chromosomes (YACs). Plasmids are the most commonly used cloning vectors and can accept DNA inserts ranging from a few base pairs to 10kb. Larger DNA fragments can be cloned using vectors based on bacteriophages, cosmids, BACs, or YACs.
CONFERENCE 5-Techniques in Genetic Engineering.pptDicksonDaniel7
This document describes genetic engineering techniques such as selective breeding, recombinant DNA, PCR, and transgenic organisms. It explains how recombinant DNA technology uses restriction enzymes and DNA ligases to insert DNA fragments into cloning vectors, which are then inserted into host bacteria for replication. This allows mass production of useful proteins like insulin. The document also discusses how Agrobacterium tumefaciens and its Ti plasmid are widely used vectors to introduce foreign DNA into plant cells and genomes. In summary, the document outlines genetic engineering methods and how they have been applied to biotechnology and agriculture.
Vectors are essential tools for genetic engineering that allow recombinant DNA to be introduced into and replicated in host organisms. Plasmids are the most commonly used bacterial cloning vectors. Plasmids are small, circular DNA molecules that can replicate independently of the bacterial chromosome. Important plasmid vectors include pBR322, which contains two antibiotic resistance genes for selection, and pUC19, which allows blue-white screening to identify recombinant clones through disruption of the lacZ gene. Plasmid vectors are useful for amplifying DNA inserts, producing recombinant proteins, and transferring genes for applications such as gene therapy.
A genetically engineered DNA molecule from bacteria , phage or yeast to carry foreign DNA for the purpose of cloning and expression of the inserted DNA of interest in RDT
Plasmid vectors are circular, self-replicating DNA molecules that are commonly used to clone DNA fragments in bacteria. The document discusses the key features of plasmid vectors including their origin of replication, selectable marker genes, and cloning sites. It also describes different types of plasmids such as F-plasmids, R-plasmids, and Ti-plasmids. Common plasmid vectors used in genetic engineering like pUC19, pBR322, and Ti-plasmids are also outlined. Finally, the applications of plasmids in genetic engineering for cloning genes and mass producing proteins are briefly mentioned.
Bacterial plasmids are small, circular, extrachromosomal DNA molecules that are able to replicate independently of the bacterial chromosome. Plasmids are commonly found in bacteria and can carry genes conferring traits such as antibiotic resistance. Plasmids are useful genetic engineering tools as they can be used to insert foreign DNA and replicate this DNA within bacterial cells. Common plasmids include R plasmids containing antibiotic resistance genes and F plasmids involved in bacterial conjugation.
DNA cloning allows for the replication and amplification of specific DNA fragments. It involves inserting DNA fragments into vector molecules, such as plasmids, which are then introduced into host cells. The vector carries the DNA fragment into the host cell and acts as a replicating molecule. Restriction enzymes and ligases are used to cut out the DNA fragment and insert it into the vector. The cells are then grown on selective media so that cells containing the vector and insert are selected for. This allows unlimited copies of the DNA fragment of interest to be produced for study and manipulation.
There are six main types of cloning vectors used to insert DNA fragments: plasmid vectors, phage lambda vectors, cosmid vectors, shuttle vectors, yeast artificial chromosomes (YACs), and bacterial artificial chromosomes (BACs). Plasmid vectors are the most commonly used as they can accept DNA fragments up to 10kb and replicate autonomously in bacteria. Lambda phage and cosmid vectors can accept larger fragments up to 52kb, while YACs and BACs are used to clone very large fragments up to 500kb and 200kb respectively.
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PLASMID CLONING VECTORS.pdf
1. PLASMID CLONING VECTORS
Dr Diptendu Sarkar
diptendu81@gmail.com
RKMV
Sources:
1. Biotechnology By B.D. Singh,
2. Gene cloning and DNA analysis By TA Brown,
3. Advance and Applied Biotechnology By Marian Patrie,
4. Molecular Biotechnology By Glick et al and
5. Principle of Gene Manipulation By sandy b Primrose et al.
2. Introduction:
• DNA molecule used for carrying an exogenous DNA into a host organism and facilitates stable integration
and replicate autonomously in an appropriate host cell is termed as Vector.
• Molecular cloning involves series of sequential steps which includes restriction digestion of DNA fragments
both target DNA and vector, ligation of the target DNA with the vector and introduction into a host
organism for multiplication. Then the fragments resulted after digestion with restriction enzymes are
ligated to other DNA molecules that serve as vectors.
In general, vectors should have following characteristics:
• Capable of replicating inside the host.
• Have compatible restriction site for insertion of DNA molecule (insert).
• Capable of autonomous replication inside the host (ori site).
• Smaller in size and able to incorporate larger insert size.
• Have a selectable marker for screening of recombinant organism.
12/10/2019 DS/RKMV/MB 2
3. Plasmids:
• Plasmids are naturally occurring extra chromosomal double-stranded circular DNA molecules which can
autonomously replicate inside bacterial cells.
• Plasmids range in size from about 1.0 kb to over 250 kb.
• Plasmids encode only few proteins required for their own replication (replication proteins) and these
proteins encoding genes are located very close to the ori.
• All the other proteins required for replication, e.g. DNA polymerases, DNA ligase, helicase, etc., are provided
by the host cell. Thus, only a small region surrounding the ori site is required for replication.
• Other parts of the plasmid can be deleted and foreign sequences can be added to the plasmid without
compromising replication.
12/10/2019 DS/RKMV/MB 3
4. • The host range of a plasmid is determined by its ori region. Plasmids whose ori region is derived from
plasmid Col E1 have a restricted host range. They only replicate in enteric bacteria, such as E. coli, Salmonella,
etc. Plasmids of the RP4 type will replicate in most gram negative bacteria, to which they are readily
transmitted by conjugation. Plasmids like RSF1010 are not conjugative but can be transformed into a wide
range of gram -ve bacteria. Plasmids isolated from Staphylococcus aureus have a broad host range and can
replicate in many other gram-positive bacteria.
• Some of the phenotypes which the naturally occurring plasmids confer on their host cells:
• Antibiotic resistance
• Antibiotic production
• Degradation of aromatic compounds
• Hemolysis production
• Sugar fermentation
• Enterotoxin production
• Heavy metal resistance
• Bacteriocin production
• Induction of plant tumors
• Hydrogen sulphide production
12/10/2019 DS/RKMV/MB 4
5. Plasmid transfer by conjugation between bacterial cells. The donor and recipient cells attach to each other by
a pilus, a hollow appendage present on the surface of the donor cell. A copy of the plasmid is then passed to
the recipient cell. Transfer is thought to occur through the pilus, but this has not been proven and transfer by
some other means (e.g. directly across the bacterial cell walls) remains a possibility.
12/10/2019 DS/RKMV/MB 5
6. • Most plasmids exist as double-stranded circular DNA molecules. However, the inter-conversion of super
coiled, relaxed covalently closed circular DNA and open circular DNA is possible.
• Not all plasmids exist as circular molecules. Linear plasmids have been found in a variety of bacteria, e.g.
Streptomyces sp. and Borrelia burgdorferi.
• However, few types of plasmids are also able to replicate by integrating into bacterial chromosomal DNA;
these are known as integrative plasmids or episomes. They are found mainly in prokaryotes but some
eukaryotes are also found to harbour them. In prokaryotes they are found in Escherichia coli, Pseudomonas
species, Agrobacterium species etc. In eukaryotes they are mainly found in Saccharomyces cerevisiae.
12/10/2019 DS/RKMV/MB 6
8. Plasmid classification
The plasmids are divided into 6 major classes as described below depending on the phenotype:
i) Resistance or R plasmids carry genes which give resistance to the bacteria from one or more chemical
agents, such as antibacterial agents. R plasmids are very important in clinical microbiology as they can have
profound consequences in the treatment of bacterial infections. Eg: RP4 plasmid, which is commonly found in
Pseudomonas and in many other bacteria.
ii) Fertility or F plasmids are conjugative plasmid found in F+ bacterium with higher frequency of conjugation.
F plasmid carries transfer gene (tra) and has the ability to form Conjugation Bridge (F pilus) with F- bacterium.
Eg: F plasmid of E. coli.
iii) Col plasmids have genes that code for colicins, proteins that kill other bacteria. Eg: ColE1 of E. coli.
iv) Degradative plasmids allow the host bacterium to metabolize unusual molecules such as toluene and
salicylic acid. Eg TOL of Pseudomonas putida.ca
12/10/2019 DS/RKMV/MB 8
9. v) Virulence plasmids confer pathogenicity on the host bacterium. Eg: Ti plasmids of Agrobacterium
tumefaciens, which induce crown gall disease on dicotyledonous plants.
vi) Cryptic Plasmids do not have any apparent effect on the phenotype of the cell harboring them. They just
code for enzymes required for their replication and maintenance in the host cell.
Based on the origin or source of plasmids, they have been divided into two major classes: such as natural and
artificial.
i) Natural plasmids: They occur naturally in prokaryotes or eukaryotes. Example: ColE1.
ii) Artificial plasmids: They are constructed in-vitro by re-combining selected segments of two or more other
plasmids (natural or artificial). Example: pBR322.
12/10/2019 DS/RKMV/MB 9
10. Figure : The use of antibiotic resistance as a
selectable marker for a plasmid. RP4 (top)
carries genes for resistance to ampicillin,
tetracycline and kanamycin.
Only those E. coli cells that contain RP4 (or a
related plasmid) are able to survive and grow
in a medium that contains toxic amounts of
one or more of these antibiotics.
12/10/2019 DS/RKMV/MB 10
11. SIZE
PLASMID NUCLEOTIDE LENGTH (kb) MOLECULAR
MASS (MDa)
ORGANISM
pUC8 2.1 1.8 E. Coli
ColE1 6.4 4.2 E. Coli
RP4 54 36 Pseudomonas and others
F 95 63 E. Coli
TOL 117 78 Pseudomonas putida
pTiAch5 213 142 Agrobacterium tumefaciens
Sizes of representative plasmids.
12/10/2019 DS/RKMV/MB 11
13. Characteristics of ideal plasmid vectors
1. Size: plasmid must be small in size. The small is helpful for easy uptake of cDNA by host cells and for the
isolation of plasmid without damage. Ideal vector should be less than or equal to 10kb. The small size is
essential for easy introduction in cell by transformation, transduction and electroporation.
2. Copy number: the plasmid must be present in multiple copies.
3. Genetic markers: plasmid must have one or few genetic markers. These markers help us for the selection
of organism that has recombinant DNA
4. Origin of replication: the plasmid must have its own orogin of replication and regulatory genes for the
self-replication.
5. Unique restriction sites: the plasmid must have unique restriction sites common restriction enzymes in
use.
6. Multiple cloning sites: This property permits the insertion of gene of interest and plasmid re-
circularization.
7. Insertional inactivation: the plasmid must have unique sites for restriction enzymes in marker genes. This
will help us for the selection of recombination by insertional inactivation method.
8. Pathogenicity: the plasmid should not have any pathogenic property.
9. Should not be transferred by conjugation: This property of vector molecule prevents recombinant DNA to
escape to natural population of bacteria.
10. Selectable make gene: Vector molecules should have some detectable traits. These traits enable the
transformed cells to be identified among the non-transformed ones. eg. antibiotic resistance gene.
12/10/2019 DS/RKMV/MB 13
14. NOTE :
• It should be kept in mind that the DNA molecule used as vectors have coevolved with their natural host
species, and hence are adopted to function well in them and in their closely related species.
• Therefore, the choice of vector depends on host species into which DNA insert or gene is to be cloned.
• In addition, most naturally occurring vectors do not have all required functions; therefore, useful vectors
have been created by joining together segments performing species functions (called modules) from two
or more natural entities.
12/10/2019 DS/RKMV/MB 14
15. Multiple cloning sites (MCS)
• MCS is a synthetic DNA segment that has a
cluster of unique sites for restriction enzymes. It
is inserted into a gene cloning vector with a view
to increasing the number of gene cloning sites.
• The size of MCS usually from 60 bp to 84 bp. The
number and arrangement of restriction sites
varies from MCS to MCS in different vectors. As
MCS is a cluster of many restriction sites, it is also
known as polylinker or polylinker sequence.
USES:
1. MCS are used to increase gene cloning sites in
vector DNAs.
2. As they have unique sites for many restriction
enzymes, DNA segments with different types of
cut-ends can be inserted into the vector.
3. Restriction enzymes of choice can be used to
insert a gene into the vector.
12/10/2019 DS/RKMV/MB 15
16. Properties of good host
A good host has the following features:
1. Be easy to transform
2. Support the replication of recombinant DNA
3. Be free from elements that interfere with replication of recombinant DNA
4. Lack active restriction enzymes, e.g., E.coli K12 substrain HB 101.
5. Should not have methylases, since, these enzymes would methylate the replicated recombinant DNA
which, as a result, would become resistant to useful restriction enzymes.
6. Be deficient in normal recombinant function, so, that, the DNA insert is not altered by recombination
events.
12/10/2019 DS/RKMV/MB 16
17. ARTIFICIAL PLASMIDS:
• Naturally occurring plasmids has several limitations; for example, some are stringent and not relaxed
(pSC101), some has poor marker genes (ColE1), and some are too large (RSF2124). To overcome the
limitations of natural vectors, artificial plasmid are designed by combining different elements from diverse
sources.
• Artificial plasmids vectors are classified into two broad types based on their use:
1. Cloning vector
2. Expression vector
• Apart from the following, there is another class of vectors known as shuttle vector. Shuttle vectors can be
propagated in two or more different host species (both in prokaryotes and eukaryotes). Hence, inserted
DNA can be manipulated and replicated in two different cellular systems.
• Cloning vectors are designed for efficient transfer of foreign DNA into the host. Expression vectors have
efficient machinery for cloning and expression of foreign gene in the host system.
• Selection of a vector depends upon various criteria decided by the experimental goal.
12/10/2019 DS/RKMV/MB 17
18. Cloning Vector:
A cloning vector is defined as a vector used for replication of a cloned DNA fragment in a host cell. These
vectors are frequently engineered to contain “ori” – origin of replication sites particular to the host organism.
Examples of commonly used cloning vectors are: pUC18, pUC19, pBluescript vectors.
12/10/2019 DS/RKMV/MB 18
19. Types of Cloning Vectors:
• Cloning vectors extensively used in molecular cloning experiments can be considered under following
types: plasmid, phage vector and cosmid.
• Different vectors have different insert size and also vary in mode of replication inside the host.
• Mammalian genes are usually too large (~100 kb), and thus suffer from restrictions in complete
inclusion, with the conventional cloning vectors, having limited insert size.
• Vectors engineered more recently, known as artificial chromosomes, have overcome this problem by
mimicking the properties of host cell chromosomes. They have much larger insert size than other vectors.
12/10/2019 DS/RKMV/MB 19
20. Different type of cloning vectors
Vector Insert size Source Application
Plasmid ≤ 15 kb Bacteria cDNA cloning and expression
assays
Phage 5-20 kb Bacteriophage λ Genomic DNA cloning, cDNA
cloning and expression
library
Cosmid 35-45 kb Plasmid containing a bacteriophage λ
cos site
Genomic library construction
BAC (bacterial
artificial
chromosome)
75-300 kb Plasmid ocntaining ori from E.coli F-
plasmid
Analysis of large genomes
YAC (yeast artificial
chromosome)
100-1000 kb
(1 Mb)
Saccharomyces cerevisiae centromere,
telomere and autonomously replicating
sequence
Analysis of large genome,
YAC transgenic mice
MAC (mammalian
artificial
chromosome)
100 kb to
> 1 Mb
Mammalian centromere, telomere and
origin of replication
Under development for use
in animal biotechnology and
human gene therapy
12/10/2019 DS/RKMV/MB 20
21. EXAMPLES OF CLONING VECTOR:
pBR322
pBR322 is a widely-used E. coli cloning vector. It was created in 1977 in
the laboratory of Herbert Boyer at the University of California San
Francisco. The p stands for "plasmid" and BR for "Bolivar" and
"Rodriguez", researchers who constructed it. ‘322’ distinguishes this
plasmid from others developed in the same laboratory (there are also
plasmids called pBR325, pBR327, pBR328, etc.).
• pBR322 is 4363 base pairs in length.
• pBR322 plasmid has the following elements:
“rep” replicon from plasmid pMB1 which is responsible for
replication of the plasmid.
“rop” gene encoding Rop protein, are associated with stability of
plasmid and also controls copy number (increase number). The
source of “rop” gene is pMB1plasmid.
“tet” gene encoding tetracycline resistance derived from pSC101
plasmid.
“bla” gene encoding β lactamase which provide ampicillin
resistance (source: transposon Tn3).
A map of pBR322 showing the
positions of the ampicillin resistance
(ampR) and tetracycline resistance
(tetR) genes, the origin of replication
(ori) and some of the most important
restriction sites
12/10/2019 DS/RKMV/MB 21
22. 12/10/2019 DS/RKMV/MB 22
1) The first useful feature of pBR322 is its size. pBR322 is 4363 bp, which means that not only can the vector
itself be purified with ease, but so too can any recombinant DNA molecules constructed with it. Even with
6 kb of additional DNA, a recombinant pBR322 molecule is still of a manageable size.
2) The second feature of pBR322 is that, it carries two sets of antibiotic resistance genes. Either ampicillin or
tetracycline resistance can be used as a selectable marker for cells containing the plasmid, and each
marker gene contains unique restriction sites that can be used in cloning experiments.
3) A third advantage of pBR322 is that it has a reasonably high copy number. Generally, there are about 15
molecules present in a transformed E. coli cell, but this number can be increased up to between 1000 and
3000 by plasmid amplification in the presence of a protein synthesis inhibitor such as chloramphenicol.
An E. coli culture therefore provides a good yield of recombinant pBR322 molecules.
Useful features of pBR322
23. 12/10/2019 DS/RKMV/MB 23
5) It has 528 restriction sites for 66 restriction enzymes. Among them 20 restriction enzymes cut it at
unique restriction sites. Tetracycline has 6 unique sites for 6 restriction enzymes. Ampicillin gene has 3
unique restriction site.
6) The sequences other than Tet and Amp genes, have unique sites for 1 restriction enzymes. There is no
restriction inactivation when gene is inserted into any one of these sites.
24. The pedigree of pBR322. (a) The
manipulations involved in construction of
pBR322. (b) A summary of the origins of
pBR322.
• The ampR gene originally
resided on the plasmid
R1, a typical antibiotic
resistance plasmid that
occurs in natural
populations of E. coli .
• The tetR gene is derived
from R6-5, a second
antibiotic resistance
plasmid.
• The replication origin of
pBR322, which directs
multiplication of the
vector in host cells, is
originally from pMB1,
which is closely related to
the colicin-producing
plasmid ColE1 .
12/10/2019 DS/RKMV/MB 24
25. 12/10/2019 DS/RKMV/MB 25
• pBR322 has several unique restriction sites that can be
used to open up the vector before the insertion of a new
DNA fragment . BamHI, for example, cuts pBR322 at just
one position, within the cluster of genes that code for
resistance to tetracycline.
• A recombinant pBR322 molecule, one that carries an
extra piece of DNA in the BamHI site, is no longer able to
confer tetracycline resistance on its host, as one of the
necessary genes is now disrupted by the inserted DNA.
• Cells containing this recombinant pBR322 molecule are
still resistant to ampicillin, but are sensitive to
tetracycline (ampR tetS).
Recombinant selection with pBR322: Insertional inactivation of an antibiotic resistance gene
26. 12/10/2019 DS/RKMV/MB 26
Screening for pBR322 recombinants is performed in the following way.
• After transformation, the cells are
plated onto an ampicillin medium and
incubated until colonies appear .
• All of these colonies are transformants
(remember, untransformed cells are
ampS and so do not produce colonies
on the selective medium) but only a
few contain recombinant pBR322
molecules; most will contain the
normal, self ligated plasmid.
27. 12/10/2019 DS/RKMV/MB 27
• To identify the recombinants the colonies are replica
plated onto agar medium that contains tetracycline
• After incubation, some of the original colonies
regrow, but others do not .
• Those that do grow, consist of cells that carry the
normal pBR322 with no inserted DNA, and
therefore a functional tetracycline resistance gene
cluster (ampRtetR).
• The colonies that do not grow on tetracycline agar
are recombinants (ampRtetS).
• Reference back to the original ampicillin agar plate
reveals the positions of these colonies, enabling
samples to be recovered for further study.
28. pUC plasmids:
• pUC plasmids are small, high copy number plasmids of size 2686bp.
• This series of cloning vectors were developed by Messing and co-workers in the University of California. The
p in its name stands for plasmid and UC represents the University of California.
• pUC vectors contain a lacZ sequence and multiple cloning site (MCS) within lacZ. This helps in use of broad
spectrum of restriction endonucleases and permits rapid visual detection of an insert.
• pUC18 and pUC19 vectors are identical apart from the fact that the MCS is arranged in opposite orientation.
• pUC vectors consists of following elements:
pMB1 “rep” replicon region derived from plasmid pBR322 with single point mutation (to increase copy
number).
“bla” gene encoding β lactamase which provide ampicillin resistance which is derived from pBR322. This
site is different from pBR322 by two point mutations.
E.coli lac operon system.
• “rop” gene is removed from this vector which leads to an increase in copy number.
12/10/2019 DS/RKMV/MB 28
29. pUC8: A Lac selection plasmid
12/10/2019 DS/RKMV/MB 29
The cloning vector pUC8: (a) the normal vector molecule; (b) a
recombinant molecule containing an extra piece of DNA inserted
into the BamHI site.
30. • An MCS is a short DNA sequence consisting of restriction sites
for many different restriction endonucleases.
• The MCS is inserted into the lacZ sequence, which encodes
the promoter and the α-peptide of β-galactosidase.
• Insertion of the MCS into the lacZ fragment does not affect
the ability of the α-peptide to mediate complementation,
while cloning DNA fragments into the MCS does.
• Therefore, recombinants can be detected by blue/white
screening on growth medium containing X gal in presence of
IPTG as an inducer.
12/10/2019 DS/RKMV/MB 30
• The native lacZ promoter (Plac) is situated just upstream of the cloned gene, allowing expression of genes
on inserts that are correctly oriented. Most of the nonessential DNA has been removed to provide the ability
to clone larger fragments. An ampicillin marker is included for selection of transformants.
31. • The pUC plasmids were engineered from the pBR322
origin of replication to include the alpha portion of the
beta-galactosidase gene (lacZ' ), complete with its
promoter. The beta portion of lacZ was included in
the chromosome of the host, so that the host
containing the plasmid was Lac+.
12/10/2019 DS/RKMV/MB 31
• The restriction enzyme sites (6 in pUC8/9; 10 in
pUC18/19) are clustered together on an
oligonucleotide (called the polylinker, or multiple
cloning site), which is in-frame within the first few
amino acids of the lacZ‘ reporter gene (thus providing
insertional inactivation, Lac+ → Lac-).
32. 12/10/2019 DS/RKMV/MB 32
Recombinant selection with pUC vectors: Alpha complementation/
blue-white screening / Insertional inactivation
• The lac-Z gene product (β-galactosidase) is a tetramer, and each monomer is made of
two parts – lacZ-alpha, and lacZ- omega.
• If the alpha fragment was deleted, the omega fragment is non-functional; however, alpha fragment
functionality can be restored in transformation via plasmid. Hence, then name alpha-complementation.
• The E. coli enzyme β-galactosidase is a homo-tetramer of the protein product of the lacZ gene.
• Certain mutations in the 5' region of lacZ prevent subunit association.
• Because monomers lack enzyme activity, the failure to assemble leads to a Lac- phenotype.
33. 12/10/2019 DS/RKMV/MB 33
• The activity of the enzyme β –galactosidase is easily monitored by inducing in the growth medium the
chromogenic substrate 5-bromo-4-chloro-3-indolyl-β–galactoside (Xgal).
• This compound is colorless, but on cleavage, releases a blue indolyl derivative.
• On solid medium, colonies that are expressing active β –galactosidase are blue in colour, while
those without the activity are white in colour.
• This is often referred as blue/white screening.
34. The rationale behind insertional inactivation of the lacZ¢ gene carried by pUC8.
12/10/2019 DS/RKMV/MB 34
(a) The bacterial and plasmid
genes complement each
other to produce a functional
b-galactosidase molecule.
(b) Recombinants are screened
by plating onto agar
containing X-gal and IPTG.
35. SHUTTLE VECTORS
• The plasmid cloning vector that can replicate in two different organisms is called shuttle vector. It has two
origin of replication, each of which is specific to a host.
• Since, shuttle vectors replicate in two different hosts, they are known as bifunctional vectors.
• A shuttle vector is a vector that can propagate in two different host species, typically E. coli and a
eukaryotic host species. For example, pMK3/4 has a gram-positive ori for cloning in Bacillus subtilis, and a
gram-negative ori for cloning in E. coli.
• In this way, genetic engineering may be done in E. coli
where it is easier and transferred to B. subtilis for final
expression and excretion.
• Many eukaryotic expression vectors are shuttle
vectors, since they are first produced in E. coli, and
then introduced into the eukaryotic host.
12/10/2019 DS/RKMV/MB 35
Advantages of shuttle vectors :
Shuttle vectors can be used to shuttle (move to and fro)
a gene between two different hosts. After expression
studies in the second host, if there is a need for further manipulation, the rDNA can be modified it in the first
host.
36. pJDB 219
• The pBR322 derivative encodes for ampicillin resistance
(AmpR) and tetracycline resistance (TetR). It has an origin
of replication essential for replication in E.coli.
• 2ųm plasmid of yeast contributes an origin of replication
of plasmid in yeast cells.
• The LEU2 gene encodes for isopropyl malate
dehydrogenase that converts pyruvic acid into leucine.
The leucine can easily be assayed by growing the
transformants in a medium lacking leucine.
• The foreign DNA segment is inserted into pJDB219 to construct an rDNA. The rDNA is introduced into E.coli
cells by bacterial transformation. The recombinant E.coli cells are selected by insertional inactivation method.
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• The pJDB219 shuttles an inserted DNA between E.coli cell and Yeast cell. It consists of the
entire sequence of a pBR322 and a 2ųm plasmid and a selectable marker gene LEU2 of
yeast chromosome.
37. • The selected E.coli cells are then cultured in a medium containing chloramphenicol to increase the copy
number of the plasmid. Then the plasmids are isolated from E.coli and introduced into LEU2 mutant yeast
cells by transformation. The transformed yeasts (Saccharomyces cerevisiae) are selected by growing them
in a medium lacking leucine.
• The LEU2 gene of the plasmid
undergoes recombination with
LEU2 gene of yeast chromosome.
As a result, the entire plasmid
(rDNA) becomes a part of the
chromosome.
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38. • pEB10 is a circular, double-stranded plasmid.
• It is 8.9 Kbp in size.
• It has two selectable markers- ampicillin resistance gene (AmpR) and kanamycin resistance gene (KanR).
• The ampicillin resistance gene determinant is derived from pBR322.
• The kanamycin resistance determinant is a derivative
of pUB110.
• It has an origin of replication for replication of
the plasmid in E,coli.
pEB10
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• It has yet another origin of replication that switches
on replication in Bacillus subtilis.
• As the pEB10 has two different origins of replication,
it can replicate both in E,coli and B.subtilis.
39. • The desired gene is first inserted into pEB10 to construct rDNA.
• The rDNA is introduced in E.coli cell by bacterial transformation.
• E.coli cells are naturally resistant to chloramphenicol that inhibits protein synthesis. But B.subtilis is
sensitive to chloramphenicol.
• The recombinant E,coli culture is treated with chloramphenicol to increase the copy number of pEB10
in the E.coli.
• The amplified rDNA is isolated, purified and introduced into Bacillus subtilis by transformation.
• The transformants are screened and mass cultured for the expression of the cloned gene.
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