This document discusses various types of cloning vectors used in genetic engineering experiments. It begins by describing the basic features a vector must possess, including the ability to self-replicate and contain selectable markers. It then focuses on plasmids, noting that E. coli is commonly used as a host and that vectors like pBR322 were early workhorses. Later sections cover lambda phage vectors, cosmids, YACs, and BACs, which can accommodate larger DNA fragments. The document provides detailed information on widely used vectors like pUC, M13, and their features to replicate, package DNA, and enable cloning experiments.
This document discusses restriction enzymes, which are important tools in genetic engineering and recombinant DNA technology. Restriction enzymes cut DNA at specific recognition sequences and are used to cut DNA into fragments that can then be recombined in new ways. The document provides details on the discovery and functions of various restriction enzymes as well as other enzymes used in genetic engineering such as DNA ligase, alkaline phosphatase, polynucleotide kinase, and reverse transcriptase. It also discusses the use of restriction enzymes to build the first synthetic bacterial genome.
Recombinant DNA technology involves isolating DNA from different species, cutting it with restriction enzymes, and splicing the pieces together to form new recombinant molecules. These molecules are then inserted into host cells like bacteria or yeast where they can be replicated in large quantities. Key aspects of the process include using restriction enzymes to cut DNA at specific recognition sequences, producing DNA fragments with cohesive or blunt ends, and inserting the fragments into plasmids - small extrachromosomal DNA molecules found in bacteria. Plasmids are often used as vectors to carry foreign DNA, and they allow selection of cells containing the recombinant DNA through the use of antibiotic resistance genes on the plasmid.
Genetic engineering involves manipulating genetic material (DNA) to achieve desired goals. The basic principles involve artificially copying DNA from one organism and joining it into the DNA of another. Molecular tools like restriction enzymes and DNA ligases are used to cut and join DNA. Methods to transfer genes include transformation, electroporation, and liposome-mediated transfer. Applications include producing human proteins like insulin, developing gene therapies, and genetically modifying plants. Gene libraries, blotting techniques like Southern blotting, and PCR are also discussed as important molecular tools in genetic engineering.
This document provides an overview of recombinant DNA technology (RDT). It discusses the history and discovery of RDT, the key steps involved which include selecting DNA inserts and vectors, introducing the DNA into host cells, and expressing the DNA. Important tools for RDT are described, including restriction enzymes, vectors, and host organisms. Common applications of RDT include producing hormones, medicines, transgenic plants and animals. Ongoing projects utilizing RDT are mentioned.
Recombinant DNA technology involves joining DNA fragments from different sources to produce novel DNA molecules. This is done by using restriction enzymes to cut DNA fragments at specific recognition sequences, and DNA ligase to join the fragments together. Vectors like plasmids are used to clone and replicate the DNA fragments of interest. Restriction enzymes recognize palindromic sequences and cut the DNA either as blunt ends or sticky ends. The DNA fragments can then be ligated into an expression vector to produce the desired protein. cDNA libraries are useful for cloning eukaryotic genes by reverse transcribing mRNA to cDNA.
This document provides information about gene cloning and cloning vectors. It discusses DNA cloning, which involves reproducing DNA fragments using either cell-based or PCR-based approaches. A vector is required to insert the DNA fragment into a host cell. Common vectors discussed include plasmids, bacteriophages, cosmids, and artificial chromosomes. The document outlines the basic plasmid cloning strategy and describes key steps like restriction enzyme digestion of the DNA and vector, ligation, transformation, and selection of recombinant clones. Important enzymes and their functions are also summarized, such as ligase, kinases, phosphatases, and reverse transcriptase.
This document provides an overview of recombinant DNA technology. It discusses the basic principles, which involve generating DNA fragments, inserting a selected fragment into a cloning vector, introducing the vector into host cells, and multiplying the recombinant molecules. Key steps include using restriction enzymes to cut DNA at specific sites, ligases to join fragments, and various vectors like plasmids and bacteria to clone the DNA. The document also outlines several applications of rDNA technology, such as producing proteins, diagnosing diseases, and developing genetically engineered plants.
This document provides information about synthesizing and cloning cDNA from mRNA. It describes how cDNA libraries are constructed by isolating mRNA, synthesizing cDNA via reverse transcription, treating cDNA ends, ligating the cDNA to vectors, and transforming the vectors into host cells. The key steps include mRNA purification, first and second strand cDNA synthesis, linker ligation, vector ligation, and transformation. cDNA libraries are useful for eukaryotic gene analysis as they contain only expressed genes without introns.
This document discusses restriction enzymes, which are important tools in genetic engineering and recombinant DNA technology. Restriction enzymes cut DNA at specific recognition sequences and are used to cut DNA into fragments that can then be recombined in new ways. The document provides details on the discovery and functions of various restriction enzymes as well as other enzymes used in genetic engineering such as DNA ligase, alkaline phosphatase, polynucleotide kinase, and reverse transcriptase. It also discusses the use of restriction enzymes to build the first synthetic bacterial genome.
Recombinant DNA technology involves isolating DNA from different species, cutting it with restriction enzymes, and splicing the pieces together to form new recombinant molecules. These molecules are then inserted into host cells like bacteria or yeast where they can be replicated in large quantities. Key aspects of the process include using restriction enzymes to cut DNA at specific recognition sequences, producing DNA fragments with cohesive or blunt ends, and inserting the fragments into plasmids - small extrachromosomal DNA molecules found in bacteria. Plasmids are often used as vectors to carry foreign DNA, and they allow selection of cells containing the recombinant DNA through the use of antibiotic resistance genes on the plasmid.
Genetic engineering involves manipulating genetic material (DNA) to achieve desired goals. The basic principles involve artificially copying DNA from one organism and joining it into the DNA of another. Molecular tools like restriction enzymes and DNA ligases are used to cut and join DNA. Methods to transfer genes include transformation, electroporation, and liposome-mediated transfer. Applications include producing human proteins like insulin, developing gene therapies, and genetically modifying plants. Gene libraries, blotting techniques like Southern blotting, and PCR are also discussed as important molecular tools in genetic engineering.
This document provides an overview of recombinant DNA technology (RDT). It discusses the history and discovery of RDT, the key steps involved which include selecting DNA inserts and vectors, introducing the DNA into host cells, and expressing the DNA. Important tools for RDT are described, including restriction enzymes, vectors, and host organisms. Common applications of RDT include producing hormones, medicines, transgenic plants and animals. Ongoing projects utilizing RDT are mentioned.
Recombinant DNA technology involves joining DNA fragments from different sources to produce novel DNA molecules. This is done by using restriction enzymes to cut DNA fragments at specific recognition sequences, and DNA ligase to join the fragments together. Vectors like plasmids are used to clone and replicate the DNA fragments of interest. Restriction enzymes recognize palindromic sequences and cut the DNA either as blunt ends or sticky ends. The DNA fragments can then be ligated into an expression vector to produce the desired protein. cDNA libraries are useful for cloning eukaryotic genes by reverse transcribing mRNA to cDNA.
This document provides information about gene cloning and cloning vectors. It discusses DNA cloning, which involves reproducing DNA fragments using either cell-based or PCR-based approaches. A vector is required to insert the DNA fragment into a host cell. Common vectors discussed include plasmids, bacteriophages, cosmids, and artificial chromosomes. The document outlines the basic plasmid cloning strategy and describes key steps like restriction enzyme digestion of the DNA and vector, ligation, transformation, and selection of recombinant clones. Important enzymes and their functions are also summarized, such as ligase, kinases, phosphatases, and reverse transcriptase.
This document provides an overview of recombinant DNA technology. It discusses the basic principles, which involve generating DNA fragments, inserting a selected fragment into a cloning vector, introducing the vector into host cells, and multiplying the recombinant molecules. Key steps include using restriction enzymes to cut DNA at specific sites, ligases to join fragments, and various vectors like plasmids and bacteria to clone the DNA. The document also outlines several applications of rDNA technology, such as producing proteins, diagnosing diseases, and developing genetically engineered plants.
This document provides information about synthesizing and cloning cDNA from mRNA. It describes how cDNA libraries are constructed by isolating mRNA, synthesizing cDNA via reverse transcription, treating cDNA ends, ligating the cDNA to vectors, and transforming the vectors into host cells. The key steps include mRNA purification, first and second strand cDNA synthesis, linker ligation, vector ligation, and transformation. cDNA libraries are useful for eukaryotic gene analysis as they contain only expressed genes without introns.
Recombinant DNA technology involves isolating DNA from different species, cutting it with restriction enzymes, and splicing the pieces together to form recombinant molecules. These molecules are multiplied in bacteria or yeast cells. Key steps include extracting DNA, cutting it with restriction enzymes, inserting a gene of interest into a plasmid, transforming bacteria with the plasmid, and using antibiotics to select bacteria containing the recombinant DNA. This allows mass production of human genes for applications like gene therapy and production of therapeutic proteins.
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.
This document discusses different types of cloning vectors. It describes that vectors are used to carry foreign DNA into host cells. There are two main types of transformation vectors - cloning vectors which are used to increase copies of cloned DNA fragments, and expression vectors which are used to express foreign genes as proteins. Some examples of commonly used cloning vectors include pBR322 and pUC18/19, while examples of expression vectors include pET28 and pRSET vectors. The document then discusses various properties desirable in vectors such as an origin of replication, antibiotic resistance genes, and regulatory elements. It describes different types of vectors including plasmid vectors, bacteriophage vectors, cosmids, BACs/YACs, and mini chromosomes.
The procedure involves placing a foreign gene into bacterial cells using restriction enzymes and vectors. The modified bacteria are then grown to produce clones. Key aspects of gene cloning include restriction endonucleases to cut DNA, vectors to allow DNA replication, and probes to identify specific clones. DNA ligase is used to join cut DNA fragments.
This document provides an overview of recombinant DNA technology. It begins by describing the central dogma of molecular biology - DNA is transcribed into RNA which is translated into protein. It then discusses various applications of recombinant DNA technology, including gene isolation, sequencing, PCR, gene therapy, and genetically modified crops. The document goes on to describe common techniques used in recombinant DNA, including restriction enzymes, vectors, transformation of host cells, and plasmid cloning. It provides examples of commonly used plasmid and phage vectors.
This document discusses different techniques for cloning DNA, including using restriction enzymes and ligases. It describes 4 main classes of DNA manipulating enzymes: nucleases that cut DNA, ligases that join DNA fragments, polymerases that copy DNA, and modifying enzymes that add or remove chemical groups from DNA. It provides examples of common restriction enzymes like EcoRI, BamHI, and HindIII that cut DNA at specific recognition sequences, generating stick or blunt ends. Ligases are then used to join the cut DNA fragments back together to form recombinant DNA molecules.
Cloning and Expression Vectors document discusses:
1) Cloning a gene of interest involves inserting it into a vector that can be replicated in host cells, producing recombinant DNA molecules.
2) Vectors contain features like replication origins, antibiotic resistance genes, and unique restriction sites to facilitate cloning and isolation.
3) Early cloning experiments demonstrated that recombinant plasmids containing both prokaryotic and eukaryotic DNA could replicate stably in bacteria, allowing genetic engineering.
Restriction enzymes are enzymes that cut DNA at specific recognition sequences. There are three main types of restriction enzymes - Type I, II, and III. Type II restriction enzymes are the most commonly used in molecular cloning and DNA manipulation. They recognize short palindromic DNA sequences of 4-8 base pairs and cleave DNA within or nearby these recognition sites. Restriction enzymes have many applications including DNA sequencing, DNA libraries, and recombinant DNA techniques which enable genetic engineering.
Biotechnology uses cells or their components to produce products. Recombinant DNA technology involves genetic engineering by inserting genes into cells to turn them into "factories" producing products. Key steps include using restriction enzymes to cut DNA for insertion into vectors like plasmids, transforming cells to take up the vector, and selecting clones containing the gene of interest, often using marker genes. Products can then be produced by growing the engineered cells in bacteria, yeast, or mammalian cells.
The document discusses various vector systems used for molecular cloning, including plasmids like pBR322 and pUC, phages such as λ phage and cosmids, and phagemids. λ phage and cosmids can accept large DNA inserts up to 20-50kb, making them useful for genomic libraries. M13 phages produce single-stranded recombinant DNA useful for sequencing and mutagenesis, while plasmids called phagemids also generate single-stranded DNA with helper phages.
Lectut btn-202-ppt-l3. gene cloning and plasmid vectors (1)Rishabh Jain
The document discusses various types of plasmid vectors and cloning vectors used for gene cloning. It describes the key properties required for a vector, including autonomous replication, small size, selectable markers, and restriction enzyme sites. Some examples of early plasmid vectors discussed are pSC101, ColE1, and pBR322. Later vectors with improved properties include the pUC series, pGEM series, and pET series. A variety of other vector types were also constructed for different applications, such as bacteriophages, cosmids, YACs, BACs, and artificial chromosomes.
This document provides an overview of recombinant DNA technology. It begins by describing the basic components and structure of DNA, including nucleotides, nitrogen bases, and how DNA encodes genetic instructions. It then defines what a gene is and explains that recombinant DNA technology involves joining DNA fragments from different sources. The key steps are described as isolating the gene of interest, inserting it into a vector like a plasmid, introducing the vector into a host cell, and amplifying the recombinant DNA. A variety of applications are mentioned, such as producing pharmaceuticals, genetically modifying crops, and using bacteria to break down environmental waste.
Recombinant DNA technology involves combining DNA from two different organisms and inserting it into a host. This is done by using restriction enzymes to cut the DNA into fragments, which are then inserted into cloning vectors like plasmids, bacteriophages, or artificial chromosomes. The recombinant DNA is then inserted into a host organism using techniques like transformation or transfection. Gel electrophoresis can be used to analyze the results and identify successful recombinant clones. While cloning has potential medical applications, reproductive cloning of humans remains unsafe and controversial.
Recombinant DNA technology involves isolating a gene of interest, inserting it into a vector, transferring the recombinant DNA into a host cell, and identifying cells that contain the recombinant DNA. Key steps include isolating the target DNA through restriction enzyme digestion or PCR, selecting a vector like a plasmid or phage, ligating the DNA insert into the vector, transforming host cells, and using selection methods like antibiotic resistance to identify recombinant cells. This allows large scale production and study of the target gene.
Cloning vectors and gene constructs were submitted to Dr. Shyamalamma.S. The document discusses various topics related to molecular cloning including vectors, vector types, cloning steps, and screening procedures. Vectors are DNA molecules that can carry foreign genetic material into cells. Common vector types include plasmids, bacteriophages, cosmids, and artificial chromosomes. The cloning process involves choosing a vector and host, inserting DNA, transforming cells, and screening for positive clones.
The document discusses genetic engineering techniques. It describes the stages of gene cloning which include generating DNA fragments, inserting them into a vector, introducing the vector into host cells, and selecting clones. It also discusses various molecular tools used in genetic engineering like restriction endonucleases, vectors, host cells, and methods of gene transfer.
This document discusses recombinant DNA technology and its various applications. Recombinant DNA is produced by splicing DNA from different sources for purposes such as cloning DNA, genetically engineering organisms, and producing proteins. Key aspects covered include restriction endonucleases, cloning strategies using vectors like plasmids and bacteriophages, cDNA and genomic libraries, DNA sequencing techniques like Sanger sequencing, PCR, expression of eukaryotic proteins in prokaryotes, microarrays, and gene silencing using siRNA.
These slides give you detailed information about Recombinant DNA Technology in simple words. Do read it, these points will help you while studying this topic.
Genetic engineering involves transferring genes between organisms using recombinant DNA techniques. This allows genes to be isolated, cloned, and moved within and between different species. Cloning a gene involves using restriction enzymes to cut DNA at specific sequences, and DNA ligase to join DNA fragments together. Cloned genes have many research uses such as determining gene sequences, altering phenotypes, and obtaining protein products of genes.
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 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.
Recombinant DNA technology involves isolating DNA from different species, cutting it with restriction enzymes, and splicing the pieces together to form recombinant molecules. These molecules are multiplied in bacteria or yeast cells. Key steps include extracting DNA, cutting it with restriction enzymes, inserting a gene of interest into a plasmid, transforming bacteria with the plasmid, and using antibiotics to select bacteria containing the recombinant DNA. This allows mass production of human genes for applications like gene therapy and production of therapeutic proteins.
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.
This document discusses different types of cloning vectors. It describes that vectors are used to carry foreign DNA into host cells. There are two main types of transformation vectors - cloning vectors which are used to increase copies of cloned DNA fragments, and expression vectors which are used to express foreign genes as proteins. Some examples of commonly used cloning vectors include pBR322 and pUC18/19, while examples of expression vectors include pET28 and pRSET vectors. The document then discusses various properties desirable in vectors such as an origin of replication, antibiotic resistance genes, and regulatory elements. It describes different types of vectors including plasmid vectors, bacteriophage vectors, cosmids, BACs/YACs, and mini chromosomes.
The procedure involves placing a foreign gene into bacterial cells using restriction enzymes and vectors. The modified bacteria are then grown to produce clones. Key aspects of gene cloning include restriction endonucleases to cut DNA, vectors to allow DNA replication, and probes to identify specific clones. DNA ligase is used to join cut DNA fragments.
This document provides an overview of recombinant DNA technology. It begins by describing the central dogma of molecular biology - DNA is transcribed into RNA which is translated into protein. It then discusses various applications of recombinant DNA technology, including gene isolation, sequencing, PCR, gene therapy, and genetically modified crops. The document goes on to describe common techniques used in recombinant DNA, including restriction enzymes, vectors, transformation of host cells, and plasmid cloning. It provides examples of commonly used plasmid and phage vectors.
This document discusses different techniques for cloning DNA, including using restriction enzymes and ligases. It describes 4 main classes of DNA manipulating enzymes: nucleases that cut DNA, ligases that join DNA fragments, polymerases that copy DNA, and modifying enzymes that add or remove chemical groups from DNA. It provides examples of common restriction enzymes like EcoRI, BamHI, and HindIII that cut DNA at specific recognition sequences, generating stick or blunt ends. Ligases are then used to join the cut DNA fragments back together to form recombinant DNA molecules.
Cloning and Expression Vectors document discusses:
1) Cloning a gene of interest involves inserting it into a vector that can be replicated in host cells, producing recombinant DNA molecules.
2) Vectors contain features like replication origins, antibiotic resistance genes, and unique restriction sites to facilitate cloning and isolation.
3) Early cloning experiments demonstrated that recombinant plasmids containing both prokaryotic and eukaryotic DNA could replicate stably in bacteria, allowing genetic engineering.
Restriction enzymes are enzymes that cut DNA at specific recognition sequences. There are three main types of restriction enzymes - Type I, II, and III. Type II restriction enzymes are the most commonly used in molecular cloning and DNA manipulation. They recognize short palindromic DNA sequences of 4-8 base pairs and cleave DNA within or nearby these recognition sites. Restriction enzymes have many applications including DNA sequencing, DNA libraries, and recombinant DNA techniques which enable genetic engineering.
Biotechnology uses cells or their components to produce products. Recombinant DNA technology involves genetic engineering by inserting genes into cells to turn them into "factories" producing products. Key steps include using restriction enzymes to cut DNA for insertion into vectors like plasmids, transforming cells to take up the vector, and selecting clones containing the gene of interest, often using marker genes. Products can then be produced by growing the engineered cells in bacteria, yeast, or mammalian cells.
The document discusses various vector systems used for molecular cloning, including plasmids like pBR322 and pUC, phages such as λ phage and cosmids, and phagemids. λ phage and cosmids can accept large DNA inserts up to 20-50kb, making them useful for genomic libraries. M13 phages produce single-stranded recombinant DNA useful for sequencing and mutagenesis, while plasmids called phagemids also generate single-stranded DNA with helper phages.
Lectut btn-202-ppt-l3. gene cloning and plasmid vectors (1)Rishabh Jain
The document discusses various types of plasmid vectors and cloning vectors used for gene cloning. It describes the key properties required for a vector, including autonomous replication, small size, selectable markers, and restriction enzyme sites. Some examples of early plasmid vectors discussed are pSC101, ColE1, and pBR322. Later vectors with improved properties include the pUC series, pGEM series, and pET series. A variety of other vector types were also constructed for different applications, such as bacteriophages, cosmids, YACs, BACs, and artificial chromosomes.
This document provides an overview of recombinant DNA technology. It begins by describing the basic components and structure of DNA, including nucleotides, nitrogen bases, and how DNA encodes genetic instructions. It then defines what a gene is and explains that recombinant DNA technology involves joining DNA fragments from different sources. The key steps are described as isolating the gene of interest, inserting it into a vector like a plasmid, introducing the vector into a host cell, and amplifying the recombinant DNA. A variety of applications are mentioned, such as producing pharmaceuticals, genetically modifying crops, and using bacteria to break down environmental waste.
Recombinant DNA technology involves combining DNA from two different organisms and inserting it into a host. This is done by using restriction enzymes to cut the DNA into fragments, which are then inserted into cloning vectors like plasmids, bacteriophages, or artificial chromosomes. The recombinant DNA is then inserted into a host organism using techniques like transformation or transfection. Gel electrophoresis can be used to analyze the results and identify successful recombinant clones. While cloning has potential medical applications, reproductive cloning of humans remains unsafe and controversial.
Recombinant DNA technology involves isolating a gene of interest, inserting it into a vector, transferring the recombinant DNA into a host cell, and identifying cells that contain the recombinant DNA. Key steps include isolating the target DNA through restriction enzyme digestion or PCR, selecting a vector like a plasmid or phage, ligating the DNA insert into the vector, transforming host cells, and using selection methods like antibiotic resistance to identify recombinant cells. This allows large scale production and study of the target gene.
Cloning vectors and gene constructs were submitted to Dr. Shyamalamma.S. The document discusses various topics related to molecular cloning including vectors, vector types, cloning steps, and screening procedures. Vectors are DNA molecules that can carry foreign genetic material into cells. Common vector types include plasmids, bacteriophages, cosmids, and artificial chromosomes. The cloning process involves choosing a vector and host, inserting DNA, transforming cells, and screening for positive clones.
The document discusses genetic engineering techniques. It describes the stages of gene cloning which include generating DNA fragments, inserting them into a vector, introducing the vector into host cells, and selecting clones. It also discusses various molecular tools used in genetic engineering like restriction endonucleases, vectors, host cells, and methods of gene transfer.
This document discusses recombinant DNA technology and its various applications. Recombinant DNA is produced by splicing DNA from different sources for purposes such as cloning DNA, genetically engineering organisms, and producing proteins. Key aspects covered include restriction endonucleases, cloning strategies using vectors like plasmids and bacteriophages, cDNA and genomic libraries, DNA sequencing techniques like Sanger sequencing, PCR, expression of eukaryotic proteins in prokaryotes, microarrays, and gene silencing using siRNA.
These slides give you detailed information about Recombinant DNA Technology in simple words. Do read it, these points will help you while studying this topic.
Genetic engineering involves transferring genes between organisms using recombinant DNA techniques. This allows genes to be isolated, cloned, and moved within and between different species. Cloning a gene involves using restriction enzymes to cut DNA at specific sequences, and DNA ligase to join DNA fragments together. Cloned genes have many research uses such as determining gene sequences, altering phenotypes, and obtaining protein products of genes.
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 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.
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 - Plasmids, Bacteriophages and Phagemids.Ambika Prajapati
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.
They allow the exogenous DNA to be inserted, stored, and manipulated mainly at DNA level.
Types -
1.Plasmid vectors.
2.Bacteriophage vectors .
3.Phagemids.
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.
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.
Gene cloning involves inserting DNA fragments into cloning vectors, which are then transferred into host cells. Some key points:
- Plasmid vectors like pBR322 were early cloning vectors but had limitations. Improved vectors like pUC18 addressed these with features like blue-white screening and expanded multiple cloning sites.
- Lambda phage vectors can clone larger DNA fragments of 5-25kb compared to plasmid vectors. The lambda phage genome is engineered to package recombinant DNA in vitro before infecting host cells.
- Different vector types are suited to different applications based on size of insert, host range, and other factors. Gene cloning allows isolation and analysis of genes and their regulation.
Gene cloning allows for the creation of identical copies of genes. It involves amplifying genes using polymerase chain reaction, cutting DNA with restriction enzymes, and joining DNA fragments together with DNA ligase. Cloning vectors like plasmids and bacteriophages are used to move genes into host cells. Transformed cells are selected using antibiotic resistance or reporter genes. Cloned genes have applications in pharmaceutical production, disease diagnosis using probes or PCR, and controlling insect pests by producing bacterial pesticides, transgenic plants, or viral pesticides.
A cloning vector is a small piece of DNA, such as a plasmid, virus, or artificial chromosome, that can accept foreign DNA and be used to clone that DNA and replicate it in a host cell. This document discusses the history and features of common cloning vectors like plasmids, bacteriophages, cosmids, and artificial chromosomes. It explains how vectors are chosen based on factors like insert size and used in molecular cloning by digesting DNA with restriction enzymes, ligating into the vector, transforming into host cells, and selecting for recombinant clones.
Recombinant DNA technology allows for the cloning and manipulation of DNA. DNA is first isolated from an organism and cut with restriction enzymes. The cut DNA fragments are then inserted into cloning vectors like plasmids or phage lambda. These recombinant DNA molecules are introduced into host cells, where they can be replicated in large quantities. Libraries of cloned DNA fragments can be generated that represent entire genomes or individual chromosomes, enabling applications such as genetic mapping, DNA sequencing, and genetic engineering.
This document provides information on various vector types used in recombinant DNA technology for cloning DNA fragments, including their features and applications. It discusses phage vectors such as lambda phages and M13 phages that can accommodate inserts over 10 kb. Other vectors described are cosmids, fosmids, phagemids, and artificial chromosomes like BACs that can stabilize very large DNA inserts of 100-300 kb in bacteria. Each vector type has unique properties like replication mechanism, copy number, and insert size capacity that make them suitable for different cloning applications.
Cloning vectors are small DNA molecules that can accept foreign DNA inserts and replicate within a host cell. They contain features like an origin of replication, antibiotic resistance genes, and restriction enzyme cleavage sites. Common vector types include plasmids, bacteriophages, cosmids, and artificial chromosomes. The choice of vector depends on factors like the size of the DNA insert and the intended use. Vectors allow amplification and manipulation of cloned DNA fragments.
Cosmids are hybrid vectors that combine features of bacteriophages and plasmids. They can clone large DNA fragments of 25-45 kb. Cosmids contain cos sites that allow packaging of the foreign DNA by lambda phage proteins. Phagemids contain both phage and plasmid replication origins, allowing replication as a plasmid and packaging as single-stranded DNA in phage particles. Bacterial artificial chromosomes (BACs) are derived from bacterial plasmids and can clone inserts of 150-350 kb in E. coli. They are more stable than yeast artificial chromosomes (YACs) but can hold smaller inserts. YACs can accommodate megabase-sized inserts in yeast but are prone to rearrange
Recombinant DNA technology allows for the isolation, cloning, and manipulation of genes. Two key advances enabled this field: genetic engineering using restriction enzymes to isolate and modify genes in vitro, and DNA sequencing to determine the order of nucleotides. Recombinant DNA is generated by joining DNA from different sources, and molecular cloning produces large quantities of a particular DNA fragment through construction of a recombinant vector, introduction into a host cell, selective propagation of cells containing the vector, and extraction of the cloned DNA.
A cloning vector is a small piece of DNA, such as a plasmid, virus, or artificial chromosome, that can accept foreign DNA and be replicated within a host cell. The summary describes the main types of cloning vectors, including plasmids, bacteriophages, cosmids, BACs, YACs, and retroviral vectors. It also outlines some key features of cloning vectors like origins of replication, cloning sites, and selectable markers. The choice of vector depends on factors like the size of the DNA insert and cloning efficiency.
This document discusses cloning vectors, which are DNA molecules used to transport cloned DNA sequences between biological hosts. It defines a cloning vector as a small piece of DNA from a virus, plasmid, or cell that can maintain foreign DNA for cloning. The summary describes the key features of cloning vectors, including an origin of replication, cloning site, selectable marker, and optional reporter gene. It also lists common vector types like plasmids, bacteriophages, cosmids, and artificial chromosomes, and factors that determine the choice of vector, such as insert size.
Objectives:
After the end of the presentation we’ll know -
What is cloning vector?
Why cloning vector?
History
Features of a cloning vector
Types of cloning vector
Plasmid
Bacteriophage
Cosmid
Bacterial Artificial Chromosome (BAC)
Yeast Artificial Chromosome (BAC)
Human Artificial Chromosome (HAC)
Retroviral Vectors
What determines choice of vector?
Vector in molecular gene cloning
Cloning vector - The molecular analysis of DNA has been made possible by the cloning of DNA. The two molecules that are required for cloning are the DNA to be cloned and a cloning vector.
A cloning vector is a small piece of DNA taken from a virus, a plasmid or the cell of a higher organism, that can be stably maintained in an organism and into which a foreign DNA fragment can be inserted for cloning purposes.
Most vectors are genetically engineered.
The cloning vector is chosen according to the size and type of DNA to be cloned.
The vector therefore contains features that allow for the convenient insertion or removal of DNA fragment in or out of the vector, for example by treating the vector and the foreign DNA with a restriction enzyme and then ligating the fragments together.
After a DNA fragment has been cloned into a cloning vector, it may be further subcloned into another vector designed for more specific use.
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.
This document discusses different types of cloning vectors. It begins by defining a cloning vector as a vector used to reproduce a DNA fragment. It then describes some key properties of good vectors, including being small in size and having an origin of replication and antibiotic resistance. The main types of vectors discussed are plasmids, bacteriophages, cosmids, and artificial chromosomes. Plasmids are described as the first vectors used, being naturally occurring and able to clone fragments up to 10kb. Lambda phage and M13 phage vectors are discussed as able to clone larger fragments. Cosmids are defined as combining plasmid and phage features to clone fragments up to 50kb.
Travis Hills' Endeavors in Minnesota: Fostering Environmental and Economic Pr...Travis Hills MN
Travis Hills of Minnesota developed a method to convert waste into high-value dry fertilizer, significantly enriching soil quality. By providing farmers with a valuable resource derived from waste, Travis Hills helps enhance farm profitability while promoting environmental stewardship. Travis Hills' sustainable practices lead to cost savings and increased revenue for farmers by improving resource efficiency and reducing waste.
ANAMOLOUS SECONDARY GROWTH IN DICOT ROOTS.pptxRASHMI M G
Abnormal or anomalous secondary growth in plants. It defines secondary growth as an increase in plant girth due to vascular cambium or cork cambium. Anomalous secondary growth does not follow the normal pattern of a single vascular cambium producing xylem internally and phloem externally.
Comparing Evolved Extractive Text Summary Scores of Bidirectional Encoder Rep...University of Maribor
Slides from:
11th International Conference on Electrical, Electronics and Computer Engineering (IcETRAN), Niš, 3-6 June 2024
Track: Artificial Intelligence
https://www.etran.rs/2024/en/home-english/
The debris of the ‘last major merger’ is dynamically youngSérgio Sacani
The Milky Way’s (MW) inner stellar halo contains an [Fe/H]-rich component with highly eccentric orbits, often referred to as the
‘last major merger.’ Hypotheses for the origin of this component include Gaia-Sausage/Enceladus (GSE), where the progenitor
collided with the MW proto-disc 8–11 Gyr ago, and the Virgo Radial Merger (VRM), where the progenitor collided with the
MW disc within the last 3 Gyr. These two scenarios make different predictions about observable structure in local phase space,
because the morphology of debris depends on how long it has had to phase mix. The recently identified phase-space folds in Gaia
DR3 have positive caustic velocities, making them fundamentally different than the phase-mixed chevrons found in simulations
at late times. Roughly 20 per cent of the stars in the prograde local stellar halo are associated with the observed caustics. Based
on a simple phase-mixing model, the observed number of caustics are consistent with a merger that occurred 1–2 Gyr ago.
We also compare the observed phase-space distribution to FIRE-2 Latte simulations of GSE-like mergers, using a quantitative
measurement of phase mixing (2D causticality). The observed local phase-space distribution best matches the simulated data
1–2 Gyr after collision, and certainly not later than 3 Gyr. This is further evidence that the progenitor of the ‘last major merger’
did not collide with the MW proto-disc at early times, as is thought for the GSE, but instead collided with the MW disc within
the last few Gyr, consistent with the body of work surrounding the VRM.
ESPP presentation to EU Waste Water Network, 4th June 2024 “EU policies driving nutrient removal and recycling
and the revised UWWTD (Urban Waste Water Treatment Directive)”
Current Ms word generated power point presentation covers major details about the micronuclei test. It's significance and assays to conduct it. It is used to detect the micronuclei formation inside the cells of nearly every multicellular organism. It's formation takes place during chromosomal sepration at metaphase.
ESR spectroscopy in liquid food and beverages.pptxPRIYANKA PATEL
With increasing population, people need to rely on packaged food stuffs. Packaging of food materials requires the preservation of food. There are various methods for the treatment of food to preserve them and irradiation treatment of food is one of them. It is the most common and the most harmless method for the food preservation as it does not alter the necessary micronutrients of food materials. Although irradiated food doesn’t cause any harm to the human health but still the quality assessment of food is required to provide consumers with necessary information about the food. ESR spectroscopy is the most sophisticated way to investigate the quality of the food and the free radicals induced during the processing of the food. ESR spin trapping technique is useful for the detection of highly unstable radicals in the food. The antioxidant capability of liquid food and beverages in mainly performed by spin trapping technique.
Deep Behavioral Phenotyping in Systems Neuroscience for Functional Atlasing a...Ana Luísa Pinho
Functional Magnetic Resonance Imaging (fMRI) provides means to characterize brain activations in response to behavior. However, cognitive neuroscience has been limited to group-level effects referring to the performance of specific tasks. To obtain the functional profile of elementary cognitive mechanisms, the combination of brain responses to many tasks is required. Yet, to date, both structural atlases and parcellation-based activations do not fully account for cognitive function and still present several limitations. Further, they do not adapt overall to individual characteristics. In this talk, I will give an account of deep-behavioral phenotyping strategies, namely data-driven methods in large task-fMRI datasets, to optimize functional brain-data collection and improve inference of effects-of-interest related to mental processes. Key to this approach is the employment of fast multi-functional paradigms rich on features that can be well parametrized and, consequently, facilitate the creation of psycho-physiological constructs to be modelled with imaging data. Particular emphasis will be given to music stimuli when studying high-order cognitive mechanisms, due to their ecological nature and quality to enable complex behavior compounded by discrete entities. I will also discuss how deep-behavioral phenotyping and individualized models applied to neuroimaging data can better account for the subject-specific organization of domain-general cognitive systems in the human brain. Finally, the accumulation of functional brain signatures brings the possibility to clarify relationships among tasks and create a univocal link between brain systems and mental functions through: (1) the development of ontologies proposing an organization of cognitive processes; and (2) brain-network taxonomies describing functional specialization. To this end, tools to improve commensurability in cognitive science are necessary, such as public repositories, ontology-based platforms and automated meta-analysis tools. I will thus discuss some brain-atlasing resources currently under development, and their applicability in cognitive as well as clinical neuroscience.
Or: Beyond linear.
Abstract: Equivariant neural networks are neural networks that incorporate symmetries. The nonlinear activation functions in these networks result in interesting nonlinear equivariant maps between simple representations, and motivate the key player of this talk: piecewise linear representation theory.
Disclaimer: No one is perfect, so please mind that there might be mistakes and typos.
dtubbenhauer@gmail.com
Corrected slides: dtubbenhauer.com/talks.html
Nucleophilic Addition of carbonyl compounds.pptxSSR02
Nucleophilic addition is the most important reaction of carbonyls. Not just aldehydes and ketones, but also carboxylic acid derivatives in general.
Carbonyls undergo addition reactions with a large range of nucleophiles.
Comparing the relative basicity of the nucleophile and the product is extremely helpful in determining how reversible the addition reaction is. Reactions with Grignards and hydrides are irreversible. Reactions with weak bases like halides and carboxylates generally don’t happen.
Electronic effects (inductive effects, electron donation) have a large impact on reactivity.
Large groups adjacent to the carbonyl will slow the rate of reaction.
Neutral nucleophiles can also add to carbonyls, although their additions are generally slower and more reversible. Acid catalysis is sometimes employed to increase the rate of addition.
1. Genetic Engineering
Chapter 3- Cloning Vectors
Hikmet Geçkil, Professor
Department of Molecular Biology and Genetics
Inonu University
2. Cloning vectors
• Vectors:
– are autonomously replicating DNA molecules that can be used to carry
foreign DNA fragments
– must possess the ability to self-replicate
– must have a selectable characteristic so that transformed cells may be
recognized fromuntransformed cells
– must contain restriction enzyme recognition sites so that DNA
fragments can be cloned into
• Most cloning experiments utilize the bacterium Escherichia coli as
the host for the propagation of cloned DNA fragments, given its:
– ease and cost to grow
– rapid growth (e.g., doubling time 20–30 min)
– well understood genetics
– being safe and having desired mutants
Genetic Engineering/Hikmet
Geckil
Chapter 3: Cloning Vectors 2
3. • Widely distributed
throughout prokaryotes
• Mostly in sizes 1 to 20
kbp.
• Relaxed plasmids at
multiple copies (10–
200), stringent plasmids
at low copies (1–2) per
cell.
• Thus, stringent plasmids
are biger and have more
genes on them.
Genetic Engineering/Hikmet
Geckil
Chapter 3: Cloning Vectors 3
Cloning vectors: plasmids
4. • The genes carried in plasmids provide bacteria with
genetic advantages, such as antibiotic resistance.
• Most plasmids in common use are based upon the
replication origin of the naturally occurring E. coli
plasmid ColE1, or its very close relative pMB1
• ColE1 is a 6646 bp closed-circular DNA molecule that
encodes a bacteriocin, colicin E1, and a bacteriocin
resistance gene
• Colicin E1 is a transmembrane protein that causes
lethal membrane depolarization in bacteria
Genetic Engineering/Hikmet
Geckil
Chapter 3: Cloning Vectors 4
5. Types of Plasmids
• Cloning Plasmids - Cloning vectors are simple, often
contain only a bacterial resistance gene, origin and
MCS.
• Expression Plasmids - Used for gene expression. They
contain promoter terminator sequences and the
inserted gene. An expression vector can also include an
enhancer sequence which increases the amount of
protein or RNA produced.
• Gene knock-down Plasmids - Used for reducing the
expression of an endogenous gene. This is frequently
accomplished through expression of an shRNA
targeting the mRNA of the gene of interest.
Genetic Engineering/Hikmet
Geckil
Chapter 3: Cloning Vectors 5
6. • Reporter Plasmids - Used for studying the function of
genetic elements. They contain a reporter gene (e.g.,,
luciferase or GFP) that offers a read-out of the activity
of the genetic element.
• Viral Plasmids - used in delivery of genetic material
into target cells just like a virus . One can use these
plasmids to create viral particles, such as lentiviral,
retroviral, and adenoviral particles.
Genetic Engineering/Hikmet
Geckil
Chapter 3: Cloning Vectors 6
7. pBR322, one of the first constructed plasmids
• pBR322 is one of the first widely used
E. coli cloning vectors, created in 1977
in the laboratory of Herbert Boyer at
the University of California, San
Francisco.
• It was named after the postdoctoral
researchers who constructed it,
"Bolivar" and "Rodriguez".
• It has all the essential requirements
for a cloning vector (i.e., relatively
small size, useful restriction enzyme
sites, an origin of replication, and
antibiotic resistance genes).
Genetic Engineering/Hikmet Geckil Chapter 3: Cloning Vectors 7
Map and important features of pBR322
8. pUC series of plasmids
• pUC (origin of name:
University of California)
cloning vectors are high copy
number and contain a
multiple cloning site at the
lacZ' region.
• Created by Joachim
Messing and co-workers in
1982.
• Their recombinants can
be verified via blue/white
colony screening using agar
plates containing IPTG and X-
Gal.
• Expression of target DNA is
enabled by the presence of
a lac promoter in the cloning
vectors.
Genetic Engineering/Hikmet
Geckil
Chapter 3: Cloning Vectors 8
Map and important features of pUC18
9. • The plasmid pBR322 has an origin of replication, or ori, a
sequence where replication is initiated by cellular enzymes.
• The plasmid contains genes that confer resistance to the
antibiotics tetracycline (TetR) and ampicillin (AmpR),
allowing the selection of cells that contain the plasmid
• Several unique recognition sequences in pBR322 are
targets for restriction endonucleases (PstI, EcoRI, BamHI,
SalI, and PvuII), providing sites where the plasmid can be
cut to insert foreign DNA.
• The small size (4,361 bp), generated simply by trimming
away many DNA segments, facilitates its entry into cells and
the biochemical manipulation of the DNA.
Genetic Engineering/Hikmet
Geckil
Chapter 3: Cloning Vectors 9
10. Genetic Engineering/Hikmet Geckil Chapter 3: Cloning Vectors 10
Use of pBR322 to clone foreign DNA. The
entire procedure is illustrated, including
both positive and negative selection.
11. Replication origin
• The replication origin
(ORI) is a specific DNA
sequence of 50 – 100
base pairs that must be
present in a plasmid for
it to replicate.
• Host-cell enzymes bind
to ORI, initiating
replication of the
circular plasmid.
Genetic Engineering/Hikmet
Geckil
Chapter 3: Cloning Vectors 11
The parental strands are shown in blue, and
newly synthesized daughter strands are shown
in red. Once DNA replication is initiated at the
origin (ORI), it continues in both directions
around the circular molecule until the
advancing growing forks merge and two
daughter molecules are produced.
14. Vectors based on bacteriophage λ
• The limitation of plasmid vector is the size of DNA that can
be introduced into the cell by transformation.
• This presents problems when you are trying to create a
genomic library of a large genome such as with plants.
• A genomic library contains all of the DNA found in the cell
of the plant (or any organism).
• Thus, you need to use a vector that can accept large
fragments of DNA.
• Examples of these are bacteriophage and cosmid vectors
and more recently yeast artificial chromosomes.
Genetic Engineering/Hikmet
Geckil
Chapter 3: Cloning Vectors 14
16. The life cycle of lambda
• Adsorption - the phage particle binds at a
maltose receptor site of the bacterial cell;
growing the cell in the presence of the sugar
increase the number of receptor sites
• Penetration - DNA is injected into the cell; at this
point it can enter one of two pathways;
– Lysogenic pathway - the phage DNA becomes
integrated into the genome and is replicated along
with the bacterial DNA; it remains integrated until it
enters the lytic pathway
– Lytic pathway - large scale production of
bacteriophage particles that eventually leads to the
lysis of the cell; base pairing at the cos site leads to a
circular molecule
Genetic Engineering/Hikmet
Geckil
Chapter 3: Cloning Vectors 16
17. • Packaging of the DNA into the lambda phage
head does not require a complete length of
wild type lambda.
• It has been determined that a lambda
molecule that is between 78% and 105% of
wild type length can be packaged. This is from
37 to 53 kb in length.
Genetic Engineering/Hikmet
Geckil
Chapter 3: Cloning Vectors 17
18. Vectors based on bacteriophage M13
Genetic Engineering/Hikmet
Geckil
Chapter 3: Cloning Vectors 18
Genetic map and important features of bacteriophage vector M13
19. Cosmids
• Cosmids are plasmid vectors that contain cos
sites.
• The cos site is required for DNA to be
packaged into a phage particle.
• Since phage particles can accept between 38
and 53 kb of DNA and since most cosmids are
about 5 kb, between 33 and 48 kb of DNA can
cloned in these vectors.
Genetic Engineering/Hikmet
Geckil
Chapter 3: Cloning Vectors 19
20. Hybrid plasmid/phage vectors
• Phagemids, similarly, are not as common as plasmids.
• The most known is pBluescript used for cloning, sequencing, site-
directed mutagenesis, or in vitro transcription purposes.
• Phagemids are essentially plasmids that contain an origin of
replication for single-stranded phages (such as M13 or f1)
• Thus bacteria which are transformed with this plasmid and infected
with a helper phage (such as M13 or f1) can produce single-
stranded copies of plasmids, which in turn can be packaged into
phage heads.
• Since this vector is a hybrid of both plasmids and single-stranded
phage vectors, it can be used to generate single-stranded DNA to be
used in sequencing reactions.
Genetic Engineering/Hikmet
Geckil
Chapter 3: Cloning Vectors 20
22. Vectors for use in eukaryotic cells
• The genomes of eukaryotes are larger and more
complex than those of bacteria, so modifications of the
techniques are needed to handle the larger amounts of
DNA and the array of different cells and life cycles of
eukaryotes.
• For instance, some eukaryotic proteins cannot be easily
expressed in large amounts in bacteria, and eukaryotic
expression systems need to be employed.
• A widely used vector–expression system for eukaryotic
proteins is insect baculovirus, into which genes are
inserted and expressed at high rates in cultured insect
cells
Genetic Engineering/Hikmet
Geckil
Chapter 3: Cloning Vectors 22
23. Genetic Engineering/Hikmet
Geckil
Chapter 3: Cloning Vectors 23
Baculovirus is a very large
DNA virus (about 150 kb)
that infects insect cells.
To express a foreign gene
in baculovirus, the gene
of interest is cloned in
place of the viral coat-
protein gene in a plasmid
carrying a small part of
the viral genome.
The recombinant plasmid
is cotransfected into
insect cells with wild-type
baculovirus DNA for
high expression of the
foreign protein.
25. Bacterial Artificial Chromosomes
• Large genome sequencing projects often require the
cloning of much longer DNA segments than cannot be
incorporated into standard plasmid cloning vectors.
• To meet this need, plasmid vectors have been
developed with special features that allow the cloning
of very long segments (typically 100,000 to 300,000 bp)
of DNA.
• Once such large segments of cloned DNA have been
added, these are large enough to be thought of as
chromosomes, and are known as bacterial artificial
chromosomes, or BACs.
Genetic Engineering/Hikmet
Geckil
Chapter 3: Cloning Vectors 25
26. • BACs are based on F plasmids which help even
distribution (aka. plasmid partitioning) of these
large-sized recombinant plasmids to the next
generation cells.
• A typical BAC vector contains ori and rep
sequences to ensure replication of the vector and
the copy number, and par sequences for even
partitioning of the DNA to daughter cells.
• Additionally selectable (e.g., amp) and screenable
(e.g., lacZ) markers and phage promoters such as
the T7 promoter are required.
Genetic Engineering/Hikmet
Geckil
Chapter 3: Cloning Vectors 26
27. Genetic Engineering/Hikmet
Geckil
Chapter 3: Cloning Vectors 27
Bacterial artificial chromosomes (BACs) as cloning vectors. After treatment with an
appropriate restriction endonuclease, a BAC and a long fragment of DNA are ligated. The
recombinant BAC is transferred into E. coli by electroporation, and colonies with
recombinant BACs are selected by growth on media containing both the antibiotic
chloramphenicol and X-gal, the substrate for –galactosidase that produces a colored
product.
28. Yeast Artificial Chromosomes
• As with E. coli, yeast genetics is a well-developed discipline.
• The genome of Saccharomyces cerevisiae contains only 14
million bp (about four times the size of the E. coli
chromosome), and its entire sequence is known.
• Plasmid vectors have been constructed for yeast,
employing the same principles that govern the use of E. coli
vectors.
• Some recombinant plasmids incorporate multiple
replication origins and other elements that allow them to
be used in more than one species (e.g., in yeast and E. coli).
Plasmids that can be propagated in cells of two or more
species are called shuttle vectors.
Genetic Engineering/Hikmet
Geckil
Chapter 3: Cloning Vectors 28
29. • Yeast artificial chromosomes, or YACs contain all the
elements needed to maintain a eukaryotic chromosome in
the yeast nucleus:
– a yeast origin of replication
– two selectable markers
– specialized sequences (derived from the telomeres and
centromere) needed for stability and proper segregation of the
chromosomes at cell division.
• In preparation for its use in cloning, the vector is
propagated as a circular bacterial plasmid. Cleavage with a
restriction endonuclease removes a length of DNA between
two telomere sequences (TEL), leaving the telomeres at the
ends of the linearized DNA. Cleavage at another internal
site divides the vector into two DNA segments, referred to
as vector arms, each with a different selectable marker.
Genetic Engineering/Hikmet
Geckil
Chapter 3: Cloning Vectors 29
30. • The genomic DNA to be cloned is prepared by partial digestion to obtain a
suitable fragment size.
• Genomic fragments are then separated by pulsed field gel
electrophoresis, a variation of gel electrophoresis that segregates very
large DNA segments.
• DNA fragments of appropriate size (up to about 2 million bp) are mixed
with the prepared vector arms and ligated.
• The ligation mixture is then used to transform yeast cells (pretreated to
partially degrade their cell walls) with these very large DNA molecules—
which now have the structure and size to be considered yeast
chromosomes.
• Culture on a medium that requires the presence of both selectable marker
genes ensures the growth of only those yeast cells that contain an artificial
chromosome with a large insert sandwiched between the two vector
arms.
Genetic Engineering/Hikmet
Geckil
Chapter 3: Cloning Vectors 30
31. Genetic Engineering/Hikmet
Geckil
Chapter 3: Cloning Vectors 31
Construction of a yeast artificial chromosome (YAC). A YAC vector includes an origin of replication
(ori), a centromere (CEN), two telomeres (TEL), and selectable markers (here designated X and Y). Two
separate DNA arms are generated by digestion with BamHI and EcoRI, each arm having a telomeric
end and one selectable marker. A large DNA fragment, produced by EcoRI digestion, is ligated to the
two arms, creating a YAC. The YAC is transferred into yeast cells (which have been prepared by
removing the cell wall to form spheroplasts). The transformed cells are selected for X and Y, and the
surviving cells propagate the DNA insert.
32. • One arm contains an autonomous replication sequence (ARS),
a centromere (CEN) and aselectable marker (trp1). The other
arm contains a second selectable marker (ura3).
• Insertion of DNA into the cloning site inactivates a mutant
expressed in the vector DNA and red yeast colonies appear.
• Transformants are identified as those red colonies which grow
in a yeast cell that is mutant for trp1 andura3. This ensures
that the cell has received an artificial chromosome with both
telomeres (because of complementation of the two mutants)
and the artificial chromosome contains insert DNA (because
the cell is red).
Genetic Engineering/Hikmet
Geckil
Chapter 3: Cloning Vectors 32
33. Getting DNA into cells
In bacteria, the passage of genetic material happens
in three ways:
1. Conjugation - transfer using sex pili
2. Transduction - transfer by bacteriophages
3. Transformation - uptake of naked DNA from outside
the cell (In mammalian cell culture, the analogous
process of introducing DNA into cells is commonly
termed transfection)
Some of the other artificial methods include protoplast
fusion, microinjection , and the use of "gene guns" to
blast DNA-coated particles through cell walls and into
plant cells.
Genetic Engineering/Hikmet
Geckil
Chapter 3: Cloning Vectors 33
34. Selection, screening, and analysis of recombinants
Genetic Engineering/Hikmet
Geckil
Chapter 3: Cloning Vectors 34
35. • The lac Z fragment,
induced by IPTG, is
capable of intra-allelic
complementation with a
defective form of β-
galactosidase enzyme
encoded by host
chromosome.
• The complementation
results an active enzyme
which hydrolyses X-gal
(5-bromo-4-chloro-3-
indolyl- beta-D-
galactopyranoside) and
form blue colonies.
Genetic Engineering/Hikmet
Geckil
Chapter 3: Cloning Vectors 35
The molecular mechanism involved for screening
recombinant cells
36. • Insertion of foreign DNA into the MCS located within the lac Z gene
causes insertional inactivation of this gene and abolishes intra-allelic
complementation.
• Thus bacteria carrying recombinant plasmids in the MCS cannot hydrolyse X-gal,
giving rise to white colonies contrary to non-recombinant cells which are blue.
Genetic Engineering/Hikmet
Geckil
Chapter 3: Cloning Vectors 36
Principle of
blue/white
selection for
the detection of
recombinant
vectors
38. Genetic Engineering/Hikmet
Geckil
Chapter 3: Cloning Vectors 38
Alpha complementation
• Plasmid encodes N-terminus of
beta galactosidase (alpha
fragment)
• Host strain encodes the C-
terminus of beta galactosidase
(omega fragment)
• Beta galactosidase function is
only seen in the presence of both
the N- and C-terminal fragments
• Beta gal function can be
monitored by the cleavage of X-gal
which yields a bright blue product
(blue colonies on a plate)
Bright blue
X-gal
39. Genetic Engineering/Hikmet
Geckil
Chapter 3: Cloning Vectors 39
• Plasmid encodes N-terminus of beta galactosidase (alpha
fragment), with an MCS
• Foreign DNA in the MCS, no alpha fragment
• No alpha fragment, no B-gal
• No B-gal, no blue color (white colonies)
pUC19
transformation
plate
Colony without foreign DNA in MCS
Colony with foreign DNA in MCS
40. Genetic Engineering/Hikmet
Geckil
Chapter 3: Cloning Vectors 40
Gel electrophoresis is a laboratory
technique used to separate nucleic
acids of different sizes.
In gel electrophoresis, a porous gel is
often made from agarose (a
polysaccharide isolated from seaweed),
which is melted in a buffer solution and
poured into a plastic mold. As it cools,
the agarose solidifies, making a gel that
looks something like stiff gelatin.