This document provides an introduction to the subject of biotechnology for a 6th semester B Pharmacy course. It discusses key topics including the objectives and learning outcomes of the course, an overview of modules to be covered such as enzyme immobilization, biosensors, protein engineering and genetic engineering. Specific techniques in these areas like methods of enzyme immobilization and applications of biosensors are explained. The benefits, applications and future potential of biotechnology in fields like medicine, agriculture, food and industry are also summarized.
This seminar discusses protein engineering, which modifies protein structure using recombinant DNA or chemical treatment. The objectives are to create superior enzymes for industrial chemical production and drugs. Proteins must be robust, stable under industrial conditions, and efficiently use non-natural substrates. Protein engineering aims to alter properties like kinetics, thermostability, pH and substrate optimization. It involves studying protein structures, using mutagenesis, selection and recombinant DNA to achieve desired functions.
This document discusses the work of Frances H. Arnold on directed evolution of enzymes. Some key points:
- Frances H. Arnold won the Nobel Prize in Chemistry in 2018 for her work developing directed evolution to design enzymes for specific functions.
- Directed evolution is an iterative process that involves introducing mutations to a starting protein, screening variants to select for desired properties, and repeating rounds of mutation and selection.
- Common methods for introducing mutations include error-prone PCR, DNA shuffling, and saturation mutagenesis.
- Directed evolution has been used to develop enzymes for applications like biocatalysis, optogenetics, energy harvesting, and more.
Protein engineering involves modifying protein structure using recombinant DNA technology or chemical treatment to improve function for use in medicine, industry, and agriculture. The objectives of protein engineering are to create superior enzymes for specific chemical production, produce enzymes in large quantities, and produce superior biological compounds. Protein engineering aims to alter properties like kinetic properties, thermostability, stability in nonaqueous solvents, substrate specificity, and cofactor requirements to meet industrial needs. Common methods for protein engineering include mutagenesis, selection, and recombinant DNA technology.
Protein engineering involves designing new proteins or enzymes with desirable functions. It requires an understanding of amino acid chemistry, protein structure at various levels, and stabilizing forces. Key prerequisites include knowledge of amino acids, primary/secondary/tertiary protein structure, protein synthesis and modification, and relevant techniques. Case studies on enzymology, glycosylation and techniques like SDS-PAGE are useful. Applications include developing oxidation-resistant proteases for detergents, engineering tissue plasminogen activator for medical use, and modifying insulin. Protein engineering also aims to design new enzymes with improved properties for industries like food and medicine.
Protein engineering is the modification of proteins using recombinant DNA technology or chemical treatment to achieve a desired function. It involves disciplines like molecular biology, protein chemistry, and structural biology. The objectives are to create superior enzymes for industrial use, produce biological compounds in large quantities, and develop more potent pharmaceuticals. Key methods are genetic modification techniques like mutagenesis and gene cloning to alter stability and activity, as well as chemical modifications like PEGylation to increase enzyme half-life. Significant progress has been made in engineering proteins like insulin, interferon, and antibodies for improved properties.
Protein engineering and its techniques himanshuhimanshu kamboj
b pharma 6th sem
pharmaceutical biotechnology
Protein engineering
Objectives of protein engineering
Rationale of protein engineering
Protein engineering methods
Rational design -site-directed mutagenesis methods
Advantages and disadvantages of rational design
Directed evolution -random mutagenesis
Advantages and disadvantages of directed evolution
Peptidomimetics
Classification of peptidomimetics
Advantages and disadvantages of peptidomimetics
Flow cytometry
Instrumentation
Principle
components
This document discusses various applications of protein engineering in different industries and fields. It describes how protein engineering is used in the food industry to modify enzymes like proteases, amylases, and lipases to improve their properties. It also discusses applications in environmental remediation, medicine like cancer treatment, biopolymer production, nanobiotechnology, and redox proteins. The document provides an overview of the wide range of uses of protein engineering across diverse domains.
This document discusses protein engineering and methods to improve protein stability. It begins by defining protein engineering as modifying protein structure using recombinant DNA technology or chemical treatment to achieve desirable functions. The objectives of protein engineering are outlined, such as creating superior enzymes for industrial use. Methods of protein engineering discussed include mutagenesis and gene modification techniques. Strategies to increase protein stability through additions like disulfide bonds or changes to amino acids are also presented. The document provides examples of protein engineering applications in medicine, industry and agriculture.
This seminar discusses protein engineering, which modifies protein structure using recombinant DNA or chemical treatment. The objectives are to create superior enzymes for industrial chemical production and drugs. Proteins must be robust, stable under industrial conditions, and efficiently use non-natural substrates. Protein engineering aims to alter properties like kinetics, thermostability, pH and substrate optimization. It involves studying protein structures, using mutagenesis, selection and recombinant DNA to achieve desired functions.
This document discusses the work of Frances H. Arnold on directed evolution of enzymes. Some key points:
- Frances H. Arnold won the Nobel Prize in Chemistry in 2018 for her work developing directed evolution to design enzymes for specific functions.
- Directed evolution is an iterative process that involves introducing mutations to a starting protein, screening variants to select for desired properties, and repeating rounds of mutation and selection.
- Common methods for introducing mutations include error-prone PCR, DNA shuffling, and saturation mutagenesis.
- Directed evolution has been used to develop enzymes for applications like biocatalysis, optogenetics, energy harvesting, and more.
Protein engineering involves modifying protein structure using recombinant DNA technology or chemical treatment to improve function for use in medicine, industry, and agriculture. The objectives of protein engineering are to create superior enzymes for specific chemical production, produce enzymes in large quantities, and produce superior biological compounds. Protein engineering aims to alter properties like kinetic properties, thermostability, stability in nonaqueous solvents, substrate specificity, and cofactor requirements to meet industrial needs. Common methods for protein engineering include mutagenesis, selection, and recombinant DNA technology.
Protein engineering involves designing new proteins or enzymes with desirable functions. It requires an understanding of amino acid chemistry, protein structure at various levels, and stabilizing forces. Key prerequisites include knowledge of amino acids, primary/secondary/tertiary protein structure, protein synthesis and modification, and relevant techniques. Case studies on enzymology, glycosylation and techniques like SDS-PAGE are useful. Applications include developing oxidation-resistant proteases for detergents, engineering tissue plasminogen activator for medical use, and modifying insulin. Protein engineering also aims to design new enzymes with improved properties for industries like food and medicine.
Protein engineering is the modification of proteins using recombinant DNA technology or chemical treatment to achieve a desired function. It involves disciplines like molecular biology, protein chemistry, and structural biology. The objectives are to create superior enzymes for industrial use, produce biological compounds in large quantities, and develop more potent pharmaceuticals. Key methods are genetic modification techniques like mutagenesis and gene cloning to alter stability and activity, as well as chemical modifications like PEGylation to increase enzyme half-life. Significant progress has been made in engineering proteins like insulin, interferon, and antibodies for improved properties.
Protein engineering and its techniques himanshuhimanshu kamboj
b pharma 6th sem
pharmaceutical biotechnology
Protein engineering
Objectives of protein engineering
Rationale of protein engineering
Protein engineering methods
Rational design -site-directed mutagenesis methods
Advantages and disadvantages of rational design
Directed evolution -random mutagenesis
Advantages and disadvantages of directed evolution
Peptidomimetics
Classification of peptidomimetics
Advantages and disadvantages of peptidomimetics
Flow cytometry
Instrumentation
Principle
components
This document discusses various applications of protein engineering in different industries and fields. It describes how protein engineering is used in the food industry to modify enzymes like proteases, amylases, and lipases to improve their properties. It also discusses applications in environmental remediation, medicine like cancer treatment, biopolymer production, nanobiotechnology, and redox proteins. The document provides an overview of the wide range of uses of protein engineering across diverse domains.
This document discusses protein engineering and methods to improve protein stability. It begins by defining protein engineering as modifying protein structure using recombinant DNA technology or chemical treatment to achieve desirable functions. The objectives of protein engineering are outlined, such as creating superior enzymes for industrial use. Methods of protein engineering discussed include mutagenesis and gene modification techniques. Strategies to increase protein stability through additions like disulfide bonds or changes to amino acids are also presented. The document provides examples of protein engineering applications in medicine, industry and agriculture.
This document discusses protein engineering, which uses recombinant DNA technology to modify protein structure and function. It describes several methods of protein engineering, including site-directed mutagenesis, error-prone PCR, and DNA shuffling. The objectives of protein engineering are to improve protein stability, modify cofactor requirements, increase enzyme activity, and modify enzyme specificity. As an example, the document discusses how site-directed mutagenesis was used to increase the stability of streptokinase by replacing lysine residues susceptible to cleavage by plasmin.
Meta-genomics is the application of modern genomics techniques to the study of communities of microbial organisms directly in their natural environments, bypassing the need for isolation and lab cultivation of individual species”
This document discusses biotransformation and microbial conversion of organic compounds. It defines biotransformation as the microbial conversion of a substrate into a product using enzymatic reactions. Various examples of biotransformation reactions are provided, including the production of gluconic acid, antibiotics, lactic acid, and acetic acid (vinegar). The document also discusses methods for biotransformation like growing, resting, and immobilized cells as well as cell-free extracts and enzymes. It provides details on the production processes of certain compounds like gluconic acid, vinegar, and steroids.
Lipase catalyzed process for biodiesel production protein engineering and lip...Melike Şeyma Kadayıfçı
This review document discusses the use of lipases in biodiesel production. It covers lipase engineering approaches like directed evolution and rational design to improve properties such as thermostability and activity. It also discusses lipase production methods like host strain selection and metabolic engineering. The goal is to develop high-performance lipases through these techniques to enable large-scale commercialization of lipase-catalyzed biodiesel production as an alternative to fossil fuels.
The document discusses metabolic pathway engineering and metabolic engineering. It provides an overview of four commercially important fermentation products, including the microorganism used, annual production levels, and applications. It then discusses the core concepts of metabolic engineering, including manipulating enzymatic and regulatory functions using recombinant DNA to improve cellular activities. Examples of applications include strain improvement for biocatalysis and bioprocessing, increasing productivity, and developing novel biosynthetic routes.
Strain development techniques of industrially important microorganismsMicrobiology
Strain improvement and development involves manipulating microbial strains to enhance their metabolic capacities for biotechnology applications. Targets of improvement include rapid growth, genetic stability, non-toxicity, large cell size, ability to use cheaper substrates, increased productivity, and reduced cultivation costs. Methods for optimization include modifying environmental conditions, nutrition, mutagenesis, transduction, conjugation, transformation, and genetic engineering. Common industrial microorganisms are bacteria such as Bacillus subtilis and yeasts such as Saccharomyces cerevisiae.
This document discusses techniques for strain improvement in microbiology. It describes the ideal characteristics of microbial strains, the purpose of strain improvement, and three main approaches: mutant selection through chemical or radiation mutagenesis, recombination through techniques like transformation and conjugation, and recombinant DNA technology. Novel technologies discussed include metabolic engineering and genome shuffling. Applications include production of medicines and industrial enzymes.
Mutagenesis; A conventional tool for strain improvement in industry Zohaib HUSSAIN
The strain improvement is the process of improvement and manipulation of microbial strains for the icreasment of metabolic level for industrial applications. The yield of microbial enzymes can be increased by using microbe specific medium for fermentation, improving the fermentation process and strain improvement for higher yield of product.
All these things lead to decrease in cost production. Microbe produce product according to its need therefore there is great need for overproduction. There is tremendous contribution of conventional Mutagenesis for strain improvement. Mutagenesis is important tool for the production of mutants which are capable to produce large product i.e. hyperactive.
The document discusses purification of recombinant proteins using affinity tags. It describes immobilized metal affinity chromatography (IMAC) as a widely used method to purify recombinant proteins fused to tags like histidine, GST or MBP. The document outlines the steps involved, including gene amplification, cloning, expression in bacteria or yeast, and purification. It focuses on using histidine tags and nickel-chelate affinity chromatography, noting the advantages of tags for simplifying purification and detection of recombinant proteins.
Chemical protein engineering synthetic and semisyntheticAli Hatami
This document summarizes various methods for chemically synthesizing and modifying peptides and proteins. It discusses solid phase peptide synthesis, native chemical ligation using peptide thioesters, and fragment condensation strategies. It also covers chemoselective ligations using oxime and hydrazone bonds and decarboxylative amide formation. Additionally, the document outlines chemical modifications like PEGylation, phosphorylation, and backbone modifications. Finally, it examines enzyme-mediated ligation techniques like sortase and biotin ligase that can link proteins and peptides in a sequence-specific manner.
Science and technology of manipulating and improving microbial strains, in order to enhance their metabolic capacities for biotechnological applications, are referred to as strain improvement.
This document discusses metabolic engineering. Metabolic engineering modifies cellular properties through recombinant DNA technology to alter metabolic pathways for production of chemicals, fuels, pharmaceuticals and medicine. It requires overexpression or downregulation of proteins in pathways so cells produce new products. The first step is understanding the host cell environment for genetic modifications, and the effect of modifications on growth should be examined. Genetic manipulation may negatively impact metabolic burden. Metabolic engineering is used to produce various compounds through methods like eliminating competing pathways, expressing foreign enzymes, and optimizing cofactors like NAD+/NADH.
This document discusses enzyme biotechnology and methods of enzyme immobilization. It begins by defining enzymes and their function in cells. It then describes the different methods of immobilizing enzymes, including adsorption, covalent bonding, entrapment, cross-linking/copolymerization, and encapsulation. The advantages and disadvantages of each method are provided. Overall, the document provides an overview of enzyme biotechnology with a focus on immobilization techniques.
This study investigated the indigoidine synthetase enzyme which contains an oxidase (Ox) domain involved in nonribosomal peptide synthesis. Through structural analysis of the Ox domain, a conserved tyrosine residue was identified that acts as an active site base. Multiple sequence alignment and biochemical assays on other Ox domains confirmed the general importance of this tyrosine residue. The findings provide mechanistic insight into how Ox domains catalyze oxidation reactions via an E2-like mechanism, utilizing the conserved tyrosine base. This work advances understanding of nonribosomal peptide synthesis which produces medically relevant natural products.
This document discusses protein engineering through directed evolution. It explains that directed evolution involves randomly recombining genes from protein libraries and screening mutant proteins for improved functions. This leaves beneficial changes up to chance but generates diversity. Rational design is also used to map important protein interactions to guide evolution. The document provides an example of using directed evolution to modify a cytochrome P450 enzyme to more efficiently convert alkanes to alcohols over multiple generations. However, it notes that expressing evolved proteins in vivo and requiring expensive cofactors limit the commercial potential of this approach.
This document provides an outline for a presentation on directed evolution. It discusses the process of directed evolution, which involves randomly introducing mutations at the genetic level followed by selection of variants with desired protein characteristics. The document also covers types of mutations, naturally evolutionary processes like random mutagenesis and gene recombination that directed evolution mimics, library size, selection and screening strategies, applications, and advantages of directed evolution over rational design.
This document discusses protein engineering techniques for modifying proteins, including rational protein design using site-directed mutagenesis and directed evolution using random mutagenesis. Site-directed mutagenesis involves introducing point mutations in a particular known area to modify a specific protein function, while directed evolution generates genetic diversity through random mutagenesis and screens variants to identify successful mutations without requiring structural information. Common random mutagenesis methods discussed are error-prone PCR and DNA shuffling, which can be used to engineer properties like protein folding, stability, binding, and catalysis.
protein engineering and site directed mutagenesisNawfal Aldujaily
This document discusses various techniques for protein engineering and site-directed mutagenesis. It describes altering the sequence of proteins through genetic engineering to improve properties like stability and changing amino acids near the active site to modify enzyme specificity. Directed evolution and DNA shuffling techniques are also discussed that introduce mutations and recombine protein domains to generate novel proteins with optimized functions.
1) G.N. Ramachandran created the Ramachandran plot in 1963, which is an essential tool for understanding protein structure. The plot analyzes allowed regions of phi and psi dihedral angles in peptide units.
2) Protein stability refers to a protein maintaining its native folded conformation rather than becoming denatured. Stability depends on a balance of forces and is important for protein function.
3) Factors that influence protein stability include pH, ligand binding, disulfide bonds, and interactions within the protein and between the protein and solvent. Chaperone proteins and proteases also help maintain stability in cells.
This document provides an introduction to the concepts of biotechnology and its applications in the pharmaceutical industry. It discusses key topics including the objective of the course, important modules to be covered, definitions of biotechnology, the stages of biotechnology development, areas and applications of biotechnology. Specific techniques discussed include enzyme immobilization, biosensors, protein engineering and genetic engineering. Methods of enzyme immobilization like adsorption, entrapment, covalent binding and cross-linking are also summarized along with their advantages and limitations.
The document discusses immobilization of enzymes. There are several reasons for immobilizing enzymes, including easy separation of the enzyme from reaction products and reuse of the enzyme. Common immobilization methods include covalent binding of the enzyme to a support, entrapment within a polymer matrix, and cross-linking. Supports can be inorganic materials like silica or organic polymers. Immobilization can impact properties of enzymes like activity, pH optimum, and stability. Reversible immobilization allows regeneration of supports and is important for labile enzymes.
This document discusses protein engineering, which uses recombinant DNA technology to modify protein structure and function. It describes several methods of protein engineering, including site-directed mutagenesis, error-prone PCR, and DNA shuffling. The objectives of protein engineering are to improve protein stability, modify cofactor requirements, increase enzyme activity, and modify enzyme specificity. As an example, the document discusses how site-directed mutagenesis was used to increase the stability of streptokinase by replacing lysine residues susceptible to cleavage by plasmin.
Meta-genomics is the application of modern genomics techniques to the study of communities of microbial organisms directly in their natural environments, bypassing the need for isolation and lab cultivation of individual species”
This document discusses biotransformation and microbial conversion of organic compounds. It defines biotransformation as the microbial conversion of a substrate into a product using enzymatic reactions. Various examples of biotransformation reactions are provided, including the production of gluconic acid, antibiotics, lactic acid, and acetic acid (vinegar). The document also discusses methods for biotransformation like growing, resting, and immobilized cells as well as cell-free extracts and enzymes. It provides details on the production processes of certain compounds like gluconic acid, vinegar, and steroids.
Lipase catalyzed process for biodiesel production protein engineering and lip...Melike Şeyma Kadayıfçı
This review document discusses the use of lipases in biodiesel production. It covers lipase engineering approaches like directed evolution and rational design to improve properties such as thermostability and activity. It also discusses lipase production methods like host strain selection and metabolic engineering. The goal is to develop high-performance lipases through these techniques to enable large-scale commercialization of lipase-catalyzed biodiesel production as an alternative to fossil fuels.
The document discusses metabolic pathway engineering and metabolic engineering. It provides an overview of four commercially important fermentation products, including the microorganism used, annual production levels, and applications. It then discusses the core concepts of metabolic engineering, including manipulating enzymatic and regulatory functions using recombinant DNA to improve cellular activities. Examples of applications include strain improvement for biocatalysis and bioprocessing, increasing productivity, and developing novel biosynthetic routes.
Strain development techniques of industrially important microorganismsMicrobiology
Strain improvement and development involves manipulating microbial strains to enhance their metabolic capacities for biotechnology applications. Targets of improvement include rapid growth, genetic stability, non-toxicity, large cell size, ability to use cheaper substrates, increased productivity, and reduced cultivation costs. Methods for optimization include modifying environmental conditions, nutrition, mutagenesis, transduction, conjugation, transformation, and genetic engineering. Common industrial microorganisms are bacteria such as Bacillus subtilis and yeasts such as Saccharomyces cerevisiae.
This document discusses techniques for strain improvement in microbiology. It describes the ideal characteristics of microbial strains, the purpose of strain improvement, and three main approaches: mutant selection through chemical or radiation mutagenesis, recombination through techniques like transformation and conjugation, and recombinant DNA technology. Novel technologies discussed include metabolic engineering and genome shuffling. Applications include production of medicines and industrial enzymes.
Mutagenesis; A conventional tool for strain improvement in industry Zohaib HUSSAIN
The strain improvement is the process of improvement and manipulation of microbial strains for the icreasment of metabolic level for industrial applications. The yield of microbial enzymes can be increased by using microbe specific medium for fermentation, improving the fermentation process and strain improvement for higher yield of product.
All these things lead to decrease in cost production. Microbe produce product according to its need therefore there is great need for overproduction. There is tremendous contribution of conventional Mutagenesis for strain improvement. Mutagenesis is important tool for the production of mutants which are capable to produce large product i.e. hyperactive.
The document discusses purification of recombinant proteins using affinity tags. It describes immobilized metal affinity chromatography (IMAC) as a widely used method to purify recombinant proteins fused to tags like histidine, GST or MBP. The document outlines the steps involved, including gene amplification, cloning, expression in bacteria or yeast, and purification. It focuses on using histidine tags and nickel-chelate affinity chromatography, noting the advantages of tags for simplifying purification and detection of recombinant proteins.
Chemical protein engineering synthetic and semisyntheticAli Hatami
This document summarizes various methods for chemically synthesizing and modifying peptides and proteins. It discusses solid phase peptide synthesis, native chemical ligation using peptide thioesters, and fragment condensation strategies. It also covers chemoselective ligations using oxime and hydrazone bonds and decarboxylative amide formation. Additionally, the document outlines chemical modifications like PEGylation, phosphorylation, and backbone modifications. Finally, it examines enzyme-mediated ligation techniques like sortase and biotin ligase that can link proteins and peptides in a sequence-specific manner.
Science and technology of manipulating and improving microbial strains, in order to enhance their metabolic capacities for biotechnological applications, are referred to as strain improvement.
This document discusses metabolic engineering. Metabolic engineering modifies cellular properties through recombinant DNA technology to alter metabolic pathways for production of chemicals, fuels, pharmaceuticals and medicine. It requires overexpression or downregulation of proteins in pathways so cells produce new products. The first step is understanding the host cell environment for genetic modifications, and the effect of modifications on growth should be examined. Genetic manipulation may negatively impact metabolic burden. Metabolic engineering is used to produce various compounds through methods like eliminating competing pathways, expressing foreign enzymes, and optimizing cofactors like NAD+/NADH.
This document discusses enzyme biotechnology and methods of enzyme immobilization. It begins by defining enzymes and their function in cells. It then describes the different methods of immobilizing enzymes, including adsorption, covalent bonding, entrapment, cross-linking/copolymerization, and encapsulation. The advantages and disadvantages of each method are provided. Overall, the document provides an overview of enzyme biotechnology with a focus on immobilization techniques.
This study investigated the indigoidine synthetase enzyme which contains an oxidase (Ox) domain involved in nonribosomal peptide synthesis. Through structural analysis of the Ox domain, a conserved tyrosine residue was identified that acts as an active site base. Multiple sequence alignment and biochemical assays on other Ox domains confirmed the general importance of this tyrosine residue. The findings provide mechanistic insight into how Ox domains catalyze oxidation reactions via an E2-like mechanism, utilizing the conserved tyrosine base. This work advances understanding of nonribosomal peptide synthesis which produces medically relevant natural products.
This document discusses protein engineering through directed evolution. It explains that directed evolution involves randomly recombining genes from protein libraries and screening mutant proteins for improved functions. This leaves beneficial changes up to chance but generates diversity. Rational design is also used to map important protein interactions to guide evolution. The document provides an example of using directed evolution to modify a cytochrome P450 enzyme to more efficiently convert alkanes to alcohols over multiple generations. However, it notes that expressing evolved proteins in vivo and requiring expensive cofactors limit the commercial potential of this approach.
This document provides an outline for a presentation on directed evolution. It discusses the process of directed evolution, which involves randomly introducing mutations at the genetic level followed by selection of variants with desired protein characteristics. The document also covers types of mutations, naturally evolutionary processes like random mutagenesis and gene recombination that directed evolution mimics, library size, selection and screening strategies, applications, and advantages of directed evolution over rational design.
This document discusses protein engineering techniques for modifying proteins, including rational protein design using site-directed mutagenesis and directed evolution using random mutagenesis. Site-directed mutagenesis involves introducing point mutations in a particular known area to modify a specific protein function, while directed evolution generates genetic diversity through random mutagenesis and screens variants to identify successful mutations without requiring structural information. Common random mutagenesis methods discussed are error-prone PCR and DNA shuffling, which can be used to engineer properties like protein folding, stability, binding, and catalysis.
protein engineering and site directed mutagenesisNawfal Aldujaily
This document discusses various techniques for protein engineering and site-directed mutagenesis. It describes altering the sequence of proteins through genetic engineering to improve properties like stability and changing amino acids near the active site to modify enzyme specificity. Directed evolution and DNA shuffling techniques are also discussed that introduce mutations and recombine protein domains to generate novel proteins with optimized functions.
1) G.N. Ramachandran created the Ramachandran plot in 1963, which is an essential tool for understanding protein structure. The plot analyzes allowed regions of phi and psi dihedral angles in peptide units.
2) Protein stability refers to a protein maintaining its native folded conformation rather than becoming denatured. Stability depends on a balance of forces and is important for protein function.
3) Factors that influence protein stability include pH, ligand binding, disulfide bonds, and interactions within the protein and between the protein and solvent. Chaperone proteins and proteases also help maintain stability in cells.
This document provides an introduction to the concepts of biotechnology and its applications in the pharmaceutical industry. It discusses key topics including the objective of the course, important modules to be covered, definitions of biotechnology, the stages of biotechnology development, areas and applications of biotechnology. Specific techniques discussed include enzyme immobilization, biosensors, protein engineering and genetic engineering. Methods of enzyme immobilization like adsorption, entrapment, covalent binding and cross-linking are also summarized along with their advantages and limitations.
The document discusses immobilization of enzymes. There are several reasons for immobilizing enzymes, including easy separation of the enzyme from reaction products and reuse of the enzyme. Common immobilization methods include covalent binding of the enzyme to a support, entrapment within a polymer matrix, and cross-linking. Supports can be inorganic materials like silica or organic polymers. Immobilization can impact properties of enzymes like activity, pH optimum, and stability. Reversible immobilization allows regeneration of supports and is important for labile enzymes.
The document discusses various methods for immobilizing microbial cells and enzymes, including carrier binding techniques like adsorption, covalent binding, cross-linking and entrapment. It describes common support materials like natural polymers, synthetic polymers and inorganic materials used for immobilization. The advantages of immobilization include recyclability, stability and potential applications in industries like food, biomedical and biodiesel production. Yeast cell immobilization using calcium alginate entrapment is provided as an example.
Application of Biological Assemblies in NanoBiotechnology PPtZohaib HUSSAIN
The document discusses several applications of biological assemblies in nanobiotechnology. It describes how biological assemblies, such as hemoglobin, are the functional forms of molecules and can be used to build larger structures. It then discusses using drug nanocrystals to improve drug solubility and bioavailability. Next, it covers using nano-containers like liposomes for targeted drug delivery. The document also discusses using protein crystals called S-layers for nanolithography and peptide templates for controlling biomineralization. Finally, it discusses the potential of utilizing biomineralization principles in nanotechnology applications like tissue engineering.
Enzyme immobilization method & application easybiologyclasslalit mathuriya
This document discusses enzyme immobilization methods and applications. It begins by defining enzyme immobilization as imprisoning cells or enzymes in a support or matrix. The main advantages listed are increased efficiency, reproducibility, and reuse of enzymes. Supports described include natural polymers like alginate and chitosan, synthetic polymers, and inorganic materials like zeolites and ceramics. The five main immobilization methods are adsorption, covalent bonding, entrapment, copolymerization, and encapsulation. Adsorption, the oldest method, involves weak bonding of enzymes to carrier surfaces.
Plant cells can be immobilized through various methods like surface attachment, entrapment in porous matrices, containment behind barriers, and self-aggregation. This allows maintaining high cell densities to increase productivity of secondary metabolites. Immobilization provides advantages like easier product separation, continuous processing, and protecting cells from shear forces. However, limitations include additional costs, complexity in understanding plant cell pathways, and potential loss of biosynthetic capacity. Applications of immobilized plant cells include production of high-value compounds, biotransformations, and synthetic seed technology.
This document discusses various methods of enzyme immobilization including physical and chemical methods. Physical methods include adsorption, entrapment, and microencapsulation. Adsorption involves binding enzymes to a carrier's surface through weak forces. Entrapment physically traps enzymes within a porous polymer matrix. Microencapsulation encloses enzymes within semi-permeable membrane capsules. Chemical methods involve covalent bonding of enzymes to carriers through functional groups, and cross-linking which uses polyfunctional reagents to create cross-links between enzymes. The document provides details on each method's process, examples, advantages, and disadvantages.
Nanotechnology involves manipulating matter at the atomic or nano scale. The document outlines the history, definitions, and approaches to nanotechnology such as top-down and bottom-up. It discusses applications of nanotechnology in food processing and packaging by using nano capsules for delivery and improving shelf life. Other applications mentioned include optics, medicine, energy, and more. The conclusion states that nanotechnology is an emerging field that will transform industries and potentially help address challenges in health, energy, and beyond.
This document discusses biodegradable polymers, including their classification and examples. It describes several types of biodegradable polymers in more detail, including polysaccharides, polylactic acid, polylactic-co-glycolic acid, polypropylene carbonate, polycaprolactone, biodegradable silk, and chitosan. It provides information on their properties, applications, and advantages of using biodegradable polymers which include being degradable at the end of use, easy to recycle, requiring less energy than non-biodegradable polymers, producing less waste, and being more environmentally friendly.
This document provides an overview of biofilms and their relevance to pharmaceutical manufacturing. It discusses how biofilms can cause both opportunities and problems in the industry. Biofilms are defined as the unwanted adhesion of microorganisms to surfaces, which can lead to manufacturing issues like product contamination, process downtime, and sampling difficulties. The document outlines the typical sequence of biofilm formation and highlights their increased resistance to antimicrobials and cleaning agents. It also introduces hurdle technology as an approach to prevent biofilms through multiple barriers in the manufacturing process.
This document discusses the biocompatibility of polymeric dental materials. It defines biocompatibility and provides historical context for testing materials' biocompatibility. The key requirements for dental materials to be biocompatible are that they must not be harmful to pulp, soft tissues, or cause toxic, allergic, carcinogenic or other systemic responses. Methods of measuring biocompatibility include in vitro tests like cytotoxicity tests and cell permeability assays, animal tests, and clinical usage tests. Common polymers used in dentistry include PMMA, BIS-GMA, UDMA, and PEEK, which are generally biocompatible but must still undergo rigorous testing.
Lecture 5_Polymers in biomedical applications (1).pptAsmaHwedi1
This document discusses various biomedical applications of polymers. It begins by noting polymers' widespread uses due to their ease of production, biocompatibility, lower cost, ability to mimic natural materials, and to prevent additional surgery. It then discusses specific applications, including medical packaging, pharmaceuticals, and drug delivery. It provides examples of polymers used for these purposes and how they can be processed. The document also discusses polymers as biomaterials for implantable medical components and their requirements. It provides examples of polymeric materials used in cochlear implants and dental implants. In summary, the document outlines the many uses of polymers in biomedical applications including packaging, drug delivery, and as implantable biomaterials.
This document provides information about enzyme immobilization. It discusses how immobilizing enzymes attaches them to an insoluble support, which allows them to be reused over multiple catalytic cycles while being easily separated from reaction products. The advantages of immobilization include improved enzyme stability under various conditions and the ability for repetitive use. Common immobilization techniques include adsorption, ionic binding, covalent binding, cross-linking, and entrapment. The choice of support material and technique can impact enzyme activity and stability. Characterization of immobilized enzymes includes measuring activity, bound protein, and specific activity of the bound protein.
The document discusses the scope of modern biology. It states that molecular cell biology now blends advanced cytology, molecular nature, genetics, biochemistry, computation, and engineering. Technological advances like automation, DNA sequencing, mass spectroscopy and microarrays allow large-scale genomic and proteomic analyses. Techniques such as PCR, FRET and RNAi have led to more sophisticated experiments. The document also discusses various topics in modern biology like bioinformatics, genetics, phytochemistry, structural biology, and synthetic biology. It notes both the potential applications and ethical risks of synthetic biology.
This document provides an introduction to nanobiotechnology. It discusses how nanotechnology involves working at the nanoscale of 1-100 nanometers to develop applications in areas like biotechnology. Nanobiotechnology uses nanotechnology techniques to develop and improve biotechnological processes and products like lab-on-a-chip devices and biosensors. The document outlines the differences between classical biotechnology, modern biotechnology, and how biotechnology is evolving into bionanotechnology through the integration of nanoscale techniques. Examples of current nanobiotechnology applications are given in areas like drug delivery, disease diagnostics, and food packaging.
introduction to Nanobiotechnology
what is nanotechnology
bionanotechnology
classical biotechnology industrial production using biological system
modern biotechnology from industrial processes to noval therapeutics
modern biotechnology immunological enzymatic and neucleic acid based technology
Dna based technology
self assembly and supramolecular chemistry
formation of ordered structure at nano scale
4th year class ppt bio.2014 final .pdfasmamawbelew
Here are the key points about immobilized enzymes:
- Enzymes are normally soluble in water, making it difficult to separate and reuse them in batch processes.
- Immobilization involves attaching or confining enzymes to an insoluble support or carrier material to localize them.
- This allows enzymes to be easily separated from reaction mixtures and reused multiple times.
- Common support materials include membranes, gels, polymers, and inorganic materials like silica.
- The method of immobilization depends on the support used but may involve adsorption, covalent binding, cross-linking, or entrapment.
- Immobilization can improve enzyme stability and allow continuous operation of biocataly
This slide is special for master students (MIBS & MIFB) in UUM. Also useful for readers who are interested in the topic of contemporary Islamic banking.
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1. INTRODUCTION TO
BIOTECHNOLOGY
Prepared by: Chamanpreet Kaur
Assistant Professor ( PHARMACEUTICS)
ASBASJSM COLLEGE OF PHARMACY, BELA.
B Pharmacy 6th sem.
Subject: Pharmaceutical Biotechnology
Subject Code: BP605T
2. Objective of Course
• Understanding the principle of biotechnology and
importance of immobilized enzymes in pharamaceutical
industries.
• Genetic engineering application in relation to production
of pharmaceuticals
• Learning Outcomes
• Students will learn about biotechnology with references to
pharmaceutical sciences.
• They will learn about biosensor and various methods used
for immobilization of enzymes.
• They will be able to implement the theoretical concept of
genetic engineering and protein engineering.
3. Module-1
Brief introduction to Biotechnology with
reference to Pharmaceutical Sciences.
Enzyme Biotechnology- Methods of
enzyme immobilization and applications.
Biosensors- Working and applications of
biosensors in Pharmaceutical Industries.
Brief introduction to Protein Engineering.
Basic principles of genetic engineering.
4. What is biotechnology?
• Biotechnology = bios (life) + logos (study of or
essence)
– Literally ‘the study of tools from livingthings’
• CLASSIC: The word "biotechnology" was first used in
1917 to describe processes using living organisms to
make a product or run a process, such as industrial
fermentations. (Robert Bud, The Uses of Life: A
History of Biotechnology)
• LAYMAN: Biotechnology began when humans began
to plant their own crops, domesticate animals,
ferment juice into wine, make cheese, and leaven
bread (AccesExcellence)
5. What are the stages of biotechnology?
• Ancient Biotechnology
• early history as related to food and shelter,
including domestication
• Classical Biotechnology
• built on ancient biotechnology
• fermentation promoted food production
• medicine
• Modern Biotechnology
• manipulates genetic information in organism
• genetic engineering
6. • Cell biology
• Structure, organization and reproduction
• Biochemistry
• Synthesis of organic compounds
•Cell extracts for fermentation (enzymes
versus whole cells)
• Genetics
• Resurrection of Gregor Mendel’s findings 1866
1900s
•Theory of Inheritance (ratios dependent on traits of
parents)
• Theory of Transmission factors
• W.H. Sutton – 1902
• Chromosomes = inheritance factors
Modern biotechnology
7. Molecular Biology
• Beadle and Tatum (Neurospora crassa)
• One gene, one enzyme hypothesis
• Charles Yanofsky colinearity
between mutations in genes and amino
acid sequence (E. coli)
• Genes determine structure of proteins
• Hershey and Chase – 1952
• T2 bacteriophage – 32P DNA, not 35S protein
is the material that encodes genetic
information
Modern biotechnology
8. • Watson, Crick, Franklin and Wilkins (1953)
• X-ray crystallography
• 1962 – Nobel Prize awarded to three men
• Chargaff – DNA base ratios
• Structural model of DNA developed
• DNA Revolution – Promise and Controversy!!!
• Scientific foundation of modern biotechnology
•based on knowledge of DNA, its replication,
repair and use of enzymes to carry out in vitro
splicing DNA fragments
Modern biotechnology
9. What are the areas of biotechnology?
• Organismic biotechnology
• uses intact organisms and does not alter genetic
material
• Molecular Biotechnology
• alters genetic makeup to achieve specific goals
Transgenic organism: an organism with artificially
altered genetic material
10. What are the benefits of
biotechnology?
• Medicine
• human
• veterinary
• biopharming
• Environment
• Agriculture
• Food products
• Industry and manufacturing
11. What are the applications of biotechnology?
•
•
•
•
•
•
•
•
•
•
Production of new and improved crops/foods,
industrial chemicals, pharmaceuticals and livestock
Diagnostics for detecting genetic diseases
Gene therapy (e.g. ADA, CF)
Vaccine development (recombinant vaccines)
Environmental restoration
Protection of endangered species
Conservation biology
Bioremediation
Forensic applications
Food processing (cheese, beer)
12. Monoclonal
Antibodies
Molecular
Biology
Cell
Culture
Genetic
Engineering
Anti-cancer drugs
Diagnostics
Culture of plants
from single cells
Transfer of new
genes into animal
organisms
Synthesis of
specific DNA
probes
Localisation of
genetic disorders
Tracers
Cloning
Gene therapy
Mass prodn. of
human proteins
Resource bank
for rare human
chemicals
Synthesis
of new
proteins
New
antibiotics
New types of
plants and
animals
New types
of food
DNA
technology
Crime solving
Banks of
DNA, RNA
and proteins
Complete
map of the
human
genome
13.
14. Enzyme immobilization may be defined as a
process of confining the enzyme molecules
to a solid support over which a substrate is
passed and converted to products.
What Is An Immobilized Enzyme?
An immobilized enzyme is one whose
movement in space has been restricted either
completely or to a small limited region.
What Is Enzyme Immobilization ?
15. Why ImmobilizeEnzymes?
Protection from degradation and deactivation.
Re-use of enzymes for many reaction cycles, lowering the total production
cost of enzyme mediated reactions.
Ability to stop the reaction rapidly by removing the enzyme from the reaction
solution.
Enhanced stability.
Easy separation of the enzyme from the product.
Product is not contaminated with the enzyme.
16. An Ideal Carrier Matrices For Enzyme
Immobilization
Inert.
Physically strong and stable.
Cost effective.
Regenerable.
Reduction in product inhibition.
17. CLASSIFICATION OF CARRIERS
CLASSIFICATION OF CARRIERS
Inorganic
Carriers
•High pressure
stability.
• May undergo
abrasion
Examples:
1.Commercialy SiO2
available materials-
oPorous glass.
oSilica.
2. Mineral materials -
(clays)
Celite ,Centonite
Inorganic
Carriers
•High pressure
stability.
•May undergo
abrasion
Examples:
1.Commercialy SiO2
available materials-
oPorous glass.
oSilica.
2.Mineral materials -
(clays)
Celite ,Centonite
Organic Natural
Carriers
•Favourable
compatibility with
proteins.
Examples:
1.cellulose derivatives-
oDEAE-cellulose
oCM-cellulose.
2. Dextran.
3.Polysacharides
Agarose, Starch
Pectine ,Chitosan
Organic Natural
Carriers
•Favourable
compatibility with
proteins.
Examples:
1.cellulose derivatives-
oDEAE-cellulose
oCM-cellulose.
2.Dextran.
3.Polysacharides
Agarose, Starch
Pectine ,Chitosan
Organic
Synthetic
Carriers
•High chemical
and mechanical
stability.
Examples:
1.Polystyrene
2.Polyvinylacetate
3. Acrylic
polymers
Organic
Synthetic
Carriers
•High chemical
and mechanical
stability.
Examples:
1.Polystyrene
2.Polyvinylacetate
3. Acrylic
polymers
19. Physical Methods For
Immobilization
ADSORPTION
Involves the physical binding of the enzyme on the surface of carrier matrix.
Carrier may be organic or inorganic.
The process of adsorption involves the weak interactions like Vander Waal
or hydrogen bonds.
Carriers: - silica, bentonite, cellulose, etc.
e.g. catalase & invertase
20. ADVANTAGE DISADVANTAGES
1. Simple and economical
2. Limited loss of activity
3.Can be Recycled,
Regenerated & Reused.
1.Relatively low surface
area for binding.
2.Exposure of enzyme to
microbial attack.
3.Yield are often low due to
inactivation and desorption.
21. Entrapment
In entrapment, the enzymes or cells are not directly attached to the support
surface, but simply trapped inside the polymer matrix.
Enzymes are held or entrapped within the suitable gels or fibres.
It is done in such a way as to retain protein while allowing penetration of
substrate. It can be classified into lattice and micro capsule types.
Inclusion in gels: Poly acrylamide gel, Poly vinyl alcohol gels.
Inclusion in fibers: Cellulose and Poly -acryl amide gels.
Inclusion in micro capsules: Polyamine, Polybasic -
acid chloride monomers.
22. Lattice-Type Entrapment
Entrapment involves entrapping enzymes within the
interstitial spaces of a cross-linked water-insoluble
polymer. Some synthetic polymers such as polyarylamide,
polyvinylalcohol, etc... and natural polymer (starch) have
been used to immobilize enzymes using this technique.
25. Covalent Binding
Based on the binding of enzymes and water-insoluble carriers by covalent
bonds
The functional groups that may take part in this binding are Amino
group, Carboxyl group, Sulfhydryl group, Hydroxyl group, Imidazole
group, Phenolic group, Thiol group, etc
Disadvantages : covalent binding may alter the conformational structure
and active center of the enzyme, resulting in major loss of activity and/or
changes of the substrate
Advantages : the binding force between enzyme and carrier is so strong
that no leakage of the enzymes occurs, even in the presence of substrate or
solution of high ionic strength.
26. CrossLinking
Cross linking involves intermolecular cross linking of enzyme molecules in the
presence/absence of solid support.
The method produces a 3 dimensional cross linked enzyme aggregate
(insoluble in water) by means of a multifunctional reagent that links covalently
to the enzyme molecules.
27. Advantages of cross linking:-
1. Very little desorption(enzyme strongly bound)
2. Higher stability (i.e. ph, ionic & substrate concentration)
Disadvantages of cross linking:-
1. Cross linking may cause significant changes in the active site.
2. Not cost effective.
30. Limitations Of Enzyme
Immobilization
Cost of carriers and immobilization.
Changes in properties (selectivity).
Mass transfer limitations.
Problems with cofactor and regeneration.
Problems with multienzymes systems.
Activity loss during immobilization.
34. 1. LINEARITY Linearity of the sensor should be high
forthe detection of high substrate
concentration.
2. SENSITIVITY Value of the electrode response per
substrate concentration.
3. SELECTIVITY Chemicals Interference must be
minimised for obtaining the correct
result.
4.RESPONSE TIME Time necessary for having 95%
of the response.
Basic Characteristics of a
Biosensor
35. 1. The Analyte (What do you want to detect)
Molecule - Protein, toxin, peptide, vitamin, sugar,
metal ion
2. Sample handling (How to deliver the analyte to the sensitive region?)
(Micro) fluidics - Concentration increase/decrease),
Filtration/selection
Biosensor
36. 4. Signal
(How do you know there was a detection)
3. Detection/Recognition
(How do you specifically recognize the analyte?)
Biosensor
39. PIEZO-ELECTRIC BIOSENSORS
.
Piezo-electric devices use gold to detect the
specific angle at which electron waves are
emitted when the substance is exposed to laser
light or crystals, such as quartz, which vibrate
under the influence of an electric field.
40. ELECTROCHEMICAL BIOSENSORS
• For applied current: Movement of e- in redox
reactions detected when a potential is applied
between two electrodes.
42. OPTICAL BIOSENSORS
•Colorimetric for color
Measure change in light adsorption
•Photometric for light intensity
Photon output for a luminescent or
fluorescent process can be detected with
photomultiplier tubes or photodiode
systems.
43. CALORIMETRIC BIOSENSORS
If the enzyme catalyzed reaction is exothermic,
two thermistors may be used to
measure the difference in resistance
between reactant and product and, hence,
the analyte concentration.
44. Electrochemical DNA Biosensor
Steps involved in electrochemical
DNA hybridization biosensors:
Formation of the DNA recognition layer
Actual hybridization event
Transformation of the hybridization event
into an electrical signal
45. Motivated by the application to clinical diagnosis
and genome mutation detection
Types DNA Biosensors
Electrodes
Chips
Crystals
DNA biosensor
46. Food Analysis
Study of biomolecules and their interaction
Drug Development
Crime detection
Medical diagnosis (both clinical and laboratory use)
Environmental field monitoring
Quality control
Industrial Process Control
Detection systems for biological warfare agents
Manufacturing of pharmaceuticals and replacement
organs
Application of Biosensor
48. Genes are the fundamental basis of all life,
determine the properties of all living forms of
life, and are defined segments of DNA.
Because DNA structure and composition in all
living forms is essentially the same, any
technology that can isolate, change or
reproduce a gene is likely to have an impact on
almost every aspect of society.
48
49. Genetic engineering has been defined as the
formation of new combinations of heritable
material by the insertion of nucleic acid
molecules, into any virus, bacterial plasmid or
other vector system so as to allow their
incorporation into a host organism in which
they do not naturally occur, but in which they
are capable of continued propagation.
49
51. gene technology is the modification of the
genetic properties of an organism by the use of
recombinant DNA technology.
Genes may be viewed as the biological
software and are the programs that drive the
growth, development and functioning of an
organism.
By changing the software in a precise and
controlled manner, it becomes possible to
produce desired changes in the characteristics
of the organism.
51
52. These techniques allow the splicing of DNA
molecules of quite diverse origin, and, when
combined with techniques of genetic
transformation etc., facilitate the introduction of
foreign DNA into other organisms.
The foreign DNA or gene construct is introduced
into the genome of the recipient organism host
in such a way that the total genome of the host
is unchanged except for the manipulated
gene(s).
52
53. While traditional plant and animal genetic breeding
techniques also change the genetic code it is
achieved in a less direct and controlled manner.
Genetic engineering will now enable the breeder to
select the particular gene required for a desired
characteristic and modify only that gene.
Although much work to date has involved bacteria,
the techniques are evolving at an astonishing rate
and ways have been developed for introducing
DNA into other organisms such as yeasts and
plant and animal cell cultures.
53
54. Life forms containing ‘foreign’ DNA are termed
transgenic
These methods potentially allow totally new
functions to be added to the capabilities of
organisms, and open up vistas for the genetic
engineering of industrial microorganisms and
agricultural plants and animals that are quite
breathtaking in their scope.
This is undoubtedly the most significant new
technology in modern bioscience and
biotechnology.
54
55. In industrial microbiology it will permit the
production in microorganisms of a wide range
of hitherto unachievable products such as
human and animal proteins and enzymes such
as insulin and chymosin (rennet)
in medicine, better vaccines, hormones and
improved therapy of diseases;
in agriculture, improved plants and animals for
productivity, quality of products, disease
resistance, etc;
55
56. in food production, improved quality, flavour,
taste and safety;
in environmental aspects, a wide range of benefits
such as pollution control can be expected.
In microbial technology these techniques will be
widely used to improve existing microbial
processes by improving stability of existing
cultures and eliminating unwanted side products.
However, there are many who view genetic
engineering as a transgression of normal life
processes that goes well beyond normal evolution.
56
57. Genetic engineering holds the potential to extend the range and
power of almost every aspect of biotechnology.
It is confidently anticipated that within this decade recombinant DNA
techniques will form the basis of new strains of microorganisms
with new and unusual metabolic properties.
In this way fermentations based on these technical advances could
become competitive with petrochemicals for producing a whole
range of chemical compounds, for example ethylene glycol (used
in the plastics industry) as well as improved biofuel production.
In the food industry, improved strains of bacteria and fungi are now
influencing such traditional processes as baking and cheese-
making and bringing greater control and reproducibility of flavour
and texture.
57
58. A full understanding of the working concepts of
recombinant DNA technology requires a good
knowledge of molecular biology.
The basic molecular techniques for the in vitro
transfer and expression of foreign DNA in a
host cell (gene transfer technology), including
isolating, cutting and joining molecules of DNA,
and inserting into a vector (carrying) molecule
that can be stably retained in the host cell,
were first developed in the early 1970s.
58
60. Cutting DNA molecules:
DNA can be cut using mechanical or enzymatic
methods.
The non-specific mechanical shearing will
generate random DNA fragments
In contrast, when specific restriction
endonuclease enzymes are used it is possible
to recognise and cleave specific target base
sequences in double-stranded (ds) DNA.
60
61. Large numbers of different restriction
endonucleases have been extracted and
classified from a wide variety of microbial
species.
Restriction endonucleases are named according
to the species from which they were first
isolated, e.g. enzymes isolated from
Haemophilus influenzae strain Rd are
designated Hind and when several different
restriction enzymes areisolated from the same
organism they are designated HindI, HindII etc.
61
62. Splicing DNA:
DNA fragments can be joined together in vitro
by the action of specific DNA ligases.
The DNA ligase that is widely used was
encoded by phage T4.
The composite molecules in which DNA has
been inserted have also been termed ‘DNA
chimaeras’
62
63. The vector or carrier system:
Two broad categories of expression vector
molecules have been developed as vehicles for
gene transfer, plasmids (small units of DNA distinct
from chromosomes) and bacteriophages (or
bacterial viruses).
Vector molecules should be capable of entering
the host cell and replicating within it.
Ideally, the vector should be small, easily prepared
and must contain at least one site where
integration of foreign DNA will not destroy an
essential function.
63
64. Introduction of vector DNA recombinants:
The new recombinant DNA can now be
introduced into the host cell and if acceptable
the new DNA will be cloned with the
propagation of the host cell.
Novel methods of ensuring DNA uptake into
cells include electroporation and mechanical
particle delivery or biolistics.
64
65. Electroporation is a process of creating transient pores in the
cell membrane by application of a pulsed electric field.
Creation of such pores in a membrane allows introduction of
foreign molecules, such as DNA, RNA, antibodies, drugs,
etc., into the cell cytoplasm.
Development of this technology has arisen from synergy of
biophysics, bioengineering and cell and molecular biology.
While the technique is now widely used to create transgenic
microorganisms, plants and animals, it is also being
increasingly used for application of therapeutics and gene
therapy.
65
66. The mechanical particle delivery or ‘gene gun’
methods deliver DNA on microscopic particles
into target tissue or cells. This process is
increasingly used to introduce new genes into a
range of bacterial, fungal, plant and
mammalian species and has become a main
method of choice for genetic engineering of
many plant species including rice, corn, wheat,
cotton and soybean.
66