This document provides an overview of biotechnology and bioprocess engineering. It discusses biomolecules like carbohydrates, proteins, lipids, and nucleic acids. It then focuses on recombinant DNA technology, explaining the process from gene to product, including gene isolation, cloning, cell transformation, fermentation and downstream processing to produce biomolecules like proteins. The main applications of biotechnology in pharmaceutical, agriculture, chemical and fuel industries are also summarized.
Agrobacterium tumefaciens is a soil bacterium that causes crown gall disease in plants. It transfers a segment of DNA called T-DNA from its Ti plasmid into the plant genome, enabling it to modify plant cell growth. The presentation discusses the bacterial characteristics, mechanism of T-DNA transfer, use of Agrobacterium for genetic engineering of plants through insertion of foreign genes in place of tumor-causing genes on the Ti plasmid, and the regeneration of transformed plants. While it is an effective tool for plant genetic engineering, some plant species are not susceptible to Agrobacterium infection.
Plants can be used as bioreactors to produce valuable proteins and chemicals. There are several types of plant bioreactors, including seed-based systems, plant suspension cultures, hairy root cultures, and chloroplast bioreactors. Plants offer advantages over traditional fermentation systems as they are inexpensive to culture and scale up, can produce properly folded and assembled proteins, and do not harbor human pathogens. However, some safety and environmental concerns must be addressed when using genetically modified plants as bioreactors.
This document discusses plant molecular pharming (PMP), which uses plants as bioreactors for producing recombinant pharmaceutical proteins. It covers the definition, history, strategies, host systems, production of antibiotics/enzymes/vaccines in plants, advantages/disadvantages of plant systems, and issues of transgene pollution. Key points include:
- PMP uses whole plants, plant cells or tissues to produce commercially valuable proteins like vaccines via recombinant DNA.
- Early work in 1986 produced human growth hormone in tobacco and sunflower. Commercial production of various proteins in plants has occurred.
- Strategies include transforming host plants, growing biomass, processing/purifying the product of interest.
- Plants,
The document discusses using plants as bioreactors to produce valuable biomolecules. Key points include:
- Plants can be genetically engineered to produce pharmaceuticals, industrial compounds, and other non-native products.
- Various plant parts like seeds, cell cultures, hairy roots, and chloroplasts can serve as bioreactors. Products are targeted to organelles or extracellular spaces.
- Examples of products made in plants include vaccines, antibodies, growth hormones, starch variants, and fatty acid modifications. Crops like tobacco, potatoes, and rice have been engineered as bioreactors.
- Cyclodextrins can be produced in potato tubers by expressing a bacterial gene encoding cyclodextrin glycos
This presentation is about chloroplast transformation, the importance of chloroplast transformation on nucleus transformation and strategies for making marker-free transplastomic plant
This document provides an overview of Agrobacterium-mediated gene transfer. It discusses that Agrobacterium tumefaciens is a soil bacterium that causes crown gall disease in plants by transferring tumor-inducing (Ti) plasmid DNA (T-DNA) into the plant genome. The Ti plasmid contains the T-DNA region flanked by borders, and virulence genes required for T-DNA transfer. Upon sensing plant signals, the virulence genes activate and the T-DNA is transferred to plant cells, where it integrates into the genome and expresses genes that cause tumor formation by increasing phytohormone levels. This natural plant genetic transformation ability makes Agrobacterium an important tool in genetic engineering
This document provides an overview of biopharming, which uses agricultural plants to produce useful molecules for non-food applications. Biopharming aims to lower production costs of therapeutic molecules like enzymes by expressing genes in plants. Current research focuses on plant-made vaccines, antibodies, and proteins to treat diseases. Risks include contamination of the food supply or environment. Suggested safeguards include making biopharmed crops sterile or detectable. Future progress requires improving yields and stability while establishing reliable biosafety. Whether biopharmed crops are further developed will depend on regulation and public perception of risks.
Agrobacterium tumefaciens is a soil bacterium that causes crown gall disease in plants. It transfers a segment of DNA called T-DNA from its Ti plasmid into the plant genome, enabling it to modify plant cell growth. The presentation discusses the bacterial characteristics, mechanism of T-DNA transfer, use of Agrobacterium for genetic engineering of plants through insertion of foreign genes in place of tumor-causing genes on the Ti plasmid, and the regeneration of transformed plants. While it is an effective tool for plant genetic engineering, some plant species are not susceptible to Agrobacterium infection.
Plants can be used as bioreactors to produce valuable proteins and chemicals. There are several types of plant bioreactors, including seed-based systems, plant suspension cultures, hairy root cultures, and chloroplast bioreactors. Plants offer advantages over traditional fermentation systems as they are inexpensive to culture and scale up, can produce properly folded and assembled proteins, and do not harbor human pathogens. However, some safety and environmental concerns must be addressed when using genetically modified plants as bioreactors.
This document discusses plant molecular pharming (PMP), which uses plants as bioreactors for producing recombinant pharmaceutical proteins. It covers the definition, history, strategies, host systems, production of antibiotics/enzymes/vaccines in plants, advantages/disadvantages of plant systems, and issues of transgene pollution. Key points include:
- PMP uses whole plants, plant cells or tissues to produce commercially valuable proteins like vaccines via recombinant DNA.
- Early work in 1986 produced human growth hormone in tobacco and sunflower. Commercial production of various proteins in plants has occurred.
- Strategies include transforming host plants, growing biomass, processing/purifying the product of interest.
- Plants,
The document discusses using plants as bioreactors to produce valuable biomolecules. Key points include:
- Plants can be genetically engineered to produce pharmaceuticals, industrial compounds, and other non-native products.
- Various plant parts like seeds, cell cultures, hairy roots, and chloroplasts can serve as bioreactors. Products are targeted to organelles or extracellular spaces.
- Examples of products made in plants include vaccines, antibodies, growth hormones, starch variants, and fatty acid modifications. Crops like tobacco, potatoes, and rice have been engineered as bioreactors.
- Cyclodextrins can be produced in potato tubers by expressing a bacterial gene encoding cyclodextrin glycos
This presentation is about chloroplast transformation, the importance of chloroplast transformation on nucleus transformation and strategies for making marker-free transplastomic plant
This document provides an overview of Agrobacterium-mediated gene transfer. It discusses that Agrobacterium tumefaciens is a soil bacterium that causes crown gall disease in plants by transferring tumor-inducing (Ti) plasmid DNA (T-DNA) into the plant genome. The Ti plasmid contains the T-DNA region flanked by borders, and virulence genes required for T-DNA transfer. Upon sensing plant signals, the virulence genes activate and the T-DNA is transferred to plant cells, where it integrates into the genome and expresses genes that cause tumor formation by increasing phytohormone levels. This natural plant genetic transformation ability makes Agrobacterium an important tool in genetic engineering
This document provides an overview of biopharming, which uses agricultural plants to produce useful molecules for non-food applications. Biopharming aims to lower production costs of therapeutic molecules like enzymes by expressing genes in plants. Current research focuses on plant-made vaccines, antibodies, and proteins to treat diseases. Risks include contamination of the food supply or environment. Suggested safeguards include making biopharmed crops sterile or detectable. Future progress requires improving yields and stability while establishing reliable biosafety. Whether biopharmed crops are further developed will depend on regulation and public perception of risks.
Plants can be used as bioreactors to produce valuable compounds. Transgenic plants and plant cell cultures can produce large quantities of proteins, vaccines, and other molecules through biochemical reactions using techniques like genetic engineering. Some key advantages of plant bioreactors are that they are cost-effective, can produce high biomass, and allow storage of products for a long time. However, differences in plant and bacterial genetics can impact expression efficiency and safety testing is required.
☺INTRODUCTION
☺Bt COTTON
☺MAJOR PESTS OF COTTON
☺MODE OF ACTION OF Bt GENE
☺ADVANTAGES
☺DISADVANTAGES
☺CONCLUSION
☺REFERENCES
Genetically modified variety of cotton that produces an insecticide whose gene has been derived from a soil bacterium called Bacillus thuringiensis (Bt).
Three types of toxins.
A total of 229 cry toxins ( cry1Aa to Cry72Aa), cyt toxins ( cyt 11Aa to cyt3Aa) and 102 vip toxins( vip1Aa1 to vip4Aa1) have been discovered.
Directed enzyme evolution is a technique that mimics natural selection to engineer proteins. It involves introducing random mutations into genes and screening proteins for modified activity. The key steps are selecting a gene, creating a library of mutant genes through error-prone PCR or other mutagenesis methods, expressing the proteins, and selecting variants with improved properties. Examples where directed evolution has been applied include improving the activity of enzymes used in producing the antibiotic cephalosporin and in the cholesterol-lowering drug atorvastatin. The goal is to leverage natural selection to develop enzymes with desired industrial applications like increased stability, activity, or substrate specificity.
The document discusses different expression vectors and systems used for recombinant protein expression. It describes key elements required for an expression vector including an origin of replication, selective marker, promoter, multiple cloning site, and terminator. It provides details on commonly used expression systems in E. coli such as the lac, tac, lambda PL, and T7 promoters. It also summarizes protein expression in yeast using the galactose-inducible GAL promoter system.
Strategies to enhance production of secondary metabolitesPrashant Singam
1. Strategies to enhance production of secondary metabolites in plant cell cultures include selecting high-productivity cell lines, using elicitors to boost productivity over short periods, and immobilizing cells to improve yields of extracellular metabolites.
2. Elicitors are compounds that induce plant cells to produce more secondary metabolites in response to stress and include fungal or bacterial extracts, polysaccharides, and physical/chemical agents like UV radiation or heavy metals.
3. Immobilizing plant cells by entrapping them in matrices like alginate or polyacrylamide anchors the cells while allowing continuous production and release of metabolites into the medium.
This ppt explains about molecular farming, history of molecular farming, importance, basic process underlying it, its application in agriculture and its limitations
This document summarizes a seminar presentation on improving fruit quality through the use of elicitors and bio-molecules. The presentation outlines the seminar topics which include introduction to elicitors and bio-molecules, their mechanisms of action, and case studies on their use in pomegranate and citrus fruits. It also summarizes results from studies showing elicitors can increase phenolic content and bioactive compounds in fruits, thereby improving quality attributes such as color, firmness, and reducing decay during storage.
Gene tagging uses recognizable DNA fragments like T-DNA or transposons to disrupt gene function and identify genes responsible for mutant phenotypes. T-DNA tagging in plants involves random integration of Agrobacterium T-DNA that can disrupt genes and create mutants. Transposon tagging relies on the ability of transposons to move within genomes and disrupt gene function. Both techniques have been used successfully to isolate numerous plant genes involved in traits like color and development.
This document discusses hairy root cell culture. Hairy root culture involves infecting plant explants with the soil bacterium Agrobacterium rhizogenes, which transfers genes to the plant genome and causes roots to form with increased cell division and elongation, producing "hairy roots". Hairy roots have properties like genotype/phenotype stability, fast growth, and high production of secondary metabolites. The process involves wounding explants, inoculating with A. rhizogenes, inducing hairy roots within 1 week to 1 month, and subculturing in antibiotic media to remove bacteria. Hairy roots can be cultured in various bioreactors and have applications like gene analysis, protein expression, and secondary metabolite production.
Plant transformation vectors and their typesZahra Naz
This document summarizes a presentation on plant transformation vectors and their types. It discusses various types of vectors used for plant transformation including plasmids, viruses, bacteriophages, and cosmids. Plasmids are the most commonly used vector for plant transformation. Agrobacterium-mediated transformation using tumor-inducing (Ti) plasmids is an effective method for genetically transforming plants. Viral vectors like cauliflower mosaic virus (CaMV) are also used but have certain limitations.
It is a part of Ti Plasmid which takes part intransfer and integration of T-DNA into plant chromosome.
The vir sequence consist of 8 operons which take part in different functions associated with virulence of Ti Plasmid. These are vir H, vir A, vir B, vir G, vir C, vir D, vir E, & vir F. ( vir H & vir F present occasionally).
The document discusses several genetically engineered plants including Bt crops, Golden Rice, and Flavr Savr tomato. Bt crops contain a gene from Bacillus thuringiensis that produces a toxin harmful to certain insects, protecting the plant. Golden Rice was engineered to produce beta-carotene in the endosperm to address vitamin A deficiency. Flavr Savr tomato was modified using antisense RNA technology to reduce polygalactouronase levels and slow fruit softening for a longer shelf life.
Gene transfer technologies can be used to treat diseases by inserting therapeutic genes into cells. There are viral and non-viral methods of gene transfer. Viral methods use viruses like retroviruses, adenoviruses, and adeno-associated viruses to efficiently deliver genes. Non-viral methods include mechanical techniques like electroporation, microinjection, and biolistics (gene gun), as well as chemical methods like liposomes, calcium phosphate, and polyethylene glycol. Each method has advantages and limitations for different applications in research and potential gene therapy.
This document summarizes research on chloroplast engineering for various applications. Chloroplasts naturally contain their own DNA and can be genetically engineered via homologous recombination. This allows for high levels of transgene expression without gene silencing effects. The document discusses how chloroplasts have been engineered for herbicide resistance, pathogen resistance, drought tolerance, and production of recombinant proteins. While chloroplast engineering holds promise, limitations include lack of expression in non-green cells and full genome sequence information for some species.
This document outlines a two-week summer course on bioprocess engineering and biofactories held in July 2010 in Malaysia. The course objectives are to provide a broad overview of the science and technology of bioprocess research and industries. The course will cover topics such as bioprocess design, operation, scaling up, facility design requirements and regulations. The schedule includes lectures and practical sessions the first week on bioprocess topics and the second week on facility design. Practical sessions involve inoculum preparation, shake flask culture, bioreactor set-up, calibration, operation, and fed-batch cultivation design for recombinant protein production.
Plants can be used as bioreactors to produce valuable compounds. Transgenic plants and plant cell cultures can produce large quantities of proteins, vaccines, and other molecules through biochemical reactions using techniques like genetic engineering. Some key advantages of plant bioreactors are that they are cost-effective, can produce high biomass, and allow storage of products for a long time. However, differences in plant and bacterial genetics can impact expression efficiency and safety testing is required.
☺INTRODUCTION
☺Bt COTTON
☺MAJOR PESTS OF COTTON
☺MODE OF ACTION OF Bt GENE
☺ADVANTAGES
☺DISADVANTAGES
☺CONCLUSION
☺REFERENCES
Genetically modified variety of cotton that produces an insecticide whose gene has been derived from a soil bacterium called Bacillus thuringiensis (Bt).
Three types of toxins.
A total of 229 cry toxins ( cry1Aa to Cry72Aa), cyt toxins ( cyt 11Aa to cyt3Aa) and 102 vip toxins( vip1Aa1 to vip4Aa1) have been discovered.
Directed enzyme evolution is a technique that mimics natural selection to engineer proteins. It involves introducing random mutations into genes and screening proteins for modified activity. The key steps are selecting a gene, creating a library of mutant genes through error-prone PCR or other mutagenesis methods, expressing the proteins, and selecting variants with improved properties. Examples where directed evolution has been applied include improving the activity of enzymes used in producing the antibiotic cephalosporin and in the cholesterol-lowering drug atorvastatin. The goal is to leverage natural selection to develop enzymes with desired industrial applications like increased stability, activity, or substrate specificity.
The document discusses different expression vectors and systems used for recombinant protein expression. It describes key elements required for an expression vector including an origin of replication, selective marker, promoter, multiple cloning site, and terminator. It provides details on commonly used expression systems in E. coli such as the lac, tac, lambda PL, and T7 promoters. It also summarizes protein expression in yeast using the galactose-inducible GAL promoter system.
Strategies to enhance production of secondary metabolitesPrashant Singam
1. Strategies to enhance production of secondary metabolites in plant cell cultures include selecting high-productivity cell lines, using elicitors to boost productivity over short periods, and immobilizing cells to improve yields of extracellular metabolites.
2. Elicitors are compounds that induce plant cells to produce more secondary metabolites in response to stress and include fungal or bacterial extracts, polysaccharides, and physical/chemical agents like UV radiation or heavy metals.
3. Immobilizing plant cells by entrapping them in matrices like alginate or polyacrylamide anchors the cells while allowing continuous production and release of metabolites into the medium.
This ppt explains about molecular farming, history of molecular farming, importance, basic process underlying it, its application in agriculture and its limitations
This document summarizes a seminar presentation on improving fruit quality through the use of elicitors and bio-molecules. The presentation outlines the seminar topics which include introduction to elicitors and bio-molecules, their mechanisms of action, and case studies on their use in pomegranate and citrus fruits. It also summarizes results from studies showing elicitors can increase phenolic content and bioactive compounds in fruits, thereby improving quality attributes such as color, firmness, and reducing decay during storage.
Gene tagging uses recognizable DNA fragments like T-DNA or transposons to disrupt gene function and identify genes responsible for mutant phenotypes. T-DNA tagging in plants involves random integration of Agrobacterium T-DNA that can disrupt genes and create mutants. Transposon tagging relies on the ability of transposons to move within genomes and disrupt gene function. Both techniques have been used successfully to isolate numerous plant genes involved in traits like color and development.
This document discusses hairy root cell culture. Hairy root culture involves infecting plant explants with the soil bacterium Agrobacterium rhizogenes, which transfers genes to the plant genome and causes roots to form with increased cell division and elongation, producing "hairy roots". Hairy roots have properties like genotype/phenotype stability, fast growth, and high production of secondary metabolites. The process involves wounding explants, inoculating with A. rhizogenes, inducing hairy roots within 1 week to 1 month, and subculturing in antibiotic media to remove bacteria. Hairy roots can be cultured in various bioreactors and have applications like gene analysis, protein expression, and secondary metabolite production.
Plant transformation vectors and their typesZahra Naz
This document summarizes a presentation on plant transformation vectors and their types. It discusses various types of vectors used for plant transformation including plasmids, viruses, bacteriophages, and cosmids. Plasmids are the most commonly used vector for plant transformation. Agrobacterium-mediated transformation using tumor-inducing (Ti) plasmids is an effective method for genetically transforming plants. Viral vectors like cauliflower mosaic virus (CaMV) are also used but have certain limitations.
It is a part of Ti Plasmid which takes part intransfer and integration of T-DNA into plant chromosome.
The vir sequence consist of 8 operons which take part in different functions associated with virulence of Ti Plasmid. These are vir H, vir A, vir B, vir G, vir C, vir D, vir E, & vir F. ( vir H & vir F present occasionally).
The document discusses several genetically engineered plants including Bt crops, Golden Rice, and Flavr Savr tomato. Bt crops contain a gene from Bacillus thuringiensis that produces a toxin harmful to certain insects, protecting the plant. Golden Rice was engineered to produce beta-carotene in the endosperm to address vitamin A deficiency. Flavr Savr tomato was modified using antisense RNA technology to reduce polygalactouronase levels and slow fruit softening for a longer shelf life.
Gene transfer technologies can be used to treat diseases by inserting therapeutic genes into cells. There are viral and non-viral methods of gene transfer. Viral methods use viruses like retroviruses, adenoviruses, and adeno-associated viruses to efficiently deliver genes. Non-viral methods include mechanical techniques like electroporation, microinjection, and biolistics (gene gun), as well as chemical methods like liposomes, calcium phosphate, and polyethylene glycol. Each method has advantages and limitations for different applications in research and potential gene therapy.
This document summarizes research on chloroplast engineering for various applications. Chloroplasts naturally contain their own DNA and can be genetically engineered via homologous recombination. This allows for high levels of transgene expression without gene silencing effects. The document discusses how chloroplasts have been engineered for herbicide resistance, pathogen resistance, drought tolerance, and production of recombinant proteins. While chloroplast engineering holds promise, limitations include lack of expression in non-green cells and full genome sequence information for some species.
This document outlines a two-week summer course on bioprocess engineering and biofactories held in July 2010 in Malaysia. The course objectives are to provide a broad overview of the science and technology of bioprocess research and industries. The course will cover topics such as bioprocess design, operation, scaling up, facility design requirements and regulations. The schedule includes lectures and practical sessions the first week on bioprocess topics and the second week on facility design. Practical sessions involve inoculum preparation, shake flask culture, bioreactor set-up, calibration, operation, and fed-batch cultivation design for recombinant protein production.
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.
This document provides an overview of bioprocessing and industrial biotechnology. It discusses the history and milestones of the industry from ancient times to present. Key topics covered include major industrial fermentation products, stages of development from 1900 to today, microbial cell bioprocessing, scaling up processes from lab to production scale, and the types of bioreactors used to produce products from mammalian, plant, insect, algal and bacterial cells. The document also briefly outlines considerations for media composition, cultivation conditions, process optimization and control, and the future potential of industrial bioprocessing.
The document provides guidelines for Good Manufacturing Practice (GMP) for processing raw-unclean and raw-clean edible-birdnest (EBN) in Malaysia. It covers specifications for raw materials, production processes like sorting and cleaning, facility requirements, safety controls, personnel hygiene, training and legal compliance. The standard aims to ensure the production of quality and safe EBN for human consumption.
Lecture 5 bioprocess technology, operation mode and scaleDr. Tan Boon Siong
This document discusses different bioprocess cultivation systems and operation modes. It covers two-phase and three-phase cultivation systems, as well as free and immobilized cell systems. Batch, fed-batch, and continuous cultivation modes are described in detail. Specific topics covered include microbial growth curves, factors affecting lag phase, kinetics of exponential and stationary phases, and product formation under different operation modes. Advantages of fed-batch cultivation like avoiding inhibition and catabolite repression are highlighted. High cell density cultivation using exponential feeding strategies is also summarized.
This document provides an overview of medium formulation for microbial growth. It discusses the basic requirements including carbon, nitrogen, mineral and vitamin sources. Key factors that affect medium design are described such as pH, temperature, oxidation-reduction potential and water activity. The document outlines different types of media including defined, complex, and industrial formulations. Overall, the document offers a comprehensive overview of the nutrients, environmental conditions, and considerations for optimizing microbial growth media.
The document analyzes the pigments and antioxidant properties of red dragon fruit (Hylocereus polyrhizus).
High Performance Liquid Chromatography analysis identified betanin as the main pigment contributing to the fruit's deep purple color. Antioxidant assays found high levels of polyphenols (86.1 mg/0.5 g) and flavonoids (2.3 mg/g), as well as strong reducing power and DPPH radical scavenging activity, indicating dragon fruit has significant antioxidant activity. The results confirm betanin is the primary pigment in dragon fruit and that the fruit contains high levels of antioxidants with potential health benefits.
Since 1957, Lord was involved in swiflet research. Till todate, more of his works has not been shared. This is the opportunity for those interested to share his works...
This document provides standards for good animal husbandry practices for edible-birdnest swiftlet ranching and its premises in Malaysia. It covers requirements for identification and record keeping, animal welfare, breeding, health care, hygiene, facilities and the environment. The standards are intended to ensure the continuous and sustainable production of edible bird's nests while protecting the health, safety and welfare of the birds and operators, and preventing environmental degradation. It includes minimum housing requirements and addresses issues like water, feeding, handling, nest building and weaning of the swiftlets. Requirements for disease prevention, treatment and reporting are also specified.
This document discusses various types of bioreactors and their key properties and design considerations. It covers topics like:
1) Desirable properties of bioreactors include simplicity of design, continuous operation, large number of organisms, and uniform distributions of oxygen and microorganisms.
2) Common bioreactor types include stirred tank, airlift, packed bed, and immobilized cell bioreactors.
3) Important design considerations for bioreactors include agitation and mixing, aeration, mass transfer, power requirements, and fluid rheology which can be Newtonian or non-Newtonian.
The document describes the process that birds' nests undergo to be cleaned and prepared for human consumption. Workers remove feathers, dirt and other debris by hand. The nests are then soaked and bleached to remove any remaining particles and turn the nests white. No chemicals are used in this process. Finally, the cleaned nests are sorted by grade before being dried and packaged for sale.
This document reviews the role of bacterial extracellular polysaccharides in biofilm formation. It discusses how extracellular polymeric substances (EPS) produced by microorganisms form the matrix of microbial aggregates and biofilms. EPS are involved in the initial attachment of cells to surfaces and provide protection from environmental stresses. The production of EPS is regulated by quorum sensing and helps mediate processes like bioremediation and bioleaching that are important in industrial applications.
1) The document discusses various types of cell growth and division, including binary fission in bacteria, budding in yeast cells, and the eukaryotic cell cycle.
2) It also covers factors that regulate cell growth in mammalian cells and yeast, such as nutrient availability and protein complexes that control translation and cell division.
3) Methods for measuring bacterial growth are described, such as direct counts, plate counts, optical density, and analyzing nutrient uptake and product formation over time. Models for bacterial growth kinetics and the calculation of specific growth rates are also presented.
This document discusses bioprocess control for cell cultivation systems. It covers various parameters that are measured for control, including cell inputs and outputs, substrate levels, oxygen, carbon dioxide, temperature, pH, dissolved oxygen, and foam. Sensors used for online measurement of these parameters in bioreactors are also outlined. The document then describes basic feedback loops and controllers for bioprocess control, including PID and model predictive control. It concludes with an overview of using a supervisory control and data acquisition (SCADA) system connected over Ethernet for monitoring and controlling bioreactor systems.
1. Sterilization eliminates all microorganisms including bacteria, viruses and endospores. Disinfection only eliminates pathogenic microorganisms.
2. Heat is the most common sterilization method and can be applied through moist heat like autoclaving or dry heat like oven heating. Chemical sterilization uses agents like phenols, alcohols, halogens, heavy metals and aldehydes to disrupt microbial membranes and proteins.
3. Other sterilization methods include filtration, irradiation using gamma rays, x-rays or UV light, and gaseous agents like ethylene oxide and hydrogen peroxide which penetrate materials to kill microbes.
This document discusses animal cell culture and its applications. It provides information on:
1) The uses of animal cell culture including production of recombinant proteins, monoclonal antibodies, and cell biology studies.
2) Characteristics of animal cell culture compared to microorganism culture, including lower growth rates and productivity.
3) Products that can be produced from mammalian cell cultures including cells, cell-derived products, and recombinant glycoproteins.
4) Types of animal cells commonly used in culture including epithelial, fibroblast, muscle, and blood/lymph cells.
Biotechnological aspects of product developmentNaveed Sarwar
This document discusses biotechnological product development. It explains that biotechnology uses biological systems to develop technologies and products for improving quality of life. Biotech drugs, also called biopharmaceuticals, are manufactured using living expression systems and differ physically from small molecule drugs. The development process involves isolating a gene of interest, transferring it to an expression vector, transforming host cells, growing the cells, and purifying the protein product. Quality control measures like sterility testing and viral/bacterial decontamination are crucial given biotech drugs cannot be sterilized as end products.
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 bio 319 lecture 6 on biotechnology production of antibiotics. It covers using recombinant DNA technology to produce new structurally unique antibiotics with increased activity, decreased side effects and cost. Streptomyces is commonly used as it produces mycelial filaments. Genes are isolated using complementation and mutant cells transformed with gene libraries. Polyketide antibiotics are synthesized like fatty acids through enzymatic condensation. Engineering production involves studying and manipulating biosynthetic pathway enzymes. Examples provided are modifying erythromycin production. Bioterrorism agents discussed include anthrax, plague, tularemia and botulism. Strategic stockpiling of antibiotics and emergency response plans are also covered.
This document provides an overview of the course contents for Biochem-700, a biochemistry course at the M.Phil/M.Sc level. The course covers topics such as the introduction and applications of biochemistry, cell structure and types, membrane transport, enzymes, metabolism, genetics, proteomics, and nanobiotechnology. It also provides definitions and comparisons of key concepts like prokaryotic and eukaryotic cells, the domains of life, aerobic and anaerobic respiration, probiotics and prebiotics, and plasma membrane structure. Recommended textbooks are also listed.
Biomolecules (Mainly Carbohydrates, Proteins, Lipids and Nucleic Acids ) Production form Microorganisms and their Industrial applications were discussed....
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 document discusses the physiology and metabolism of bacteria. It explains that bacteria metabolize organic and inorganic substrates to generate energy through catabolic pathways, while using this energy for anabolic pathways to synthesize cellular components. The four main components of bacterial cells are water, organic matter like proteins and carbohydrates, and inorganic minerals. Bacteria are classified based on their nutritional requirements, oxygen usage, and optimal temperature for growth. Enzymes play a key role in bacterial metabolism by catalyzing biochemical reactions. Bacterial growth occurs through binary fission and follows a characteristic growth curve with lag, logarithmic, stationary, and death phases.
This document discusses the physiology and metabolism of bacteria. It explains that bacteria metabolize organic and inorganic substrates to generate energy through catabolic pathways, while using this energy for anabolic pathways to synthesize cellular components. The four main components of bacterial cells are water, organic matter like proteins and carbohydrates, and inorganic minerals. Bacteria are classified based on their nutritional requirements, oxygen usage, and optimal temperature for growth. Enzymes play a key role in bacterial metabolism by catalyzing biochemical reactions. Bacterial growth occurs through binary fission and follows a characteristic growth curve with lag, logarithmic, stationary, and death phases.
This document summarizes Michael Buschmann's work on nanomedicine at Ecole Polytechnique. It discusses how nanomedicine uses nano-sized tools for diagnosis, prevention and treatment of disease. Some key applications of nanomedicine include drug delivery via liposomes and polymeric nanoparticles. The document also outlines the requirements for successful nanomedicine research and development, including efficacy, safety, manufacturing and regulatory approval. Buschmann's group works on developing chitosan-based nanoparticles for gene delivery applications.
This document provides an overview of biotechnology, including its definitions, history, techniques, and applications. It discusses how biotechnology has evolved from ancient uses of microbes in fermentation to modern applications of genetic engineering. Key areas of biotechnology addressed include medicine, agriculture, forestry, and environmental management. The document also examines some societal and ethical issues raised by biotechnology developments.
1) Biopharmaceuticals are proteins or nucleic acids produced through biotechnology rather than direct extraction. The production process involves identifying the gene of interest, developing a host cell, producing and purifying the protein, and formulating the final product.
2) Key steps include establishing a cell bank, growing the cells through fermentation, disrupting the cells, purifying the protein through techniques like centrifugation, chromatography, and concentrating the product.
3) The final product is then formulated, filtered, and packaged while ensuring proper storage and handling to maintain stability and biological activity until use. The production process is carefully controlled and monitored to produce consistent, safe and effective biopharmaceutical drugs.
This document discusses the extraction of proteases from proteolytic bacteria and their industrial applications. It begins with an introduction to proteases and their catalytic properties. Key steps to extract proteases from bacteria are described, including culturing the bacteria, harvesting cells, disrupting cells, extracting and purifying the proteases. Methods to improve protease yield are also summarized. The document concludes by outlining several major industrial applications of proteolytic enzymes, such as in detergents, waste treatment, pharmaceuticals, food processing and more.
This document discusses topics related to industrial biotechnology including fermentation products, microorganisms used in fermentation, and historical and future applications of industrial biotechnology. It provides classifications of microorganisms including prokaryotes and eukaryotes. Details are given on bacterial cell structure, essential and non-essential components. Methods for classifying bacteria such as gram stain and morphological characteristics are also summarized.
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.
Bioprocessing uses living cells or their components to produce foods, pharmaceuticals, flavors, and other products. It involves the growth of microorganisms, plant, or animal cells in a bioreactor using a biocatalyst. Downstream processing is then required to remove impurities, concentrate the product, and reduce volume. Bioprocessing provides opportunities in areas like biopharmaceuticals, gene therapy, biopesticides, transgenic organisms, renewable energy, environmental applications, and more. It is an important field for addressing problems in healthcare, agriculture, industry, and the environment through biologically-based manufacturing processes.
Plants can be used as bioreactors to produce valuable products through biochemical reactions. Transgenic plants and plant cell cultures allow for large-scale, low-cost production of recombinant proteins, vaccines, antibodies, and other pharmaceuticals using genetic engineering techniques. Common types of plant bioreactors include seed-based systems, hairy root cultures, suspension cultures, and chloroplast systems, each offering different advantages for stable recombinant protein expression and storage. While plant bioreactors provide cost-effective production of various products, there are also some disadvantages such as less efficient protein expression than microbial systems and potential safety and environmental concerns.
This document discusses biopolymers and biomaterials, including definitions of biopolymers as renewable and sustainable polymers derived from biological sources like carbohydrates, proteins, lipids, and nucleic acids. Key properties and applications of common biopolymers like carbohydrates, proteins, and lipids are described. The document also provides an overview of biomaterials, their types and properties, as well as guidelines for evaluating biocompatibility.
This document summarizes a seminar presentation on using transgenic plants as bioreactors. Some key points:
- Genetically modified plants can be engineered to produce important compounds like carbohydrates, lipids, and proteins for industrial and medical use.
- Transgenic plants have potential as bioreactors because genetic manipulation is easy, and there is no risk of human pathogen contamination. Plant cells are a good host for molecular production, especially proteins.
- Compared to other production systems, plant bioreactors have lower costs, allow post-translational modification, and require less infrastructure than microbial fermentation.
- Examples of biomolecules that can be produced in transgenic plant bioreactors include cyclodextrins, vaccines
This document discusses the production of recombinant therapeutic proteins. It outlines three main methods: microbial bioreactors like E. coli, mammalian cell culture bioreactors like CHO cells, and transgenic animal bioreactors. Transgenic animals are produced via DNA microinjection into embryos to incorporate expression vectors for target proteins. Their milk can then produce large quantities of complex proteins through scale-up. While advantageous for production scale, transgenic systems have limitations regarding animal health effects and post-translational modifications. Examples of therapeutic proteins produced include antithrombin in transgenic goats and alpha-1-antitrypsin in transgenic sheep.
Similar to Lecture 3 biofactories in the biotechnology industry – introduction(2) (20)
The document discusses the history and production of bird's nests in Southeast Asia. It details that bird's nests have been consumed for over 1000 years in China and were an expensive luxury item. Today, Malaysia is a major producer of bird's nests, which are created from the saliva of swiftlets for building nests. There are two main types of nests - cave nests found naturally and house nests built in man-made structures. The document also outlines traditional and improved processing methods for cleaning bird's nests and analyzes their nutritional composition and health benefits.
UGSOLAR TECHNOLOGY SDN BHD is a solar energy company that has been operating since 1997 in Taiwan and has since expanded to China, Malaysia, Vietnam, Cambodia, and Bangladesh. They offer a wide range of solar products and systems including micro solar lighting kits, mini solar power kits, solar home systems, solar water pumps, and solar surveillance units. Their hybrid solar power systems combine solar power with battery storage and backup generators to provide reliable off-grid power for telecom towers and other applications.
Palm oil production has significantly contributed to global vegetable oil supply, with Malaysia and Indonesia being major exporters. Palm oil cultivation uses less land than other oilseed crops to produce higher yields, making it more sustainable. The palm oil industry in Malaysia has adopted various green technologies over the past two decades such as zero burning practices and integrated pest management to reduce environmental impacts. Palm oil biomass is also being utilized through applications like power generation and waste treatment to further improve sustainability.
1) The document analyzes the thermal degradation of three natural polymers - sodium hyaluronate, xanthan, and methylcellulose - using thermogravimetric analysis and infrared spectroscopy.
2) The results show sodium hyaluronate and xanthan, which are charged polysaccharides, have lower thermal stability than the neutral polysaccharide methylcellulose.
3) Kinetic parameters like activation energy were determined using the Ozawa and Freeman-Carroll methods, which suggest changes in the degradation mechanism with increasing mass loss. Activation energies are generally higher for methylcellulose indicating greater thermal stability.
This document summarizes research on the effects of gamma irradiation on the viscoelastic properties of sodium alginate polysaccharides. The researchers found that:
1) Irradiating sodium alginate solutions decreased their apparent viscosity and consistency, suggesting the gamma rays broke down the macromolecular structure.
2) Higher irradiation doses and lower polysaccharide concentrations led to greater decreases in viscosity.
3) The non-Newtonian, pseudoplastic behavior of the solutions was maintained after irradiation, though trends moved toward Newtonian behavior at higher doses.
The document summarizes a study on the thermal degradation of three natural polymers: sodium hyaluronate, xanthan, and methylcellulose. Thermogravimetric analysis showed sodium hyaluronate and xanthan had lower thermal stability than methylcellulose. Kinetic parameters like activation energy were determined using the Ozawa and Freeman-Carroll methods, which suggested changes in degradation mechanism with mass loss. Activation energies from Freeman-Carroll were higher, accounting for thermal history effects. Infrared spectroscopy observed scission of side groups at low temperatures and backbone links at high temperatures, agreeing with kinetic parameters.
The document outlines the Credit Accumulation and Transfer Scheme (CATS) used by the university. It discusses (1) how credits are weighted and equated to student learning hours, (2) how modules are assigned levels, (3) the credit structures required for different qualifications, (4) assessment methods, (5) how qualifications can be awarded, and (6) limits on credit that can be transferred from other institutions.
This document contains technical data for an unknown purpose. It includes a string of characters that may be an identifier but does not seem to contain any clear language, titles, topics or other summarizable information.
Brand Guideline of Bashundhara A4 Paper - 2024khabri85
It outlines the basic identity elements such as symbol, logotype, colors, and typefaces. It provides examples of applying the identity to materials like letterhead, business cards, reports, folders, and websites.
Andreas Schleicher presents PISA 2022 Volume III - Creative Thinking - 18 Jun...EduSkills OECD
Andreas Schleicher, Director of Education and Skills at the OECD presents at the launch of PISA 2022 Volume III - Creative Minds, Creative Schools on 18 June 2024.
🔥🔥🔥🔥🔥🔥🔥🔥🔥
إضغ بين إيديكم من أقوى الملازم التي صممتها
ملزمة تشريح الجهاز الهيكلي (نظري 3)
💀💀💀💀💀💀💀💀💀💀
تتميز هذهِ الملزمة بعِدة مُميزات :
1- مُترجمة ترجمة تُناسب جميع المستويات
2- تحتوي على 78 رسم توضيحي لكل كلمة موجودة بالملزمة (لكل كلمة !!!!)
#فهم_ماكو_درخ
3- دقة الكتابة والصور عالية جداً جداً جداً
4- هُنالك بعض المعلومات تم توضيحها بشكل تفصيلي جداً (تُعتبر لدى الطالب أو الطالبة بإنها معلومات مُبهمة ومع ذلك تم توضيح هذهِ المعلومات المُبهمة بشكل تفصيلي جداً
5- الملزمة تشرح نفسها ب نفسها بس تكلك تعال اقراني
6- تحتوي الملزمة في اول سلايد على خارطة تتضمن جميع تفرُعات معلومات الجهاز الهيكلي المذكورة في هذهِ الملزمة
واخيراً هذهِ الملزمة حلالٌ عليكم وإتمنى منكم إن تدعولي بالخير والصحة والعافية فقط
كل التوفيق زملائي وزميلاتي ، زميلكم محمد الذهبي 💊💊
🔥🔥🔥🔥🔥🔥🔥🔥🔥
Creative Restart 2024: Mike Martin - Finding a way around “no”Taste
Ideas that are good for business and good for the world that we live in, are what I’m passionate about.
Some ideas take a year to make, some take 8 years. I want to share two projects that best illustrate this and why it is never good to stop at “no”.
Lecture 3 biofactories in the biotechnology industry – introduction(2)
1. Biofactories in the OUTLINE
Biotechnology Industry • Introduction - Biotechnology
• Biomolecules
• From Gene to Product (Protein)
From Gene to Bioproducts
• Recombinant DNA Technology
Bioprocess Engineering Workshop
Prof. S. T. Yang
Dept. Chemical & Biomolecular Eng
The Ohio State University
Biotechnology Pharmaceuticals
Industrial Markets for Biotechnology
drugs, healthcare
• Pharmaceutical industry - over $400 billion
A Diagnostics
P • Agriculture and food industry
P Human Biomedical – transgenic crops, animals
artificial organs, body parts
L – recombinant bovine somatotropin (BSA)
I Plant tissue cultures, • Chemical industry – over $2 trillion on sales worldwide
C Transgenic plants – commodity chemicals, specialty chemicals, consumer
A care products, and pharmaceuticals; plastics/polymers
Agriculture
T Transgenic animals – uses nearly $24 billion worth of hydrocarbon feedstocks
I annually in the US.
O Biochemicals • Fuel and energy
N
S Industrial – 4.5 billion gallons of ethanol from corn in 2006
Environment
pollution control – 75 million gallons of biodiesel from soybean oil in 2005
1
2. Biotechnology Cell
What is biotechnology?
“Biotechnology, broadly defined, includes any technique
that uses living organisms (or parts of organisms) to make
or modify products, to improve plants or animals, or to
develop microorganisms for specific uses.”
-- Office of Technology Assessment, 1984
Biotechnology is “the integrated use of biochemistry,
microbiology, and engineering sciences in order to achieve
technological (industrial) applications of the capabilities of
microorganisms, cultured tissue cells, and parts thereof”
DNA account for about 0.25% cell weight for a typical mammalian cell and
-- E u r o p e a n F e d e r a t i o n o f B i o t e c h no lo gy , 1 98 5 1% for a typical bacterial cell.
Biomolecules Carbohydrates
• Carbohydrates • Contains oxygen, hydrogen and carbon atoms,
and no others. Some not
• Proteins • (CH2O)n or derivations
• Function as storage and transport form of
• Lipids energy
• Classified based on the number or sugar units:
• Nucleic acids Monosaccharide, Disaccharides,
Oligosaccharides, and Polysaccharides
2
3. Protein (Polypeptides) Sizes
• Discovered in 1838 by Jons Berzelius • Total molecular mass in daltons or kDa
• From Greek, protas: of primary importance
• Yeast proteins average 466 a.a. and 53
• Consists of amino acids arranged in a
linear chain linked by peptide bonds kDa in mass.
• 20 standard amino acids • The largest known protein are the titins, a
• Plants and microorganisms can synthesize component of muscle sarcomere, MM of
all the 20 a.a., animal cannot 3,000 kDa and 27,000 a.a.
• Essential amino acids from diet
Classification Functions of Proteins
• Globular proteins • Enzymes: catalyze biochemical reactions,
– Most are soluble metabolism and biosynthesis
– Mostly enzymes, antibodies, hormones • Cell cytoskeleton, i.e. scaffolding, ECM
• Fibrous proteins
• Cell signaling (i.e. immune response, cell
– Structural
adhesion, cell cycles)
• Membrane proteins
• Antibody
– Mostly in cell membranes
– Receptors • Denaturation; Folding
– Channels
3
4. Biosynthesis of Proteins
Central Dogma: DNA mRNA Protein
• Transcription: gene sequence mRNA
• Translation: mRNA amino acids:
– Codons – same for both prokaryotes and
eukaryotes, but with different frequencies
• Post translational modifications
– Different between prokaryotes and eukaryotes
Protein Structures
• Primary structure: a.a. sequence
– N terminus
– C terminus
• Secondary structure: local structures
stabilized by hydrogen bonds
– Alpha helix
– Beta sheet
– Random coil
Alpha Helix
4
5. Protein Structures (cont’d)
• Tertiary Structure (fold): overall shape of a
single protein molecule
– Stabilized by nonlocal interactions
– Hydrophobic core
– Salt bridges
– Hydrogen bonds
Parallel Anti parallel – Disulfide bonds
– Post-translation modification
Beta Sheet
Protein Structures (cont’d) N terminus
Protein Structures
• Quaternary Structure: interactions of more
than one protein molecules.
– Protein subunits
– Protein complex
– Active sites
• Monomer, dimer, trimer, tetramer, etc.
C terminus
5
6. LIPID Characteristics
• Hydro-carbon based biomolecules that are
• Ampiphilic/ampiphatic
hydrophobic (some are amphiphilic or
amphiphatic) • Alipathic/aromatic
• Water insoluble and soluble in nonpolar
organic solvents • Cyclic/acyclic
• Consists of triglycerides • Branched/straight
• Possess a broad and diverse range of
structures • Flexible/rigid
• Alipathic or aromatic
Chromosome
The genome size or
C (constant)-value of
an organism is
defined as the total
DNA & RNA amount of DNA
contained
within its haploid
chromosome set.
Genome sizes vary dramatically among species. Current eukaryotic genome sizes
are known to vary by more than five orders of magnitude; the genome of Amoeba
dubia is roughly 200 times larger than that of humans and more than 200000 times
larger than that of the microsporidium Encephalitozoon cuniculi.
6
7. DNA Structure
DNA: Deoxyribonucleic acid
Units of DNA: nucleotides,
Each nucleotide consists of:
a deoxyribose,
• Right handed double helix a nitrogen containing base,
a phosphate group.
• 3.4 nm per helical turn
• 2 nm diameter for the helical width
• 10 base pairs per turn
• Two polynucleotide chains
• Running in opposite directions
• Held together by Hydrogen bond
between base pairs Phosphodiester
bond
This structure was first described by James Watson and Francis Crick in 1953.
RNA
• Messenger RNA (mRNA): bound to ribosomes
and translated to protein with the help of tRNA
• Transfer RNA (tRNA): a small RNA chain of 74-
Pyrimidine ring Purine ring
95 nucleotides that transfers a specific amino
acid to a growing polypeptide chain at ribosomal
site
• Ribosomal RNA (rRNA): catalytic component of
the ribosomes, abundant, at least 80% of the
RNA molecules in a typical eukaryotic cell
GC pair has 3 Hydrogen bonds AT pair has 2 hydrogen bonds
7
8. Characteristics DNA RNA
Structural differences 1. Deoxyribonucleic acid ✔
between DNA and RNA 2. Ribonucleic acid ✔
molecules: 3. Ribose sugar present ✔
4. Deoxyribose sugar present ✔
RNA has OH group at 2'
position vs. DNA has only a 5. It’s sugar is linked to a phosphate group at one end and a ✔ ✔
nitrogenous base at the other end
hydrogen
6. Polymer of nucleotides ✔ ✔
RNA bases are A, U, G,
7. Nitrogenous bases:
C, while DNA bases are A,
Adenine (A) present ✔ ✔
T, G, C.
Thymine (T) present ✔
Uracil (U) present ✔
Cytosine (C) present ✔ ✔
Guanine (G) present ✔ ✔
8. Two (2) chains held in a double helix by hydrogen bonds ✔
9. Single-stranded ✔
10. Self-replication and transcription. ✔
DNA lacks the 2'-OH and will not be hydrolyzed. Thus, it is a more stable
•
11. Translation and reverse transcription. ✔
polymer and better suited for storage of genetic information.
Creation or Evolution ? From Gene (DNA) to
Gene Products (Proteins)
• Chance to synthesize one small protein:
– 1 Polypeptide with 30 amino acids
Cost of Synthetic Peptides
– 90 base pairs in DNA sequence
– Possible combination: 490 = 1.5 x 1054 Peptide No of amino Cost / gram
(Ohio Super Lotto: 456 = 8.3 x 109 ) acids
– Probability for each mutation: 10-6 Amino acid 1 $ 0.05
– Cell (E. coli) doubling time: 30 minutes (0.5 hrs) Aspartame 2 $ 0.55
Brodykinin 9 $ 1,600
– Time required for evolution:
Leutinizing releasing 10 $ 2,000
(0.5 hrs)(106)(1.5x1054) = 7.5 x 1059 hrs = 8.56 x 1055 years
hormone (LRH)
>>> earth age ? Beta-Endorphin $20,000
31
8
9. Recombinant DNA Technology
Bioprocessing Applications
Recombinant DNA Technology
• Produce gene products (human proteins) not normally
found in host cells (microorganisms)
• Construct novel biochemical pathways in cells for:
– wider substrate utilization
– small molecule (e.g., antibiotics) modification
• Increase gene dosage and concentration of gene products -
increase metabolic rate
• Increase product yield
• Put interested gene under control of known regulatory
mechanisms
• Utilize gene products with special physicochemical
properties (e.g., thermostability)
r-DNA Technology:
Protein Synthesis some website references
• http://present.smith.udel.edu/biotech/rDNA.html (This one has very
good introductory movie/animation on rDNA technology that would
be fun for you to watch)
• http://www.bio.miami.edu/dana/250/25003_10print.html
• http://cwx.prenhall.com/horton/medialib/media_portfolio/23.html
• http://www.biology.arizona.edu/molecular_bio/problem_sets/Recom
binant_DNA_Technology/recombinant_dna.html
• http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/R/Recombin
antDNA.html
9
10. Genetic Engineering Genetic Engineering
Biosynthetic processes from gene to protein
• Mutation - point mutation: 10-6
• DNA Replication
• Sexual Hybridization - Meiosis in DNA DNA
eukaryotes • RNA Transcription
• Parasexual Processes - – mRNA modification (eucaryotic cells)
RNA
– Conjugation - cell to cell contact • Protein Translation
– Transduction - phage • Post-translational modifications Protein
– Transformation - plasmid – N-terminal Methionine removal Central Dogma
• Mitotic recombination - filamentous fungi – Disulfide bond between 2 Cysteines
• Protoplast Fusion – Pre-Pro-Protein
– Glycosylation
• Cell Fusion - Hybridoma
Recombinant DNA Technology Recombinant DNA Technology
Four Main Steps in Cloning Strategies of Cloning
• How to get the foreign genetic code (DNA
• Obtaining DNA
sequence) for the protein product?
fragments (genetic
– Direct cloning
code)
– Indirect cloning - Complimentary DNA technique
• Joining to vector – PCR
• Introduction to host – Chemical DNA synthesis
cells ♦ Human genome - 46 chromosomes
• Selection of mutant – cut with EcoRI - 700,000 DNA fragments
10
11. Synthesis of cDNA Polymerase Chain Reaction (PCR)
The steps in the preparation of
cDNA are to: (1) copy the mRNA
using reverse transcriptase and a
primer with dNTPs (2) digest with
RNase H to nick the mRNA bound
to DNA (3) add polymerase I, which
will carry out nick translation,
The sequence to be amplified is shown in blue. (1) The duplex
removing the RNA with its 5'-->3'
DNA is melted by heating and cooled in the presence of a large
exonuclease activity. (4) Finally, the
excess of two primers (red and yellow) that flank the region of
double stranded DNA is cut with
interest. (2) A heat-stable DNA polymerase catalyzes extension
restriction endonucleases to
of these primers, copying each DNA strand. Successive cycles
generate the "sticky ends" needed to
of heating and cooling in the presence of the primers allow the
insert the DNA into a vector.
desired sequence to be repeatedly copied until, after 20 to 30
cycles, it represents most of the DNA in the reaction mixture.
Construction of Genomic DNA Library
Screening DNA Library
Colonies of cells containing recombinant molecules are grown
on petri plates. A replica of the colonies is made by overlaying
a filter disk on the plate. DNA and protein are released from
the cells in situ and immobilized to the filter. The filter is then
incubated with labeled probe under conditions in which the
probe specifically recognizes the desired DNA or protein.
To generate additional copies of the fragment. After nonspecifically bound probe is washed away, specifically
bound probe is detected by a method appropriate for the label
Before PCR, this was a key mechanism for (in this case, autoradiography). Duplicate filters are used to
amplifying fragments of DNA. A "restriction distinguish false positives from true positives. By aligning the
fragment" is a portion of the genome generated by filters with the original plates, cells containing the recombinant
of interest can be identified.
digestion with restriction endonucleases.
11
12. Chromosome Walking Recombinant DNA Technology
How to Join DNA Fragment to Vector
• Homopolymer tailing
• Ligation of cohesive termini produced by
restriction endonucleases
• Blunt-end ligation (no linker)
• Linker molecules
The restriction endonuclease sites (indicated by vertical arrows) are mapped on the starting recombinant. Based on this map, the terminal
fragment (1) of the starting recombinant is isolated and used to probe a genomic library. The recombinants that hybridize to this fragment are
restriction-mapped to identify the recombinant that extends furthest into the region of the chromosome adjacent to the first recombinant. The
process is then repeated, using the restriction fragment furthest removed from the starting recombinant as the next probe.
Recombinant DNA Technology Restriction Fragment Length
Restriction Enzymes Polymorphisms (RFLPs)
• Restriction endonucleases: e.g. EcoR1,
BamH1, HindIII, etc.
– sticky ends
– blunt ends
• DNA ligase
• DNA polymerase I RFLPs are used in many
• Reverse transcriptase ways, such as for disease
mapping, DNA
• Terminal transferase fingerprinting and for
examining genetic
relatedness of organisms.
12
13. Vectors (Cloning Vehicles) Recombinant DNA Technology
Cloning Vehicles (Vectors)
• Autonomous replication - origin of replication
• Ability to accommodate foreign DNA
• Easy insertion (transformation) in host
cells
• Selection markers
– antibiotic resistance gene
– nutrient gene for auxotroph mutant
• Contain specific target site for each of the
E. coli Plasmid pBR322 Yeast artificial chromosome (YAC) multiple restriction endonuclease sites
Recombinant DNA Technology Eukaryotic Shuttle Vector
Cloning Vehicles (Vectors)
• Plasmids that contain a cassette of genes for
expression in eukaryotic cells as well as elements
• Wide range hosts - Shuttle vectors that allow plasmid replication (under the control of
a bacterial origin) in bacteria and selection of
• Secretion vector plasmids plasmid-containing bacteria
• Regulation systems of expression of
• With shuttle vectors, the initial cloning steps are
cloned genes (expression vectors) conducted with E. coli before the fully developed
construct is introduced into a different host cell.
• Amplification - high copy number
• Be maintained stably in the host cells • Additionally, a number of vectors with a single
broad-host-range origin of DNA replication are
developed instead of a narrow-host-range origin,
suitable for a variety of microorganisms instead of
just E. coli.
13
14. Eukaryotic Shuttle Vector Recombinant DNA Technology
Introducing Vector into Host Cell
• Multiple cloning site for
a gene of interest • Transfection with recombinant phage DNA
• Eukaryotic selectable
marker
• Transformation with recombinant plasmid
• Origin of replication in DNA
the eukaryotic cell
• Origin of replication in
• In vitro packaging into phage coat:
bacterial cell transduction with recombinant phage or
• Bacterial selectable
marker gene
cosmid
Lambda vectors
•λ phages are viruses which can
infect bacteria
•Very High transformation
efficiency
•Have a linear genome of ~50 kb
•Insert Size 15-20 kb
From:http://wilkes1.wilkes.edu/~terzaghi/BIO-226/lectures/39.html
14
15. Other Viral Vectors Cosmid Vectors
• Combination of plasmids and “cos” sites of λ phages
• Bacteriophage M 13 • High Transformation efficiency
– Single stranded DNA • Insert Size can be up to ~45 kb
• Retroviruses • The vector size is ~6 kb
• Uses the same packaging technique as
• Adenoviruses bacteriophage lambda
• Therefore insert size can be high.
• Cut open cosmid using ScaI and BamH1
• Insert foreign DNA
• Only cosmids with inserts will form infective viruses
Yeast Artificial Chromosomes BACs and PACs
Essential components Bacterial artificial chromosomes (BACs)
• Yeast Centromeres: DNA without centromeres often get lost during • Based on the E. coli F’ plasmid: can be propagated in E.
mitosis coli
• Telomeres: Protect ends of DNA
• Can accommodate up to 500 kb
• Autonomous Replicating sequences: analogous to “ori” in plasmids
• DNA is more stable
• Ampicillin resistance gene
P1 derived artificial chromosomes (PACs)
• Markers like TRP1 or URA 3
• RE sites
• Modified bacteriophage P1 to accept inserts up to 400 kb
Insert size : UP TO 2 mega bases
• Much more efficient than BACs at infecting hosts
Very Low transformation efficiency
Unstable insert (gets deleted or rearranged)
15
16. Methods of Transformation Recombinant DNA Technology
Characterization of Cloned DNA
• Prokaryotic cells • Eukaryotic cells
– Ca treatment – Ca3(PO4)2 treatment • Insertional inactivation (negative selection)
– lacZ gene for enzyme to hydrolyze Xgal (blue colonies vs. white
– Electroporation – Electroporation
colonies)
– Viruses – Viruses
– F plasmid – Ballistic method • in situ colony hybridization (P32 probe)
– Conjugation (BIOLISTICS) • Plasmid DNA isolation
– DEAE dextran
• Southern Blotting (DNA), Northern (RNA)
– Lipofection
– microinjection • Immunochemical methods (antibody probe)
• DNA sequencing - up to ~1000 nucleotides
Recombinant Protein Therapeutics
• Colony-stimulating factors (CSFs). Immune system growth factors that control the
Recombinant Protein differentiation, growth, and activity of white blood cells. GM-CSF stimulates the
production of both granulocytes and macrophages, helping to overcome immune
deficiencies and fight infection. G-CSF; M-CSF.
Therapeutics • Erythropoietin (EPO). A protein produced in the kidney that stimulates red blood cell
production. It is used to treat anemia linked with renal failure and also find use in
anemia resulting from chemotherapy or therapy for AIDS.
• Blood factors. Proteins involved in the multi-step process of blood clotting. Some, such
as Factor VIII, is deficient in persons with hemophilia A.
• Human growth hormone (HGH)
• Growth factors. Proteins responsible for directing the differentiation and production of
various cell types. Epidermal growth factor (EGF) for wound healing; Platelet-derived
growth factor (PDGF) for collagen deposition in tissue repair; Insulin-like growth factor
(IGF) for promoting tissue growth.
• Interferons. Broad-acting agents that interfere with viral infection (e.g., AIDS) and
control the spread of some cancers and infectious diseases. α-, β-, γ-interferons.
• Interleukins (ILs). Immune system hormones, or cytokines, that stimulate and regulate
Recombinant protein therapeutics—success rates, market trends the growth and function of a wide variety of white blood cell types, can be used in
treating cancer, wound healing, immune deficiencies, and AIDS. IL-1, IL-2, IL-3.
and values to 2010
• Monoclonal antibodies (MAbs). Widely used in biodiagnostics and treatments of
http://www.nature.com/news/2004/041206/fig_tab/nbt1204-1513_F1.html infectious diseases and cancers.
16
17. Biopharmaceutical Products 1. Target new 2. Justify new
properties of
7. Product
protein formulation
existing
• Market: increased from $12 billion in 2000 products products
to over $40 billion in 2006
8. Toxicology
3. Genetic
• 122 products approved in the US and engineering 4. Protein
studies
Europe: cloning
expression
engineering
– 50 Mammalian cells (CHO, NS0, etc.) 9. FDA
acceptance
– 39 prokaryotic cells (mainly E. coli) 5. Small-scale
fermentation
– 21 yeast cells (mainly Saccharomyces and purifica- 6. Pilot-scale
tion fermentation
cerevisiae) and purifica- 10. Commercial
tion production
– 12 undisclosed systems
Flow chart for commercial development
500 undergoing clinical evaluation – high demand for cGMP of recombinant protein products 11. Marketing
manufacturing on small, pilot and large scales
Major Biologics
There are at least 23 protein therapeutics with sales of $1 billion or more.
17