This document discusses different expression systems for producing recombinant proteins, including prokaryotic, yeast, insect cell, and mammalian systems. It provides details on some commonly used expression vectors such as pGEX-3X plasmid for prokaryotic expression in E. coli, Saccharomyces cerevisiae and Pichia pastoris yeast expression systems using episomal and integrating plasmids, and baculovirus expression in insect cells using the polyhedrin promoter to drive expression of the gene of interest. The key advantages and limitations of different expression systems are also summarized.
The document discusses various host systems for expressing recombinant proteins, including bacterial and yeast systems. It provides details on commonly used bacterial hosts like E. coli DH5α, BL21, and BL21(DE3)pLysS, which are suitable for protein expression due to their rapid growth, high yields, and protein secretion abilities. The document also discusses yeast expression systems like Saccharomyces cerevisiae and Pichia pastoris, which allow for eukaryotic post-translational modifications but require more complex processes than bacteria. Key factors in choosing an expression system include the protein properties, yield, and processing needs.
Strain improvement in microbial genetics .pptxHamdiMichaelCC
Strain improvement involves manipulating microbial strains to enhance their metabolic capacities for biotechnology applications. Targets of strain improvement include rapid growth, genetic stability, non-toxicity, large cell size, ability to use cheaper substrates, increased productivity, and reduced cultivation costs. Traditional methods of strain improvement include mutagenesis, classical genetics, and rational selection. Modern techniques utilize genetic engineering and recombinant DNA technology to introduce desired mutations for traits like increased thermostability or altered substrate range.
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 provides an overview of plant biotechnology techniques. It discusses how genes can be manipulated by identifying genes that control traits of interest or modifying existing genes. Genes are then introduced into organisms using transformation methods like Agrobacterium or gene guns. Transformation cassettes containing the gene of interest and selection markers are used. The document explains this process and provides examples like making crops resistant to herbicides or increasing vitamin levels. It also notes there is public controversy around developing and releasing transgenic organisms.
The document discusses techniques for gene manipulation and introduction used in plant biotechnology. It describes how genes can be identified and modified, such as isolating a gene that controls a desired trait or creating a new allele. The modified gene is then introduced into an organism using transformation methods, creating transgenic plants. Specific examples discussed include creating Roundup-resistant crops by introducing a bacterial gene and developing Golden Rice by adding genes to produce vitamin A in rice.
This presentation gives an insight into the mammalian cell being used as an expression system, it also includes a brief introduction to the strong promoters.
gene cloning in eukaryotes (gene transfer).pdfNetHelix
Gene cloning recently faced difficulties associated with
bacteria, especially when dealing with
genes from eukaryotic organisms so we should to employ the eukaryotic expression in this PDF we will learn about gene cloning in eukaryotes, types of yeast plasmids and the importance of each one
systems
This document discusses different expression systems for producing recombinant proteins, including prokaryotic, yeast, insect cell, and mammalian systems. It provides details on some commonly used expression vectors such as pGEX-3X plasmid for prokaryotic expression in E. coli, Saccharomyces cerevisiae and Pichia pastoris yeast expression systems using episomal and integrating plasmids, and baculovirus expression in insect cells using the polyhedrin promoter to drive expression of the gene of interest. The key advantages and limitations of different expression systems are also summarized.
The document discusses various host systems for expressing recombinant proteins, including bacterial and yeast systems. It provides details on commonly used bacterial hosts like E. coli DH5α, BL21, and BL21(DE3)pLysS, which are suitable for protein expression due to their rapid growth, high yields, and protein secretion abilities. The document also discusses yeast expression systems like Saccharomyces cerevisiae and Pichia pastoris, which allow for eukaryotic post-translational modifications but require more complex processes than bacteria. Key factors in choosing an expression system include the protein properties, yield, and processing needs.
Strain improvement in microbial genetics .pptxHamdiMichaelCC
Strain improvement involves manipulating microbial strains to enhance their metabolic capacities for biotechnology applications. Targets of strain improvement include rapid growth, genetic stability, non-toxicity, large cell size, ability to use cheaper substrates, increased productivity, and reduced cultivation costs. Traditional methods of strain improvement include mutagenesis, classical genetics, and rational selection. Modern techniques utilize genetic engineering and recombinant DNA technology to introduce desired mutations for traits like increased thermostability or altered substrate range.
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 provides an overview of plant biotechnology techniques. It discusses how genes can be manipulated by identifying genes that control traits of interest or modifying existing genes. Genes are then introduced into organisms using transformation methods like Agrobacterium or gene guns. Transformation cassettes containing the gene of interest and selection markers are used. The document explains this process and provides examples like making crops resistant to herbicides or increasing vitamin levels. It also notes there is public controversy around developing and releasing transgenic organisms.
The document discusses techniques for gene manipulation and introduction used in plant biotechnology. It describes how genes can be identified and modified, such as isolating a gene that controls a desired trait or creating a new allele. The modified gene is then introduced into an organism using transformation methods, creating transgenic plants. Specific examples discussed include creating Roundup-resistant crops by introducing a bacterial gene and developing Golden Rice by adding genes to produce vitamin A in rice.
This presentation gives an insight into the mammalian cell being used as an expression system, it also includes a brief introduction to the strong promoters.
gene cloning in eukaryotes (gene transfer).pdfNetHelix
Gene cloning recently faced difficulties associated with
bacteria, especially when dealing with
genes from eukaryotic organisms so we should to employ the eukaryotic expression in this PDF we will learn about gene cloning in eukaryotes, types of yeast plasmids and the importance of each one
systems
With the advancement of biotechnology, Genetic engineering also become an important tool. Transgenic crops are the crops which are produced through genetic engineering by altering desirable traits into plant genome.
Expression and purification of recombinant proteins in Bacterial and yeast sy...Shreya Feliz
This presentation gives the information about bacterial and yeast system as host for expressing recombinant proteins, suitable vectors, strains of host, Pros and cons of this system, different purification techniques and commercially available proteins produced so far by this system.
This presentation covers a general introduction to expression vector, its components, types, and its application. Then it covers some of the expression system with examples.
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.
All about genes oncogenes mutations-cloning-gene therapyAhmed Amer
1) DNA contains the genetic code and is located in chromosomes within the nucleus. DNA is transcribed into RNA and translated into proteins, which allows genes to be expressed.
2) Mutations in genes can be caused by errors in DNA replication or exposure to mutagens and can have neutral, harmful, or beneficial effects depending on where they occur. Mutations in proto-oncogenes can transform them into oncogenes and promote cancer development.
3) Cloning techniques allow for the duplication of DNA, whole organisms, or embryonic stem cells for research and potential therapies. Gene cloning is used to study and modify genes, while reproductive and therapeutic cloning are more controversial due to ethical concerns.
This document discusses applications of genetic technologies including recombinant protein production, transgenic plants, and transgenic animals. Recombinant DNA technology is used to produce proteins like insulin on a large scale by inserting genes into bacteria or other hosts. Transgenic plants are created by inserting foreign genes to improve traits like disease resistance or nutritional quality. Transgenic animals carry inserted genes to increase growth rates, improve disease resistance, or produce recombinant proteins in milk. Examples of transgenic crops and animals are also provided.
This document discusses yeast expression systems for producing recombinant proteins. It describes some limitations of prokaryotic expression systems, such as improper folding and post-translational modifications (PTMs). Eukaryotic expression systems allow for PTMs like glycosylation and disulfide bond formation. The document focuses on the yeast Saccharomyces cerevisiae and Pichia pastoris as expression systems. S. cerevisiae allows cytoplasmic and secreted expression of proteins and can perform many PTMs. P. pastoris has advantages like simpler protein purification and more human-like glycosylation. Vector systems for S. cerevisiae include episomal, integrating, and yeast artificial chromosomes. Overall, yeast expression systems provide PTMs
Protein expression and purification services from creative biomartAnne Ehlert
Creative BioMart is committed to providing advanced tools for protein expression and purification. As a leading supplier for reagents in the biotechnology field, we understand the importance of convenient and easy-to-use systems for high level expression and sample purification. We invite you to review our growing range of expression systems resulting from our experience in cloning, overexpression and purification.
Foreign genes can be expressed in both prokaryotic and eukaryotic systems. Recombinant proteins have many pharmaceutical and industrial uses. Prokaryotic expression is faster and cheaper but some eukaryotic proteins may lack proper processing. Eukaryotic systems like yeast, insect cells and mammalian cells allow for proper post-translational modifications but are more complex and expensive. The choice of expression system depends on the desired protein properties and costs involved.
Corynex is a novel Protein Expression system that overcomes some of the most frustrating challenges with common microbial expression systems by secreting fully folded and active proteins directly into the cell medium with minimal host proteins and impurities. This greatly simplifies the entire purification process, lowers costs, and ultimately speeds time to market.
The document summarizes a study that constructed a baculovirus-silkworm multigene expression system for producing virus-like particles. Key points:
- Researchers developed a new baculovirus vector using Bombyx mori nuclear polyhedrosis virus (BmNPV) that could simultaneously express multiple foreign genes in silkworm larvae.
- Using this system, they co-expressed three fluorescent proteins and three structural proteins from rotavirus in silkworms. Electron microscopy confirmed the structural proteins self-assembled into virus-like particles.
- The system efficiently produced high yields of functional multisubunit complexes and virus-like particles in silkworms at low cost
The document discusses recombinant proteins, including their production through recombinant DNA technology. It describes how genes can be isolated and inserted into expression vectors, which are then transferred into host cells to produce the recombinant protein. Various methods are covered, including molecular cloning and polymerase chain reaction. Examples of recombinant proteins discussed include insulin, interferons, hormones, and monoclonal antibodies that are used for medical applications.
The document discusses recombinant proteins, including their production through recombinant DNA technology. It describes how genes can be isolated and inserted into expression vectors, which are then transferred into host cells to produce the recombinant protein. Various methods are covered, including molecular cloning and polymerase chain reaction. Examples of recombinant proteins discussed include insulin, interferons, hormones, and monoclonal antibodies that are used for medical applications.
Chloroplast transformation allows for the integration of foreign genes into the chloroplast genome. This is beneficial as it provides high levels of transgene expression without epigenetic effects or position effects. Chloroplast transformation requires a chloroplast specific expression vector, a method for DNA delivery such as biolistics, and an efficient selection marker such as spectinomycin resistance. Successful transformation is confirmed by PCR and Southern blot analysis showing integration of the transgene into the chloroplast genome. Applications of chloroplast transformation include production of biopharmaceuticals, vaccines, industrial enzymes, and biomaterials as well as phytoremediation.
This document provides an overview of microbial genetics. It begins by defining key terms like genetics, gene, genome, and phenotype. It then discusses DNA structure and replication, explaining how DNA is copied semiconservatively. The processes of transcription and translation are described, showing how DNA is transcribed into mRNA and then translated into proteins. The document outlines gene regulation in bacteria through operons and repression and induction. It also covers mutations, mutagens, horizontal gene transfer through transformation, conjugation and transduction, and mobile genetic elements like plasmids and transposons.
This document discusses genetic engineering techniques such as transferring genes between organisms using vectors like plasmids, creating transgenic organisms like bacteria that express human genes to produce proteins like insulin, and potential benefits and disadvantages of genetic engineering including more affordable medicines, crop improvements, but also environmental, economic, health, and social/ethical risks.
Base editing, prime editing, Cas13 & RNA editing and organelle base editingNetHelix
Welcome back to Genome Editing and CRISPR Cas system playlist
in this pdf, we will continue our explanation for CRISPR Cas9 and will discover together:
-cytosine base editing
-Adenine base editing
-prime editing
-CRISPR/Cas13 RNA targeting (RESCUE & REPAIR) systems
-mitochondrial and chloroplast base editing using TALE
Agrobacterium-mediated transformation is one of the most successful biological methods of gene transfer in plants that is used for crop improvement and the production of GMOs it is a revolutionary method that allows scientists to introduce foreign DNA into plant cells, enabling the modification of plant genomes for various purposes, including crop improvement, pharmaceutical production, and basic research.
Whether you're a student, researcher, educator, or simply curious about the fascinating world of plant biotechnology, our session on Agrobacterium-mediated transformation offers valuable insights and information to help you understand this powerful technique and its implications for plant science and beyond.
Contenu connexe
Similaire à Problems associated with the production of recombinant protein.pdf
With the advancement of biotechnology, Genetic engineering also become an important tool. Transgenic crops are the crops which are produced through genetic engineering by altering desirable traits into plant genome.
Expression and purification of recombinant proteins in Bacterial and yeast sy...Shreya Feliz
This presentation gives the information about bacterial and yeast system as host for expressing recombinant proteins, suitable vectors, strains of host, Pros and cons of this system, different purification techniques and commercially available proteins produced so far by this system.
This presentation covers a general introduction to expression vector, its components, types, and its application. Then it covers some of the expression system with examples.
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.
All about genes oncogenes mutations-cloning-gene therapyAhmed Amer
1) DNA contains the genetic code and is located in chromosomes within the nucleus. DNA is transcribed into RNA and translated into proteins, which allows genes to be expressed.
2) Mutations in genes can be caused by errors in DNA replication or exposure to mutagens and can have neutral, harmful, or beneficial effects depending on where they occur. Mutations in proto-oncogenes can transform them into oncogenes and promote cancer development.
3) Cloning techniques allow for the duplication of DNA, whole organisms, or embryonic stem cells for research and potential therapies. Gene cloning is used to study and modify genes, while reproductive and therapeutic cloning are more controversial due to ethical concerns.
This document discusses applications of genetic technologies including recombinant protein production, transgenic plants, and transgenic animals. Recombinant DNA technology is used to produce proteins like insulin on a large scale by inserting genes into bacteria or other hosts. Transgenic plants are created by inserting foreign genes to improve traits like disease resistance or nutritional quality. Transgenic animals carry inserted genes to increase growth rates, improve disease resistance, or produce recombinant proteins in milk. Examples of transgenic crops and animals are also provided.
This document discusses yeast expression systems for producing recombinant proteins. It describes some limitations of prokaryotic expression systems, such as improper folding and post-translational modifications (PTMs). Eukaryotic expression systems allow for PTMs like glycosylation and disulfide bond formation. The document focuses on the yeast Saccharomyces cerevisiae and Pichia pastoris as expression systems. S. cerevisiae allows cytoplasmic and secreted expression of proteins and can perform many PTMs. P. pastoris has advantages like simpler protein purification and more human-like glycosylation. Vector systems for S. cerevisiae include episomal, integrating, and yeast artificial chromosomes. Overall, yeast expression systems provide PTMs
Protein expression and purification services from creative biomartAnne Ehlert
Creative BioMart is committed to providing advanced tools for protein expression and purification. As a leading supplier for reagents in the biotechnology field, we understand the importance of convenient and easy-to-use systems for high level expression and sample purification. We invite you to review our growing range of expression systems resulting from our experience in cloning, overexpression and purification.
Foreign genes can be expressed in both prokaryotic and eukaryotic systems. Recombinant proteins have many pharmaceutical and industrial uses. Prokaryotic expression is faster and cheaper but some eukaryotic proteins may lack proper processing. Eukaryotic systems like yeast, insect cells and mammalian cells allow for proper post-translational modifications but are more complex and expensive. The choice of expression system depends on the desired protein properties and costs involved.
Corynex is a novel Protein Expression system that overcomes some of the most frustrating challenges with common microbial expression systems by secreting fully folded and active proteins directly into the cell medium with minimal host proteins and impurities. This greatly simplifies the entire purification process, lowers costs, and ultimately speeds time to market.
The document summarizes a study that constructed a baculovirus-silkworm multigene expression system for producing virus-like particles. Key points:
- Researchers developed a new baculovirus vector using Bombyx mori nuclear polyhedrosis virus (BmNPV) that could simultaneously express multiple foreign genes in silkworm larvae.
- Using this system, they co-expressed three fluorescent proteins and three structural proteins from rotavirus in silkworms. Electron microscopy confirmed the structural proteins self-assembled into virus-like particles.
- The system efficiently produced high yields of functional multisubunit complexes and virus-like particles in silkworms at low cost
The document discusses recombinant proteins, including their production through recombinant DNA technology. It describes how genes can be isolated and inserted into expression vectors, which are then transferred into host cells to produce the recombinant protein. Various methods are covered, including molecular cloning and polymerase chain reaction. Examples of recombinant proteins discussed include insulin, interferons, hormones, and monoclonal antibodies that are used for medical applications.
The document discusses recombinant proteins, including their production through recombinant DNA technology. It describes how genes can be isolated and inserted into expression vectors, which are then transferred into host cells to produce the recombinant protein. Various methods are covered, including molecular cloning and polymerase chain reaction. Examples of recombinant proteins discussed include insulin, interferons, hormones, and monoclonal antibodies that are used for medical applications.
Chloroplast transformation allows for the integration of foreign genes into the chloroplast genome. This is beneficial as it provides high levels of transgene expression without epigenetic effects or position effects. Chloroplast transformation requires a chloroplast specific expression vector, a method for DNA delivery such as biolistics, and an efficient selection marker such as spectinomycin resistance. Successful transformation is confirmed by PCR and Southern blot analysis showing integration of the transgene into the chloroplast genome. Applications of chloroplast transformation include production of biopharmaceuticals, vaccines, industrial enzymes, and biomaterials as well as phytoremediation.
This document provides an overview of microbial genetics. It begins by defining key terms like genetics, gene, genome, and phenotype. It then discusses DNA structure and replication, explaining how DNA is copied semiconservatively. The processes of transcription and translation are described, showing how DNA is transcribed into mRNA and then translated into proteins. The document outlines gene regulation in bacteria through operons and repression and induction. It also covers mutations, mutagens, horizontal gene transfer through transformation, conjugation and transduction, and mobile genetic elements like plasmids and transposons.
This document discusses genetic engineering techniques such as transferring genes between organisms using vectors like plasmids, creating transgenic organisms like bacteria that express human genes to produce proteins like insulin, and potential benefits and disadvantages of genetic engineering including more affordable medicines, crop improvements, but also environmental, economic, health, and social/ethical risks.
Similaire à Problems associated with the production of recombinant protein.pdf (20)
Base editing, prime editing, Cas13 & RNA editing and organelle base editingNetHelix
Welcome back to Genome Editing and CRISPR Cas system playlist
in this pdf, we will continue our explanation for CRISPR Cas9 and will discover together:
-cytosine base editing
-Adenine base editing
-prime editing
-CRISPR/Cas13 RNA targeting (RESCUE & REPAIR) systems
-mitochondrial and chloroplast base editing using TALE
Agrobacterium-mediated transformation is one of the most successful biological methods of gene transfer in plants that is used for crop improvement and the production of GMOs it is a revolutionary method that allows scientists to introduce foreign DNA into plant cells, enabling the modification of plant genomes for various purposes, including crop improvement, pharmaceutical production, and basic research.
Whether you're a student, researcher, educator, or simply curious about the fascinating world of plant biotechnology, our session on Agrobacterium-mediated transformation offers valuable insights and information to help you understand this powerful technique and its implications for plant science and beyond.
in this PDF, we will discover:
-introduction to RNA organic extraction
- trizol method
- uses and application
- importance of extraction in the medical field
hello everyone in our course.
now you are watching Level TWO Episode 1 in the practical molecular biology course from A to Z.
in this video, we will discover:
-introduction to PCR
-Main steps in PCR
in this PDF we will discover
- What is organic extraction
-Uses of the isolated DNA
- Steps of the phenol-chloroform method
- Disadvantages of this method
- overall view
hello everyone, in this PDF we will learn about:
- introduction to nucleic acid structures
- how to choose the best method for extraction
-types of these methods
- importance of extraction in the medical field
Unlock the mysteries of life with our latest episode on DNA sequencing! Join us on a captivating journey into the world of genetics as we delve deep into the fascinating process of decoding the fundamental building blocks of life.
🔍 In this episode, we demystify the complexities of DNA sequencing, exploring the cutting-edge technologies and methodologies that scientists use to unravel the secrets hidden within our genetic code. From the revolutionary Sanger sequencing to the high-throughput wonders of Next-Generation Sequencing (NGS), we break down the techniques that have shaped our understanding of genetics.
🔬 Get ready to witness the incredible precision and innovation behind modern DNA sequencing machines, as we showcase how they read, analyze, and interpret the four-letter alphabet that comprises our genetic information. We'll explore the significance of DNA sequencing in various fields, from medicine and forensics to evolutionary biology and personalized genomics.
🌐 Join us as we interview leading experts in the field, gaining insights into the latest advancements and future possibilities of DNA sequencing technology. Learn about the impact of sequencing on medical diagnostics, disease research, and the development of personalized therapies.
📊 Dive into the world of bioinformatics, where powerful algorithms make sense of the vast amount of data generated by DNA sequencing. Discover how this information is transforming our understanding of human evolution, biodiversity, and the interconnectedness of all living organisms.
👩🔬 Whether you're a science enthusiast, student, or simply curious about the intricacies of life, this episode promises to unravel the wonders of DNA sequencing in an accessible and engaging manner. Don't miss out on this illuminating exploration of the code that defines us all!
Dive into the fascinating world of genomics with our latest video! 🧬 In this exploration, we unravel the mysteries of Sequence Tagged Sites (STS) and their crucial role in understanding the intricate language of DNA.
🧬 What are STS, you ask? Join us as we break down the concept of Sequence Tagged Sites, which are unique landmarks along the DNA sequence that help researchers navigate the vast genomic landscape. Discover how STS play a pivotal role in gene mapping, genome sequencing, and unraveling the secrets encoded in our DNA.
🔬 Whether you're a student, a science enthusiast, or just curious about the wonders of genetics, this video is designed to make complex genomic concepts accessible and engaging. We'll explore the significance of STS in genetic research, discuss their applications in molecular biology, and showcase real-world examples of how STS are revolutionizing our understanding of the human genome.
🚀 Join us on this scientific journey as we delve into the world of Sequence Tagged Sites – from their discovery to their impact on modern genomic research. Get ready to unlock the mysteries of DNA, one sequence at a time!
👩🔬👨🔬 Don't forget to hit the like button, subscribe to our channel, and ring the notification bell to stay updated on our latest scientific explorations. Share this video with fellow enthusiasts and let's embark on a thrilling adventure into the heart of genomics together!
#Genomics #DNA #ScienceExplained #SequenceTaggedSites #MolecularBiology #GeneticResearch #ScienceEducation #ExploreWithUs
Embark on a journey into the microscopic world of genetics with our latest PDF, "Fluorescent In Situ Hybridization (FISH) Demystified"! 🧬✨ Join us as we unravel the intricacies of this groundbreaking molecular biology technique that allows us to visualize and understand genetic information at a whole new level.
🌈 Dive into the fascinating realm of genetics as we explore how FISH works, highlighting its crucial role in identifying and locating specific DNA sequences within cells. Witness the magic of fluorescent probes binding to target DNA, illuminating the intricate patterns that make up the blueprint of life.
🧫 Our expert molecular biologists guide you through the step-by-step process of conducting FISH experiments, from sample preparation to imaging. Gain insights into the applications of FISH across various fields, including cancer research, genetic diagnostics, and uncovering chromosomal abnormalities.
🎓 Whether you're a student, researcher, or simply curious about the wonders of molecular biology, "Fluorescent In Situ Hybridization (FISH) Demystified" provides a comprehensive and accessible overview of this revolutionary technique.
🎥 Don't forget to hit the subscribe button, give us a thumbs up, and ring the notification bell to stay updated on our scientific explorations. Delve into the world of genetic research with us – it's a journey that promises to illuminate the mysteries of life at the cellular level!
🔬🧪
This PDF document provides a comprehensive overview of restriction mapping, a foundational technique in molecular biology that allows researchers to decipher the intricate genetic architecture of DNA molecules. Focusing on the utilization of restriction enzymes, this guide elucidates the principles, methods, and applications of restriction mapping, serving as a valuable resource for researchers, students, and enthusiasts in the field.
This PDF document aims to demystify the intricacies of restriction mapping, offering both beginners and seasoned researchers an in-depth understanding of the first physical mapping technique and its continued relevance in the era of advanced genomic technologies.
Embark on a genomic adventure with our latest PDF guide, "AFLP Odyssey." In this exploration of Amplified Fragment Length Polymorphisms (AFLPs), we unveil the power of this sophisticated technique in deciphering the mysteries of the genome. Join us as we navigate the genomic seas, charting the course of genetic discovery with AFLPs as our compass.
Set sail on a genomic adventure with "AFLP Odyssey." Whether you're an intrepid genetic explorer or a curious learner, this PDF promises to guide you through the turbulent seas of the genome, revealing the treasures hidden within DNA fragments amplified by AFLPs. Let the genomic odyssey begin!
Prepare for a genetic face-off as we unravel the intriguing world of microsatellites in our latest PDF, "SSR vs. ISSR Showdown." Dive into the microscopic realm of Simple Sequence Repeats (SSR) and Inter-Simple Sequence Repeats (ISSR), as we dissect their unique features, applications, and the genetic tales they unveil.
RFLPs (Restriction Fragment Length Polymerase).pdfNetHelix
Dive into the intricate world of molecular genetics with our comprehensive guide to Restriction Fragment Length Polymorphism (RFLP). This PDF unfolds the underlying principles, applications, and significance of RFLP as a molecular marker, offering a thorough exploration of its role in genetic research.
Embark on a journey of genetic discovery with our comprehensive guide to RFLP. Whether you are a seasoned researcher or a curious enthusiast, this PDF promises to unravel the mysteries of RFLP and its enduring impact on genetic research.
Plasmids are circular, self-replicating DNA molecules that are commonly used as cloning vectors. Key properties of plasmids that make them useful for cloning include their ability to replicate independently of the bacterial chromosome, carry genes for antibiotic resistance or other selectable markers, and accept foreign DNA at specific restriction sites. Common plasmids used for cloning are pBR322, which has features like a high copy number and blue-white screening, and pUC plasmids, which have even higher copy numbers. Plasmids can shuttle DNA between bacterial and yeast cells, and RNA production plasmids contain promoters to transcribe cloned DNA into RNA for applications like Northern blotting.
we will explore an intriguing and fascinating topic: how DNA is packaged into chromosomes. Together, we will delve into the magical process that occurs inside our cells, where complex genetic codes are transformed into organized and tightly regulated structures known as chromosomes.
We will show you the amazing process of how DNA is intricately folded inside the cell nucleus and shaped into a chromosomal structure. You will learn about the proteins and incredible mechanisms involved in this process, and how the precise arrangement of genes within cells is achieved.
Join us on this exciting journey into the small and intricate world inside our cells, and uncover the secret behind how DNA is packaged into chromosomes. Don't forget to subscribe to the channel and hit the notification bell to stay updated with all the latest and exciting content. Thank you for your continuous support and for watching us.
genome structure and repetitive sequence.pdfNetHelix
Welcome to our channel, where science meets discovery! In today's enlightening video, we unravel the mysteries of life at its most fundamental level - the chromosomes.
Join us on an exhilarating journey deep within the human cell as we explore the intricate architecture and organization of these tiny yet immensely powerful structures.
Don't forget to subscribe to the channel and hit the notification bell to stay updated with all the latest and exciting content. Thank you for your continuous support and for watching us.
The document discusses enzymes used for DNA manipulation, which fall into four main categories: DNA polymerases, nucleases, ligases, and end-modification enzymes. DNA polymerases are template dependent and include DNA polymerase 1. Nucleases include exonucleases like Bal31 and endonucleases like DNase I. Ligases involve T4 DNA ligase. End-modification enzymes include alkaline phosphatase. Restriction endonucleases recognize specific DNA sequences and have various applications in genetic engineering.
DNA polymerases (DNA manupliation Enzymes).pdfNetHelix
the Secrets of DNA Manipulation: A Comprehensive Exploration of DNA Polymerase and Enzymes
In this PDF presentation entitled "Enzymes that Manipulate DNA, Specially DNA Polymerase," we delve deep into the mechanisms and functions of these remarkable enzymes that play a pivotal role in the realm of molecular biology.
🧬 Key Highlights:
Introduction to DNA Polymerase:
Uncover the fundamental aspects of DNA polymerase, a key player in DNA replication and repair. Explore its structure, functions, and the indispensable role it plays in maintaining the genetic integrity of living organisms.
Types of DNA Polymerases:
Delve into the diverse landscape of DNA polymerases, ranging from prokaryotic to eukaryotic systems. Understand how different types of DNA polymerases contribute to the precision and efficiency of DNA synthesis.
Examples of polymerases:
•DNA polymerase 1
•klenow fragment
•sequenase
•Taq polymerase
•Reverse Transcriptase
DNA Replication
Take a closer look at the intricate dance of enzymes during DNA replication. Follow the step-by-step process, and gain insights into how DNA polymerase ensures the accurate transmission of genetic information from one generation to the next.
Technological Applications:
Unleash the potential of DNA polymerase in various biotechnological applications. From PCR (Polymerase Chain Reaction) to DNA sequencing, discover how these enzymes have revolutionized molecular biology and genetic research.
Emerging Trends and Future Prospects:
Stay ahead of the curve by exploring the latest advancements and emerging trends in DNA manipulation. Witness the ongoing research that promises to unlock new possibilities in the field.
🎓 Who Should Explore This Presentation?
Students and researchers in molecular biology and genetics
Biotechnologists and professionals in the field of genetic engineering
Enthusiasts curious about the molecular machinery behind DNA manipulation
Does Over-Masturbation Contribute to Chronic Prostatitis.pptxwalterHu5
In some case, your chronic prostatitis may be related to over-masturbation. Generally, natural medicine Diuretic and Anti-inflammatory Pill can help mee get a cure.
Travel Clinic Cardiff: Health Advice for International TravelersNX Healthcare
Travel Clinic Cardiff offers comprehensive travel health services, including vaccinations, travel advice, and preventive care for international travelers. Our expert team ensures you are well-prepared and protected for your journey, providing personalized consultations tailored to your destination. Conveniently located in Cardiff, we help you travel with confidence and peace of mind. Visit us: www.nxhealthcare.co.uk
Mercurius is named after the roman god mercurius, the god of trade and science. The planet mercurius is named after the same god. Mercurius is sometimes called hydrargyrum, means ‘watery silver’. Its shine and colour are very similar to silver, but mercury is a fluid at room temperatures. The name quick silver is a translation of hydrargyrum, where the word quick describes its tendency to scatter away in all directions.
The droplets have a tendency to conglomerate to one big mass, but on being shaken they fall apart into countless little droplets again. It is used to ignite explosives, like mercury fulminate, the explosive character is one of its general themes.
5-hydroxytryptamine or 5-HT or Serotonin is a neurotransmitter that serves a range of roles in the human body. It is sometimes referred to as the happy chemical since it promotes overall well-being and happiness.
It is mostly found in the brain, intestines, and blood platelets.
5-HT is utilised to transport messages between nerve cells, is known to be involved in smooth muscle contraction, and adds to overall well-being and pleasure, among other benefits. 5-HT regulates the body's sleep-wake cycles and internal clock by acting as a precursor to melatonin.
It is hypothesised to regulate hunger, emotions, motor, cognitive, and autonomic processes.
- Video recording of this lecture in English language: https://youtu.be/Pt1nA32sdHQ
- Video recording of this lecture in Arabic language: https://youtu.be/uFdc9F0rlP0
- Link to download the book free: https://nephrotube.blogspot.com/p/nephrotube-nephrology-books.html
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Cell Therapy Expansion and Challenges in Autoimmune DiseaseHealth Advances
There is increasing confidence that cell therapies will soon play a role in the treatment of autoimmune disorders, but the extent of this impact remains to be seen. Early readouts on autologous CAR-Ts in lupus are encouraging, but manufacturing and cost limitations are likely to restrict access to highly refractory patients. Allogeneic CAR-Ts have the potential to broaden access to earlier lines of treatment due to their inherent cost benefits, however they will need to demonstrate comparable or improved efficacy to established modalities.
In addition to infrastructure and capacity constraints, CAR-Ts face a very different risk-benefit dynamic in autoimmune compared to oncology, highlighting the need for tolerable therapies with low adverse event risk. CAR-NK and Treg-based therapies are also being developed in certain autoimmune disorders and may demonstrate favorable safety profiles. Several novel non-cell therapies such as bispecific antibodies, nanobodies, and RNAi drugs, may also offer future alternative competitive solutions with variable value propositions.
Widespread adoption of cell therapies will not only require strong efficacy and safety data, but also adapted pricing and access strategies. At oncology-based price points, CAR-Ts are unlikely to achieve broad market access in autoimmune disorders, with eligible patient populations that are potentially orders of magnitude greater than the number of currently addressable cancer patients. Developers have made strides towards reducing cell therapy COGS while improving manufacturing efficiency, but payors will inevitably restrict access until more sustainable pricing is achieved.
Despite these headwinds, industry leaders and investors remain confident that cell therapies are poised to address significant unmet need in patients suffering from autoimmune disorders. However, the extent of this impact on the treatment landscape remains to be seen, as the industry rapidly approaches an inflection point.
Osteoporosis - Definition , Evaluation and Management .pdfJim Jacob Roy
Osteoporosis is an increasing cause of morbidity among the elderly.
In this document , a brief outline of osteoporosis is given , including the risk factors of osteoporosis fractures , the indications for testing bone mineral density and the management of osteoporosis
8. 1
Determining DNA, RNA,
and protein sequence
3
2
Cloning of correct gene
into the expression vector
Transformation of vector
into host cell and
expression
9. Prokaryotic cells Eukaryotic cells
Easy to:
➢ Grow
➢ Genetically manipulated
Not all genes are able to be expressed in
prokaryotic cells
Antibiotic resistance genes for increased selectivity
of transformed bacteria
Has all necessary promoters and terminators in
gDNA already
Lack of before and after translation protein
modification pathways for correct protein
manufacturing
10.
11. Problems associated with the production of
recombinant protein
Loss of expression
• Plasmid-Based Systems
• Chromosomal Integration
• Viral vectors
Posttranslational
processing
• Folding, aggregation &
solubility
• Proteolytic processing
• Glycosylation
• Other Posttranslational
Modifications
Transport and
Localization
12. Problems associated with the production of
recombinant protein
Loss of expression
•Plasmid-Based Systems
•Chromosomal Integration
•Viral vectors
13. G.R: Loss of expression?
• expression can be lost due to:
➢structural changes in the recombinant gene
➢disappearance of the gene from host cells
Gene of interest
In Plasmid
delivered by a virus
integrated into the host’s chromosome
14. Plasmid-Based Systems
• Plasmid:
➢extra-chromosomal self-replicating cytoplasmic DNA
elements
➢found in prokaryotes and eukaryotes.
➢Used as molecular vehicles for recombinant genes
➢most popular choice when using prokaryotes as hosts?
▪ as genetic manipulation of plasmids is easy
15. Formation of a single dimeric circle with 2 ori
• As plasmid copies have the same sequence, they can recombine
and form a single dimeric circle with two origins of replication.
This results in fewer independent units to be segregated between
daughter cells, and consequently plasmid loss can increase
17. Metabolic load and plasmid
• the size of the insert
• temperature,
• expression level
➢Constitutive gene expression may increase plasmid instability because
the metabolic load of recombinant protein production is constantly
present.
• recombinant protein yield
• toxicity of the expressed protein toward the host
18. Gene dosage
• refers to the number of copies of a specific gene present within an
organism's genome
• Gene dosage is higher than when the recombinant gene is
integrated into the host’s chromosome
• Medium to high copy number plasmids:
➢Relaxed replication
➢Random distribution
➢Relatively low loss: Continuously growing and toxic genes/gene products
will lead to plasmid loss.
19. How to increase gene dosage in E.coli
genome?
Method How does is work? Limitations
RecA duplication of insert (Olson
et al. 1998)
• By RecA protein, which plays a
crucial role in homologous
recombination to duplicate the
insert
• Using this method leads to an
increased gene dosage.
• The reported range of copies
was 15 to 40.
• unstable without a selectable
marker.
Tn1545 site-specific
recombination (Peredelchuck
and Bennett 1997)
• site-specific recombination
mediated by transposon
Tn1545.
Time consuming
20. Plasmid copy number
For applications such as DNA
production for gene therapy, high
plasmid copy number is an
important objective function
21. Plasmid copy number
• copy-number control genes regulate plasmid copy-number.
• Plasmid copy number is an inherent property of each expression system
• Plasmid copy number depends on:
➢ the number of plasmid copies at the time of cell division and their random distribution
between daughter cells
➢The host
➢Culture conditions
• How to ensure plasmid survival in the cell population?
• low-copy-number plasmids guarantee their persistence by multimer
resolution through:
❑ site-specific recombination systems (cer sequence)
❑active partition mechanisms, such as the par sequences.
22. Although high plasmid copy numbers are generally desired protein
yield, this might not always be true. G.R?
• Because it may drive for improving recombinant
high protein production rates, which can result in:
• protein aggregation
• Deficient posttranslational modification.
• Reduced translation efficiency despite high
plasmid copy number
Low recombinant protein
yields can also occur in
cells with a high plasmid
copy number:
• Reduced Translation
Efficiency
• Metabolic Burden
• Cell Stress
23. Plasmid structural instability
A complete elimination of
recombinant protein
production
accumulation of aberrant
recombinant proteins with
minor changes in the original
amino acid sequence
(deletions, additions, or
substitutions)
To
Solve
this we
preform
25. Increased Plasmid stability by using
selectable markers
Genes for
antibiotic
resistance
Complementation
Genes or repressors
that lead to cell
death upon plasmid
loss.
26. Antibiotics are expensive, and their presence is
undesirable in food and therapeutic products
deletion of an essential
gene from the bacterial
chromosome and its
inclusion in the plasmid
the introduction of a growth
repressor in the bacterial
genome and its antidote in
the plasmid
To
Solve
this we
preform
27. Problems associated with the production of
recombinant protein
Loss of expression
•Plasmid-Based Systems
•Chromosomal Integration
•Viral vectors
28. Problems associated with the production of
recombinant protein
Loss of expression
•Plasmid-Based Systems
•Chromosomal Integration
•Viral vectors
29. Chromosomal integration
• A powerful alternative for overcoming problems of expression
stability in plasmid-based systems
• suitable for metabolic engineering of the host
30. Chromosomal integration Vs. Plasmid-
Based Systems
ADVANTAGES DISADVANTAGES
Reduce metabolic burden Labor intensive
Stable inheritance Time consuming
Improved genetic stability Low copy number
Reduced risk of Horizontal Gene Transfer (HGT) Integration of gene of interest into an inactive region
of chromosome
Long term expression
The recombinant cells obtained are able to grow in
the absence of antibiotics without any reduction of
recombinant protein yields.
To overcome
this
use of locus control
regions (LCRs), which
ensures transcriptional
regulation of the
transgene.
31. Problems associated with the production of
recombinant protein
Loss of expression
•Plasmid-Based Systems
•Chromosomal Integration
•Viral vectors
32. Problems associated with the production of
recombinant protein
Loss of expression
•Plasmid-Based Systems
•Chromosomal Integration
•Viral vectors
33. Viral vectors
• Easy and very effective way of delivering the gene of interest
• Viruses have evolved to deliver their genetic material to the host in an
efficient and non-destructive way.
• Useful for production in higher eukaryotes because of its simplicity
• Retroviruses: promote integration of the viral genome into the cell’s
chromosome.
• Viral expression systems are a niche for industrial protein production.
• Ex →(BEVS):
➢utilized to commercially produce several recombinant proteins.
➢suitable for the production of vaccines.
➢application for viral vectors is gene therapy.
the insect cell
baculovirus
expression
vector system
34. Viral vectors
• transient expression:
➢ refers to the temporary expression of genes in the host organism for a
limited duration
➢Allow rapid production of proteins or other gene products without the
need for stable integration into the host organism
➢utilized for rapidly generating sufficient amounts of protein for laboratory-
scale applications or preliminary testing of drug candidates
Once a promising molecule is identified, a stable cell line can be generated
35. Problems associated with the production of
recombinant protein
Loss of expression
•Plasmid-Based Systems
•Chromosomal Integration
•Viral vectors
36. Problems associated with the production of
recombinant protein
Loss of expression
• Plasmid-Based Systems
• Chromosomal Integration
• Viral vectors
Posttranslational
processing
• Folding, aggregation &
solubility
• Proteolytic processing
• Glycosylation
• Other Posttranslational
Modifications
Transport and
Localization
37. Problems associated with the production of
recombinant protein
Posttranslational processing
• Folding, aggregation & solubility
• Proteolytic processing
• Glycosylation
• Other Posttranslational Modifications
38. Post-Translational Modifications
(PTM)
the extent of modification depends on the host utilized, being the
modifications performed by higher eukaryotic cells closer to
those found in human proteins
39. Folding, aggregation and solubility
Foldases Chaperons
Key player molecules of
folding
Inclusion bodies → misfolded
proteins that accumulate in
intracellular aggregates
40. Example on human pathologies characterized by
intracellular protein aggregation and accumulation
Alzheimer’s
disease
Parkinson’s
disease
Huntington’s
disease
41. Folding, aggregation and solubility
• One of the main causes of incorrect protein folding is cell stress,
which may be caused by:
➢ heat shock
➢nutrient depletion
➢or other stimuli
• How do cells respond to stress?
• by increasing the expression of various chaperones, some of them of the
hsp70 and hsp100 families.
42. Strategies to reduce aggregation protein
engineering
Changing the
extent of
hydrophobic
regions
Using fusion
proteins
43. Strategies to reduce aggregation protein
engineering
Using fusion
proteins
• Recombinant fusion proteins are created artificially
by recombinant DNA technology for use in biological
research or therapeutics.
• The purpose of creating fusion proteins in drug
development is to impart properties from each of
the "parent" proteins to the resulting chimeric
protein.
• Fused proteins often contain a peptide native to the
host used. For example, fusing single chain
antibodies to an E. coli maltose-binding protein
allows the production of soluble functional protein
in E. coli cytoplasm
44. Problems associated with the production of
recombinant protein
Posttranslational processing
• Folding, aggregation & solubility
• Proteolytic processing
• Glycosylation
• Other Posttranslational Modifications
45. Problems associated with the production of
recombinant protein
Posttranslational processing
• Folding, aggregation & solubility
• Proteolytic processing
• Glycosylation
• Other Posttranslational Modifications
46. Proteolytic Processing
• Proteolysis: breakdown of proteins into smaller
polypeptides or amino acids through the hydrolysis of
peptide bonds by a protease.
• Signal peptides: needed to direct protein to the various
cellular compartments Signal peptides
must be cleaved?
To obtain a
functional protein
47. • Endoproteases or endopeptidases: proteolytic peptidase that
break peptide bonds of nonterminal amino acids (within the
molecule)
• There is some protein smust be expressed as proproteins
because prodomains act as folding catalysts → cells utilize
endoproteases to produce the mature active protein.
• Example:
➢Proteases
➢Insulin
➢penicillin acylase
48. Removal of the N-terminal methionine
• occurs only in proteins in which the second amino acid is:
➢ alanine
➢Glycine
➢Proline
➢Serine
➢Threonine
➢Valine
• This processing is performed by a methionine aminopeptidase
(MAP)
49. Reduction of yield of secreted proteins
To
Solve this
Overxpression
E.Coli signal
peptidase I
Bacillus subtilis
peptidase I
50. Glycosylation
• Complex form of protein modification occurring in the secretory
pathway
• requires several consecutive steps
• involves tens of enzymes and substrates.
• It usually occurs in the endoplasmic reticulum and Golgi
apparatus of eukaryotic cells
• N-glycosylation has been detected in proteins produced by
bacteria
51. Glycosylation
• In many cases, glycosylation determines protein:
➢ stability
➢Solubility
➢Antigenicity
➢Folding
➢Localization
➢biological activity
➢circulation half-life.
52. 3 types of glycosylation
N-glycosylation O-glycosylation
C-glycosylation
53. 3 types of glycosylation
• the most studied and is
considered as the most relevant
for recombinant protein
production.
• N-glycans linked to an
asparagine (Asn) of consensus
sequence
Asn – X – (Ser/Thr)
N-glycosylation
54. 3 types of glycosylation
N-glycosylation O-glycosylation
C-glycosylation
55. 3 types of glycosylation
• O-glycans linked to oxygen atom of:
➢Serine (Ser)
➢Threonine (Thr)
O-glycosylation
56. 3 types of glycosylation
N-glycosylation O-glycosylation
C-glycosylation
57. 3 types of glycosylation
• Attachment of glycans to
trptophan
• C-linked glycosylation has
hardly been studied and
little is known about its
biological significance .
C-glycosylation
58. 3 types of glycosylation
N-glycosylation O-glycosylation
C-glycosylation
60. Glycosylation
• Glycosylation profiles are protein-, tissue-, and animal specific .
Nonauthentic glycosylation → may trigger immune responses
when present in proteins for human or animal use.
• Therefore, authentic glycosylation is especially relevant for
recombinant proteins to be utilized as drugs.
61. Problems associated with the production of
recombinant protein
Loss of expression
• Plasmid-Based Systems
• Chromosomal Integration
• Viral vectors
Posttranslational
processing
• Folding, aggregation &
solubility
• Proteolytic processing
• Glycosylation
• Other Posttranslational
Modifications
Transport and
Localization