This document describes a study on expressing recombinant nanobodies in E. coli cells, extracting the proteins, and purifying them. E. coli WK6 cells were transformed with a plasmid containing the nanobody gene and expression was induced with IPTG. The cells were lysed and the proteins in the periplasmic space were extracted. Purification was done using immobilized metal affinity chromatography to bind the histidine-tagged nanobody, which was then eluted with imidazole buffer. SDS-PAGE and Western blot were used to analyze the expression and purity of the nanobody.
J. Ingram, D. Poulcharidis - Adv. Topics of Chem. Bio. - Dr. Webb - Prof. S. ...JDIngram
A presentation on the O'Connor group, given as part of the Advanced Topics of Chemical Biology module. Yet to be awarded a mark, I presented from slide 6 onwards.
The document discusses the isolation and purification of genomic DNA and plasmid DNA. It begins by outlining the history of DNA isolation and describes current methods. The key steps in isolating genomic DNA are cell growth and lysis, followed by DNA purification using techniques like phenol-chloroform extraction and ethanol precipitation to remove proteins and RNA. Plasmid DNA isolation involves bacterial growth, lysis, and purification of plasmids from chromosomal DNA using alkaline lysis and centrifugation. Purification methods like silica binding columns are also described to further clean DNA samples.
Transformation of saccharomyces cerevisiae and other fungiCAS0609
This document reviews methods for transforming various fungi, including Saccharomyces cerevisiae, with a focus on improvements since 2001. It summarizes the original spheroplast and lithium acetate methods for S. cerevisiae transformation, noting key steps and findings. The document then presents a proposed model for the mechanism of S. cerevisiae transformation based on recent reports, suggesting DNA attaches to the cell wall and enters via endocytosis, with polyethylene glycol playing a role in attachment and membrane effects.
- The study investigated the effect of calcium chloride concentration on the transformation efficiency of E. coli with plasmids pUC19 and pBR322, which differ in size.
- Maximum transformation efficiency was observed at 0.15M CaCl2 for pUC19 and 0.1M CaCl2 for the larger pBR322 plasmid.
- Increasing calcium chloride concentration above these levels decreased transformation efficiency for both plasmids, with no transformants observed above 0.2M, possibly due to decreased cell viability in hypertonic conditions.
This document summarizes a study on the heterologous expression and characterization of the c1 dioxygenase enzyme from Tetranychus urticae. Key points:
- The c1 dioxygenase gene was inserted into a plasmid and transformed into E. coli cells for expression. However, initial expression attempts did not show overexpression of the protein.
- Additional expression trials were conducted by varying culture conditions and bacterial clones. SDS-PAGE analysis showed some potential overexpression in new clones induced overnight at 37°C, though further optimization is still needed.
- Once expression is optimized, the goal is to purify the recombinant protein using its His-tag and proceed with structural characterization through crystallization,
This document discusses various mechanisms for transforming and transfecting cells, including prokaryotic, eukaryotic, plant, and fungal cells. It describes the history of bacterial transformation and mechanisms such as natural competence, artificial competence using calcium chloride or electroporation, and lipofection. For eukaryotic transfection, it discusses lipofection, dendrimers, and nucleofection. It also outlines various mechanisms for transforming plants, including Agrobacterium, electroporation, viral transformation, and particle bombardment.
Preparation of plasmid dna by M.Waqas & Noman Hafeez KhosaNoman-Hafeez khosa
The document describes the process of preparing plasmid DNA from bacterial cells. It involves growing cells, harvesting them, lysing them using enzymes to release DNA, and then purifying the plasmid DNA. Purification involves phenol-chloroform extraction to separate DNA from other cell components, followed by column chromatography or silica binding to further purify the DNA. The purified plasmid DNA can then be analyzed using gel electrophoresis to separate DNA fragments by size.
J. Ingram, D. Poulcharidis - Adv. Topics of Chem. Bio. - Dr. Webb - Prof. S. ...JDIngram
A presentation on the O'Connor group, given as part of the Advanced Topics of Chemical Biology module. Yet to be awarded a mark, I presented from slide 6 onwards.
The document discusses the isolation and purification of genomic DNA and plasmid DNA. It begins by outlining the history of DNA isolation and describes current methods. The key steps in isolating genomic DNA are cell growth and lysis, followed by DNA purification using techniques like phenol-chloroform extraction and ethanol precipitation to remove proteins and RNA. Plasmid DNA isolation involves bacterial growth, lysis, and purification of plasmids from chromosomal DNA using alkaline lysis and centrifugation. Purification methods like silica binding columns are also described to further clean DNA samples.
Transformation of saccharomyces cerevisiae and other fungiCAS0609
This document reviews methods for transforming various fungi, including Saccharomyces cerevisiae, with a focus on improvements since 2001. It summarizes the original spheroplast and lithium acetate methods for S. cerevisiae transformation, noting key steps and findings. The document then presents a proposed model for the mechanism of S. cerevisiae transformation based on recent reports, suggesting DNA attaches to the cell wall and enters via endocytosis, with polyethylene glycol playing a role in attachment and membrane effects.
- The study investigated the effect of calcium chloride concentration on the transformation efficiency of E. coli with plasmids pUC19 and pBR322, which differ in size.
- Maximum transformation efficiency was observed at 0.15M CaCl2 for pUC19 and 0.1M CaCl2 for the larger pBR322 plasmid.
- Increasing calcium chloride concentration above these levels decreased transformation efficiency for both plasmids, with no transformants observed above 0.2M, possibly due to decreased cell viability in hypertonic conditions.
This document summarizes a study on the heterologous expression and characterization of the c1 dioxygenase enzyme from Tetranychus urticae. Key points:
- The c1 dioxygenase gene was inserted into a plasmid and transformed into E. coli cells for expression. However, initial expression attempts did not show overexpression of the protein.
- Additional expression trials were conducted by varying culture conditions and bacterial clones. SDS-PAGE analysis showed some potential overexpression in new clones induced overnight at 37°C, though further optimization is still needed.
- Once expression is optimized, the goal is to purify the recombinant protein using its His-tag and proceed with structural characterization through crystallization,
This document discusses various mechanisms for transforming and transfecting cells, including prokaryotic, eukaryotic, plant, and fungal cells. It describes the history of bacterial transformation and mechanisms such as natural competence, artificial competence using calcium chloride or electroporation, and lipofection. For eukaryotic transfection, it discusses lipofection, dendrimers, and nucleofection. It also outlines various mechanisms for transforming plants, including Agrobacterium, electroporation, viral transformation, and particle bombardment.
Preparation of plasmid dna by M.Waqas & Noman Hafeez KhosaNoman-Hafeez khosa
The document describes the process of preparing plasmid DNA from bacterial cells. It involves growing cells, harvesting them, lysing them using enzymes to release DNA, and then purifying the plasmid DNA. Purification involves phenol-chloroform extraction to separate DNA from other cell components, followed by column chromatography or silica binding to further purify the DNA. The purified plasmid DNA can then be analyzed using gel electrophoresis to separate DNA fragments by size.
1. The document describes an experiment to isolate plasmid DNA from E. coli bacteria transformed with the pGLO plasmid.
2. The plasmid DNA was isolated using a modified alkaline lysis method. Samples of the isolated plasmid DNA were run on a gel electrophoresis along with a ladder for comparison but the ladder showed an unusual single band.
3. While single bands were observed for the plasmid DNA samples, the abnormal ladder prevented further analysis of the isolated plasmid DNA.
The content is about the general description of genetic material and further two techniques of biotechnology. The content includes two topics.
Firstly with introduction to biotechnology it describe about DNA, recombinant DNA (rDNA) technology, history, goals, procedure of rDNA technology, tools, techniques, application, demerits and products of rDNA technology.
Second portion entitiled as Hybridoma technology. this includes the basic principle, production of monoclonal antibodies, merits demerits and drugs from monoclonal antibody.
New pharmaceuticals derived from biotechnology is covered in last. All the content is referred from books and internet sources.
This document outlines a lecture on microbial genetics. It discusses the genetic engineering processes used to produce human insulin and growth hormone (HGH) in bacteria. For insulin, the human gene is isolated and inserted into bacterial plasmid DNA using restriction enzymes. Transformed bacteria then produce insulin. A similar process is used for HGH, isolating its mRNA from human pituitary gland cells, generating cDNA, and inserting it into bacteria to produce HGH. Lastly, it notes other human hormones can be genetically engineered using these techniques.
This document describes a study that investigated the effects of an extract from the cyanobacteria Aphanizomenon flos-aquae (AFAe) on human natural killer (NK) cells in vitro. The key findings were:
1) AFAe directly activated NK cells, as shown by increased expression of the activation markers CD69 and CD25 on CD3-CD56+ NK cells after 18 hours of exposure.
2) The low-molecular-weight fraction (<5,000 Da) of AFAe induced the strongest NK cell activation, suggesting novel activating compounds.
3) NK cell activation by AFAe required the presence of other immune cells such as monocytes, as
This document provides an overview of techniques for exploring genes, including DNA purification, restriction enzymes, recombinant DNA technology, molecular cloning using vectors, DNA libraries, blotting techniques, DNA sequencing, PCR, and gene expression studies. Key concepts covered include the types of DNA, plasmid purification, DNA conformations, nucleic acid quantitation, gel electrophoresis, restriction enzyme recognition sites and nomenclature, recombinant DNA technology tools, molecular cloning steps and components of cloning vectors. Applications of these techniques such as genetically modified organisms, recombinant proteins, gene therapy, and CRISPR are also discussed.
Recombinant DNA technology uses restriction enzymes and DNA ligase to cut and join DNA molecules from different species, generating novel genetic combinations. This document outlines the history, basic steps, tools, and applications of recombinant DNA technology. Key applications include producing human insulin, growth hormone, interferon, hepatitis B vaccine, tissue plasminogen activator, and erythropoietin to treat various diseases. Recombinant DNA technology has generated billions in pharmaceutical revenue and provides substantial quantities of therapeutic proteins that were previously unavailable.
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.
Recombinant DNA Technology - A Perforated Insight By Rxvichu !!RxVichuZ
Hello friends................................this is me....Vishnu,back after a gap of almost three weeks, with another powerpoint presentation on RECOMBINANT DNA TECHNOLOGY....
Prepared as per Pharmacology syllabus for thrird year Pharm.D students, this 40-slide ppt involves precise details on the following aspects:
1. DEFINITION
2. PRINCIPLES INVOLVED
3. ENZYMES AND VECTORS USED
4. METHODS OF GENE TRANSFER
5. APPLICATIONS OF RECOMBINANT DNA TECHNOLOGY
Not just for Pharm.D , i do sincerely hope that it will be useful for others, on a study or reference cum reading basis too...............
Do post ur feedbacks....
Suggestions for further improvement will be gratefully acknowledged..........
For further details, connect to me in :
Facebook : Rx Vishnu
Gmail: rxvichu623@gmail.com
watsapp and hike: 8086948729
communication: the same number as above.
Keep reading well....study well.................keep rocking!!!
@rxvichu-live 4 more!!!!
:) :)
This document provides an overview of recombinant DNA technology. It defines recombinant DNA technology as procedures that allow DNA from different species to be isolated, cut, and spliced together to form new recombinant molecules. The key steps described are using restriction enzymes to cut DNA at specific sites, inserting genes into bacterial plasmids, transforming bacteria, replicating the recombinant DNA as the bacteria divide, and collecting the amplified genes. Applications discussed include producing insulin, vaccines, human growth hormones, diagnosing infectious diseases, developing novel crop varieties, and industrial strain improvement.
The document discusses the principles and processes of biotechnology. It describes two core techniques that enabled modern biotechnology: genetic engineering and maintaining sterile environments for microbial growth. It then discusses various tools used in recombinant DNA technology, including restriction enzymes, vectors, competent hosts, PCR amplification, and downstream processing to obtain recombinant products.
Genetic engineering and Recombinant DNAHala AbuZied
Genetic engineering involves altering the DNA of living organisms using biotechnology. It includes techniques like changing single DNA base pairs, deleting or adding genes, or combining DNA from different species. Recombinant DNA technology is used to create recombinant DNA molecules by manipulating DNA in vitro and introducing them into host organisms. This allows bacteria to be engineered to produce human insulin through inserting the human insulin gene into bacterial plasmids. Genomic libraries can be created by ligating fragmented genomic or cDNA into plasmid vectors to transform bacteria and clone the entire genome.
1. Recombinant DNA technology involves manipulating DNA from different species and combining them to form new recombinant DNA molecules.
2. Key steps include using restriction enzymes to cut DNA at specific sites, and DNA ligase to join DNA fragments together into vectors like plasmids.
3. The recombinant DNA can then be replicated in host cells like bacteria to produce multiple copies for analysis.
This document discusses recombinant DNA technology. It begins by defining recombinant DNA as DNA molecules formed by combining genetic material from multiple sources using genetic engineering techniques. The key steps involved are isolating genetic material, restriction enzyme digestion, amplification via PCR, ligating DNA molecules, inserting the recombinant DNA into a host, and isolating recombinant cells. The document then discusses each step in more detail and provides examples of applications like insulin production and Bt cotton. It concludes by noting some limitations like potential environmental impacts and vulnerability of cloned populations.
Recombinant DNA technology involves manipulating DNA from different sources to produce novel DNA molecules. It has several key steps: isolating the desired DNA and vector, joining them using enzymes to create recombinant DNA, introducing this into a host cell, and selecting cells that express the gene. This technology has many applications including producing human insulin and growth hormones through bacteria, developing vaccines by cloning genes for antigens, and creating monoclonal antibodies. It allows mass production of important biological substances that were previously difficult to obtain.
This document summarizes the process of bacterial transformation. It explains that most bacteria can uptake DNA from the environment, but some require physical or chemical treatment to become competent. For E. coli, soaking cells in calcium chloride solution makes them competent by inducing DNA-binding proteins. Transformed cells are selected by plating on media containing antibiotics that plasmid genes confer resistance to. Recombinant plasmids are then identified by screening for disruption of plasmid genes like antibiotic resistance or beta-galactosidase expression.
The document reports on DNA and protein techniques practicals performed by Kariuki Samuel Mundia. It describes cloning the nanobody gene using restriction digestion of the nanobody PCR fragment and PHEN6c plasmid with Eco911 and PstI enzymes. The DNA fragments were purified and ligated together. It also details plasmid isolation from E. coli cells and generating calcium chloride competent E. coli cells for heat shock transformation of the ligation mixture. The goal was to amplify the nanobody gene by inserting it into the PHEN6c plasmid vector and transforming it into E. coli cells.
Recombinant DNA technology has led to many advancements in medicine and biotechnology. Key developments include:
1. The production of human insulin through recombinant DNA techniques, approved for use in 1982. This provided an effective treatment for diabetes and was the first commercial product of rDNA technology.
2. Vaccines produced using recombinant DNA techniques, such as vaccines for hepatitis B. This involves inserting the gene for the hepatitis B surface antigen protein into yeast which then produces the protein for the vaccine.
3. Production of other proteins through recombinant DNA like cytokines and interferons, which help modulate the immune system and can be used as treatments for various conditions. Overall, recombinant DNA technology has generated many commercially useful proteins and
Transfection is a technique used to insert foreign nucleic acids like DNA or RNA into cells to alter their properties. There are various biological, chemical, and physical methods to accomplish transfection, either transiently or stably. In stable transfection, the foreign DNA integrates into the cellular genome and is passed to daughter cells, while transient transfection only expresses the DNA for a short time without integration. Genetic engineering techniques are used to transfer genes between organisms, like retrovirus-mediated gene transfer where the retrovirus acts as a vector to deliver transgenes into host cells. Embryonic stem cell-mediated gene transfer involves introducing DNA into stem cells that can integrate randomly or through homologous recombination and be passed to offspring. Liposome
Recombinant DNA technology involves combining DNA molecules from different sources and introducing them into host organisms. Some key points:
- Recombinant DNA is produced by joining DNA fragments from different sources using restriction enzymes and DNA ligase.
- Plasmids and bacterial cells are commonly used as vectors to replicate and express recombinant DNA. Foreign DNA is inserted into plasmids which are then introduced into bacterial cells.
- Restriction enzymes from bacteria are used to cut DNA at specific sequences. This allows insertion of foreign DNA. DNA ligase joins the DNA fragments back together.
- Applications include production of therapeutic proteins, genetic testing, gene therapy, and genetically modified crops. Recombinant DNA technology
This study investigates the localization and function of VDAC4, a porin protein in Tetrahymena thermophila mitochondria. Bioinformatics analysis predicted VDAC4 contains a conserved Porin3 domain found in mitochondrial porins and Tom40 proteins. The researcher amplified and cloned the VDAC4 gene, created a YFP fusion construct, transformed Tetrahymena cells, and observed YFP localization using microscopy. YFP localized to punctate structures consistent with mitochondrial localization, supporting VDAC4 involvement in mitochondrial membrane processes.
1. The document describes an experiment to isolate plasmid DNA from E. coli bacteria transformed with the pGLO plasmid.
2. The plasmid DNA was isolated using a modified alkaline lysis method. Samples of the isolated plasmid DNA were run on a gel electrophoresis along with a ladder for comparison but the ladder showed an unusual single band.
3. While single bands were observed for the plasmid DNA samples, the abnormal ladder prevented further analysis of the isolated plasmid DNA.
The content is about the general description of genetic material and further two techniques of biotechnology. The content includes two topics.
Firstly with introduction to biotechnology it describe about DNA, recombinant DNA (rDNA) technology, history, goals, procedure of rDNA technology, tools, techniques, application, demerits and products of rDNA technology.
Second portion entitiled as Hybridoma technology. this includes the basic principle, production of monoclonal antibodies, merits demerits and drugs from monoclonal antibody.
New pharmaceuticals derived from biotechnology is covered in last. All the content is referred from books and internet sources.
This document outlines a lecture on microbial genetics. It discusses the genetic engineering processes used to produce human insulin and growth hormone (HGH) in bacteria. For insulin, the human gene is isolated and inserted into bacterial plasmid DNA using restriction enzymes. Transformed bacteria then produce insulin. A similar process is used for HGH, isolating its mRNA from human pituitary gland cells, generating cDNA, and inserting it into bacteria to produce HGH. Lastly, it notes other human hormones can be genetically engineered using these techniques.
This document describes a study that investigated the effects of an extract from the cyanobacteria Aphanizomenon flos-aquae (AFAe) on human natural killer (NK) cells in vitro. The key findings were:
1) AFAe directly activated NK cells, as shown by increased expression of the activation markers CD69 and CD25 on CD3-CD56+ NK cells after 18 hours of exposure.
2) The low-molecular-weight fraction (<5,000 Da) of AFAe induced the strongest NK cell activation, suggesting novel activating compounds.
3) NK cell activation by AFAe required the presence of other immune cells such as monocytes, as
This document provides an overview of techniques for exploring genes, including DNA purification, restriction enzymes, recombinant DNA technology, molecular cloning using vectors, DNA libraries, blotting techniques, DNA sequencing, PCR, and gene expression studies. Key concepts covered include the types of DNA, plasmid purification, DNA conformations, nucleic acid quantitation, gel electrophoresis, restriction enzyme recognition sites and nomenclature, recombinant DNA technology tools, molecular cloning steps and components of cloning vectors. Applications of these techniques such as genetically modified organisms, recombinant proteins, gene therapy, and CRISPR are also discussed.
Recombinant DNA technology uses restriction enzymes and DNA ligase to cut and join DNA molecules from different species, generating novel genetic combinations. This document outlines the history, basic steps, tools, and applications of recombinant DNA technology. Key applications include producing human insulin, growth hormone, interferon, hepatitis B vaccine, tissue plasminogen activator, and erythropoietin to treat various diseases. Recombinant DNA technology has generated billions in pharmaceutical revenue and provides substantial quantities of therapeutic proteins that were previously unavailable.
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.
Recombinant DNA Technology - A Perforated Insight By Rxvichu !!RxVichuZ
Hello friends................................this is me....Vishnu,back after a gap of almost three weeks, with another powerpoint presentation on RECOMBINANT DNA TECHNOLOGY....
Prepared as per Pharmacology syllabus for thrird year Pharm.D students, this 40-slide ppt involves precise details on the following aspects:
1. DEFINITION
2. PRINCIPLES INVOLVED
3. ENZYMES AND VECTORS USED
4. METHODS OF GENE TRANSFER
5. APPLICATIONS OF RECOMBINANT DNA TECHNOLOGY
Not just for Pharm.D , i do sincerely hope that it will be useful for others, on a study or reference cum reading basis too...............
Do post ur feedbacks....
Suggestions for further improvement will be gratefully acknowledged..........
For further details, connect to me in :
Facebook : Rx Vishnu
Gmail: rxvichu623@gmail.com
watsapp and hike: 8086948729
communication: the same number as above.
Keep reading well....study well.................keep rocking!!!
@rxvichu-live 4 more!!!!
:) :)
This document provides an overview of recombinant DNA technology. It defines recombinant DNA technology as procedures that allow DNA from different species to be isolated, cut, and spliced together to form new recombinant molecules. The key steps described are using restriction enzymes to cut DNA at specific sites, inserting genes into bacterial plasmids, transforming bacteria, replicating the recombinant DNA as the bacteria divide, and collecting the amplified genes. Applications discussed include producing insulin, vaccines, human growth hormones, diagnosing infectious diseases, developing novel crop varieties, and industrial strain improvement.
The document discusses the principles and processes of biotechnology. It describes two core techniques that enabled modern biotechnology: genetic engineering and maintaining sterile environments for microbial growth. It then discusses various tools used in recombinant DNA technology, including restriction enzymes, vectors, competent hosts, PCR amplification, and downstream processing to obtain recombinant products.
Genetic engineering and Recombinant DNAHala AbuZied
Genetic engineering involves altering the DNA of living organisms using biotechnology. It includes techniques like changing single DNA base pairs, deleting or adding genes, or combining DNA from different species. Recombinant DNA technology is used to create recombinant DNA molecules by manipulating DNA in vitro and introducing them into host organisms. This allows bacteria to be engineered to produce human insulin through inserting the human insulin gene into bacterial plasmids. Genomic libraries can be created by ligating fragmented genomic or cDNA into plasmid vectors to transform bacteria and clone the entire genome.
1. Recombinant DNA technology involves manipulating DNA from different species and combining them to form new recombinant DNA molecules.
2. Key steps include using restriction enzymes to cut DNA at specific sites, and DNA ligase to join DNA fragments together into vectors like plasmids.
3. The recombinant DNA can then be replicated in host cells like bacteria to produce multiple copies for analysis.
This document discusses recombinant DNA technology. It begins by defining recombinant DNA as DNA molecules formed by combining genetic material from multiple sources using genetic engineering techniques. The key steps involved are isolating genetic material, restriction enzyme digestion, amplification via PCR, ligating DNA molecules, inserting the recombinant DNA into a host, and isolating recombinant cells. The document then discusses each step in more detail and provides examples of applications like insulin production and Bt cotton. It concludes by noting some limitations like potential environmental impacts and vulnerability of cloned populations.
Recombinant DNA technology involves manipulating DNA from different sources to produce novel DNA molecules. It has several key steps: isolating the desired DNA and vector, joining them using enzymes to create recombinant DNA, introducing this into a host cell, and selecting cells that express the gene. This technology has many applications including producing human insulin and growth hormones through bacteria, developing vaccines by cloning genes for antigens, and creating monoclonal antibodies. It allows mass production of important biological substances that were previously difficult to obtain.
This document summarizes the process of bacterial transformation. It explains that most bacteria can uptake DNA from the environment, but some require physical or chemical treatment to become competent. For E. coli, soaking cells in calcium chloride solution makes them competent by inducing DNA-binding proteins. Transformed cells are selected by plating on media containing antibiotics that plasmid genes confer resistance to. Recombinant plasmids are then identified by screening for disruption of plasmid genes like antibiotic resistance or beta-galactosidase expression.
The document reports on DNA and protein techniques practicals performed by Kariuki Samuel Mundia. It describes cloning the nanobody gene using restriction digestion of the nanobody PCR fragment and PHEN6c plasmid with Eco911 and PstI enzymes. The DNA fragments were purified and ligated together. It also details plasmid isolation from E. coli cells and generating calcium chloride competent E. coli cells for heat shock transformation of the ligation mixture. The goal was to amplify the nanobody gene by inserting it into the PHEN6c plasmid vector and transforming it into E. coli cells.
Recombinant DNA technology has led to many advancements in medicine and biotechnology. Key developments include:
1. The production of human insulin through recombinant DNA techniques, approved for use in 1982. This provided an effective treatment for diabetes and was the first commercial product of rDNA technology.
2. Vaccines produced using recombinant DNA techniques, such as vaccines for hepatitis B. This involves inserting the gene for the hepatitis B surface antigen protein into yeast which then produces the protein for the vaccine.
3. Production of other proteins through recombinant DNA like cytokines and interferons, which help modulate the immune system and can be used as treatments for various conditions. Overall, recombinant DNA technology has generated many commercially useful proteins and
Transfection is a technique used to insert foreign nucleic acids like DNA or RNA into cells to alter their properties. There are various biological, chemical, and physical methods to accomplish transfection, either transiently or stably. In stable transfection, the foreign DNA integrates into the cellular genome and is passed to daughter cells, while transient transfection only expresses the DNA for a short time without integration. Genetic engineering techniques are used to transfer genes between organisms, like retrovirus-mediated gene transfer where the retrovirus acts as a vector to deliver transgenes into host cells. Embryonic stem cell-mediated gene transfer involves introducing DNA into stem cells that can integrate randomly or through homologous recombination and be passed to offspring. Liposome
Recombinant DNA technology involves combining DNA molecules from different sources and introducing them into host organisms. Some key points:
- Recombinant DNA is produced by joining DNA fragments from different sources using restriction enzymes and DNA ligase.
- Plasmids and bacterial cells are commonly used as vectors to replicate and express recombinant DNA. Foreign DNA is inserted into plasmids which are then introduced into bacterial cells.
- Restriction enzymes from bacteria are used to cut DNA at specific sequences. This allows insertion of foreign DNA. DNA ligase joins the DNA fragments back together.
- Applications include production of therapeutic proteins, genetic testing, gene therapy, and genetically modified crops. Recombinant DNA technology
This study investigates the localization and function of VDAC4, a porin protein in Tetrahymena thermophila mitochondria. Bioinformatics analysis predicted VDAC4 contains a conserved Porin3 domain found in mitochondrial porins and Tom40 proteins. The researcher amplified and cloned the VDAC4 gene, created a YFP fusion construct, transformed Tetrahymena cells, and observed YFP localization using microscopy. YFP localized to punctate structures consistent with mitochondrial localization, supporting VDAC4 involvement in mitochondrial membrane processes.
The document describes electrosomes, which are a novel surface display system composed of enzymes attached to a scaffoldin protein. This allows for multiple electron release from fuel oxidation. In the anode, an ethanol oxidation cascade is assembled using alcohol dehydrogenase and formaldehyde dehydrogenase enzymes attached to the scaffoldin. In the cathode, copper oxidase is attached for oxygen reduction. The electrosomes provide advantages as a fuel cell and drug delivery system by catalyzing chemical energy conversion to electricity and providing controlled drug release.
The electrosomes, a novel surface-display system based on the specific
interaction between the cellulosomal scaffoldin protein and a cascade of
redox enzymes that allows multiple electron-release by fuel oxidation. The
electrosomes is composed of two compartment:(i) a hybrid anode, which
consists of dockerin-containing enzymes attached specifically to cohesin sites
in the scaffoldin to assemble an ethanol oxidation cascade, and (ii) a hybrid
cathode, which consists of a dockerin-containing oxygen-reducing enzyme
attached in multiple copies to the cohesin-bearing scaffoldin.
Temp-Sensitive Inhibition of Development in Dictyostelium - Dev Bio 251 18-26...James Silverman
1) The Dictyostelium mutant HSB1 is temperature-sensitive for development, aggregating and forming fruiting bodies below 18°C but not above.
2) HSB1 cells have a defective G protein-linked adenylyl cyclase that is not stimulated by GTPγS in vitro but can be rescued by adding wild-type cytosol.
3) Transfection with the wild-type piaA gene rescued the HSB1 mutant phenotype, and sequencing revealed a point mutation in the HSB1 piaA gene resulting in a single amino acid change.
The document discusses cloning and expressing the envelope (E) protein gene from West Nile virus. Key points:
- The E gene was amplified from virus cDNA using PCR and cloned into a plasmid vector. It was then inserted into an expression vector and transformed into E. coli cells.
- Expression was induced and the recombinant E protein was purified. It had a molecular weight of 73 kDa as shown by SDS-PAGE.
- Western blot analysis confirmed the protein was antigenic. Rabbits were immunized with the protein to evaluate its ability to stimulate an immune response against West Nile virus.
This document discusses materials and methods used in a study involving the chemical fipronil and zinc. Twenty male albino rats were divided into four groups of five rats each: a control group, a zinc group that received zinc supplementation, a fipronil group exposed to the insecticide fipronil, and a combination group exposed to both zinc and fipronil. Biochemical assays were conducted to assess oxidative stress markers like superoxide dismutase, catalase, glutathione peroxidase, glutathione-S-transferase, glutathione, lipid peroxidation, and total protein in the rats. Chemicals used including fipronil and zinc sulfate were obtained from reputable suppliers. Kits for the biochemical assays were purchased from a diagnostic
The study aimed to test the specificity of two antibodies (033 and 1-9E10) for binding the c-myc protein. Human colon carcinoma cells were extracted to obtain cytosolic, nuclear, and nuclear pellet extracts. The 033 antibody bound c-myc in the cytosolic extract at approximately 110 kDa, but the 9E10 antibody did not bind c-myc. This suggests c-myc is not restricted to the nucleus and more research is needed to fully understand c-myc and its role in cancer.
This document summarizes a novel micro-emulsion technology called Phage Emulsion, Secretion, and Capture (ESCape) that can be used for the directed evolution of antibodies. The technology utilizes water-in-oil emulsions to compartmentalize individual phage clones displaying antibodies so that they can be queried against antigens individually. This allows for finer discrimination of binding kinetics compared to traditional phage display methods. The document demonstrates that the technology can distinguish antibodies with a 300-fold difference in binding affinity and can be used to select antibodies with improved thermal stability.
Molecular Cloning of the Structural Gene for ExopolygalacturonateAlan Brooks
This document summarizes research on the cloning and characterization of a gene (pelX) from Erwinia chrysanthemi that encodes an exopolygalacturonate lyase (ExoPL). The pelX gene was cloned from a mutant strain lacking known pectate lyase genes. ExoPL was purified from a recombinant E. coli strain and characterized. A pelX mutant was constructed in E. chrysanthemi but retained pathogenicity, indicating ExoPL does not contribute to tissue maceration ability.
This document summarizes a plasmid lab report that used pUC19 plasmids as the vector for E. coli transformation due to its small size, high uptake efficiency, and fast replication time. Key features of pUC19 include an origin of replication and multiple cloning sites. Transformed E. coli were able to grow on agar plates containing ampicillin due to the plasmid containing an ampicillin resistance gene. Non-recombinant E. coli colonies were blue due to expression of the lacZ gene, while recombinant colonies were white due to insertion of the CIH-1 gene within the multiple cloning sites.
DNA Barcoding of Stone Fish Uranoscopus Oligolepis: Intra Species Delineation...journal ijrtem
Abstract: The present study addresses this issue by examining the patterning of Cytochrome Oxidase I diversity in the stone fish Uranoscopus oligolepis the structurally diverse group of Family Uranoscopidae. The sequences were analyzed for their species identification using BOLD’s identification engine. The COI sequences of U. oligolepis from different geographical regions were extracted from NCBI for intra species variation analysis. All sequences were aligned using Clustal W. The sequences were trimmed using software and phylogenetic tree was constructed with bootstrap test. The results showed that the cytosine content was high (31%). The least molar concentration was observed in guanine (19.5%) and Adenine (19.6%). Thymine was the second predominant in molar concentration next to thymine which is followed by adenine. The G+C content was found to be 49.6% and A+T content was 50.4%. Leucine and Alanine content was high in the amino acid composition. From the study it is assumed that the mitochondrial gene COI can be the potential barcoding region to identify an organism up to the species level. Keywords: COI, intra species, Uranoscopus oligolepis, barcoding, phylogenetic
This is an internship report on molecular biology techniques, which was performed at PERD center under the guidance of Dr. Anshu Srivastava. This pdf contains all the basic information which is a preliminary requisite to know while approaching the molecular biology experimentally.
This document provides an overview of DNA cloning including:
1. The basic steps in DNA cloning including isolation of vector and gene source DNA, insertion into the vector, and introduction into cells.
2. Uses of polymerase chain reaction and restriction enzymes in cloning.
3. Applications of cloning such as recombinant protein production, genetically modified organisms, DNA fingerprinting, and gene therapy.
Biolostic transformation of a procaryote, bacillus megateriumCAS0609
This document describes a new method for transforming the bacterium Bacillus megaterium using biolistic transformation. Key findings include:
1) Plasmid DNA was coated onto tungsten microprojectiles and accelerated into B. megaterium cells using a helium-driven biolistic device.
2) Over 104 transformants per treated plate were obtained after optimizing biological and physical parameters of the biolistic process.
3) All strains of B. megaterium tested were successfully transformed, though efficiency varied between strains. This is the first report of biolistic transformation of a prokaryote.
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Similar to Kariuki group nanobody expression artical (20)
1. VRIJE UNIVERSITEIT BRUSSEL
INSTITUTE OF MOLECULAR BIOLOGY AND
BIOTECHNOLOGY
TITLE: FINAL ARTICLE PRACTICAL REPORT (SEMESTER 1, 2011)
NAMES: Kariuki S., E. Kamani and A. Garba
INSTRUCTOR: Steven Odongo
DATE OF SUBMISSION: 13/1/2012
2. Recombinant Nanobody™ expression in E. coli, extraction and purification.
Authors names: Kariuki S., E. Kamani and A. Garba.
Institute of Molecular Biology and Biotechnology, Building E, Faculty of Science, Vrije Universiteit Brussel,
Brussels, Belgium.
ABSTRACT:
Nanobody is a fragment antibody consisting of a single monomeric variable antibody domain lacking the
light chains and the CH1 domain of the heavy chain derived from camelides (dromedaries, camels,
Llamas and alpacas). They are less lipophilic and better soluble in water owing to their CDR3 which
forms an extended loop covering the lipophilic site that normally binds to light chains. This property
makes it easier to grow them in bacteria cells and contributes to their therapeutic usefulness.
Escherichia coli WK6 were transformed with pHEN6c containing the nanobody gene with 1M IPTG
(Isopropyl β-D-1 thiogalactopyranoside) to induce expression of the gene and ampicillin-containing LB
media for selection. The cells were harvested by using Beckman Coulter Avanti J-E centrifuge with rotor
JA-10 before lysing through osmotic shock since our protein was expressed in the periplasmic space of
E.coli cells.
Protein purification was done using immobilized metal affinity chromatography (IMAC) with nickel bead
slurry that binds the C-terminal histidine tail tag of our nanobody followed by elution through
competitive action of imidazole buffer to the nickel beads.
Finally the nanobody was analyzed using SDS-PAGE and Western blot. Since SDS-PAGE separates
proteins based primarily on their molecular weight it was possible to determine the molecular weight of
the nanobody. Western blot was used to confirm expression of nanobody in WK6 E.coli by probing with
an antihis-antibody, the 6x histidine tail attached to the C-terminal end of the nanobody.
Key Words: Nanobody, Recombinant, Affinity Chromatography, Western blot, SDS-PAGE,
Coomassie blue.
Abbreviations: HRP, Horse Radish Peroxidase, IPTG,Isopropyl β-D-1-thiogalactopyranoside,
SDS-PAGE, Sodium Dodecyl Sulfate-Polyacrylamide Agarose Gel Electrphoresis, HIS, Histidine,
BSA, Bovine Serum Albumin, TB, Terrific broth, LB, Luria Broth, IMAC, Immobilized Metal Affinity
Chromatography.
3. 1: INTRODUCTION.
Recombinant protein expression is an extension gene into E.coli plasmid (Cohen, S.; Chang, A.;
of gene expression through transcription, Boyer, H.; Helling, R.,1973). In our experiment,
translation and eventual folding of the protein. E.coli, WK6 was used.
Recombinant proteins show large variability in
terms of their expression, solubility, stability, To achieve a high gene dosage, the cDNA is
and functionality making them difficult targets typically cloned in a plasmid that replicate in a
for large scale production and analyses. relaxed fashion inside a bacteria cell. It is
However greater advancement has been made usually engineered to contain a regulatory
towards solving to improve these features. sequence that act as an enhancer and a
Among them is addition of protein fusion tags promoter region which lead to the efficient
transcription of the gene carried by the
which has improved expression, solubility and
production of biologically active proteins expression vector. In addition a selectable
especially those difficult-to-express-proteins. marker in form of antibiotic resistance, reporter
Genetically engineered tags allow the and a multiple cloning site are required (Amann
purification of the protein without prior E, Brosius J, Ptashne M., 1983). The multiple
knowledge of its biochemical activity (Esposito cloning sites has restriction site with various
D, Chatterjee DK.2006 and Arnau J, Lauritzen C, restriction endonucleases which are molecular
scapels that cut double stranded DNA at
Petersen GE, Pedersen, J.2006). We used a
genetically engineered histidine tag not only for particular recognition nucleotide sequences.
the purification purpose but also in probing These enzymes found in bacteria and archea are
with antihis-antibody. The 6x histidine tag thought to have evolved as defence mechanism
attached to the C-terminal end of the nanobody against vises (Roberts RJ; Murray, Kenneth,
bound to immobilized nickel beads acted as an 1976). The host protects itself by methylation
electron donor thereby detaining the protein in by modification enzyme methylase (Kobayashi
the column and latter eluted by addition of I.,2001). In our experiment the nanobody gene
imidazole which dislodged the histidine tails was ligated to e pHEN6c plasmid which was
used to transform WK6 E.Coli.
from the nickel beads in a competitive fashion.
Escherichia coli is one of the most widely used SDS-PAGE, a technique used to separate
for production of recombinant proteins and its proteins based on their molecular weight was
genetics is the better studied than any other used in analyzing the nanobody. Sodium
microorganism. The understanding of its Dodecyl Sulfate (SDS) is a detergent used to
transcription, translation and gene expression denature the proteins allowing the proteins to
has positioned it as valuable bacterium in exist stably in an extended conformation hence
expression of complex eukaryotic proteins. In they migrate through the pores of the gel
irrespective of their hydrodynamic properties.
addation, E.coli grows rapidly and at high
density in relatively an inexpensive In addition SDS covers all the protein with
media.Expresiion of eukaryotic proteins in negative charges which allows them to migrate
bacteria started with the pioneering work done to the positive electrode. Since polyacrylamide
by Boyer and Cohen when they inserted a frog gel is not solid but rather a meshwork of
4. labyrinth of tunnels, the protein is able to go 2.2 Extraction of the expressed protein
through with the help of electric currents.
After overnight expression, the cells of the WK6
Western blotting was used to confirm E.coli were harvested; 330ml of WK6 E.coli
expression of the nanobody protein. This culture Was centrifuged at 8000rpm for 8
technique detects proteins in minute quantities minutes at the temperature of 140C before
after immobilizing on a gel followed by transfer discarding the supernatant, and centrifugation
to a nitrocellulose membrane. repeated until all cells were harvested. The
pellets were Re-suspended using 12ml Tris
Antibodies and antibody fragments are EDTA sucrose (TES) per pellet, from WK6. E.coli
exclusively applied in human therapy and overnight culture and the mixture incubated for
diagnosis. They are highly specific making them 1 hour on ice while shaking at 200rpm on a
suitable for their uses. However, production of
table shaker. The mixture was supplemented
antibodies via hybridoma technology with 100ml 2M Mgcl2 and centrifuged at
discovered by Cesar Miltein and Georges J. F. 8000rpm for 30 minutes and the periplasmic
Kohler in 1975 (Nelson, PN et all, 2000) remains extract was pipetted into 50ml falcon tube.
expensive and difficult. Camelidae produced a
substancial proportion of their functional 2.3 Protein purifications; Analysis of
immunoglobulins as homodimer of heavy expression and purity of protein sample by
chains, lacking light chains (S. Muyldermans, SDS-PAGE
2001). Since the discovery of camelide antidoby
lacking light chains and CH1 groups domains, The 6x his-tagged Nanobody was purified by
their variable heavy chain domains (VHH) have affinity chromatography,
been proposed as valuable potential tools for HIS-select solution was solubilized to slurry
biotechnology (Hamer C. et all, 1993). and together with periplasmic extract incubated
for 1 hour with shaking. The mixture of
periplasmic extract and HIS-select solution was
2.0 METHODOLOGY loaded onto the column, after which HIS-select
column was washed with 20ml PBS. The PBS
2.1 Expression of Nanobodies in WK6 E.coli buffer was allowed to drain and solution
cells periplasm. collected. The Nanobody was eluted using PBS
WK6 E.coli cells were used for expression of Buffer supplemented with 4ml, 0.5M imidazole
Nanobody. Bacteria were grown at standard and the elute absorbance measured at 280nm.
conditions, at the temperature of 37˚c and The 12% running gel was constituted using
incubated overnight using TB media 4.0ml, 30% Acrylamide/bisacrylamide, 2.5ml
supplemented with ampicillin, glucose and ,1.5M Tris HCl, pH 8.8,3.4ml distilled water,
magnesium chloride in a baffle shaker flask. 100µl 10% SDS,100µl 10% APS, and 5µl TEMED.
IPTG was used to induce expression of the The solution was carefully introduced into gel
Nanobody gene. sandwich, until 0.5cm below the level where
teeth of the comb will reach.1-5mm layer of
5. water was made on top of the separating gel. combs were removed. The gel was placed into
After the gel polymerized the water discarded. electrophoresis chamber and electrophoresis
buffer was added to the inner and outer
Also 4% stacking gel was constituted using 30% reservoir.
0.650 Acrylamide/bisacrylamide, 0.650 1.0M
Tris pH 6.8, 3.645 distilled water, 50µl 10% SDS, The wells were marked 1-6, with different
25µl 10% APS and 5µl TEMED. The stacking gel sample added as; Maker sample, uninduced
was introduced into gel sandwich until solution sample unpurified protein sample, flow through
reached the top of front plate. sample, wash sample, and Eluted sample. 20µl
each of these samples mixed with 5µl of 1x
The combs later were carefully inserted and the sample buffer heated at 100˚c for 5 minutes
gel allowed to polymerize. After which the and added to the wells.
3: RESULTS.
3.1 Expression and protein extraction from WK6 E.coli
Following expression usind IPTG and protein extraction through osmotic shock, the concentration of the
protein was measured using a Nanodrop™ at 280nm and found to be 119µg/µl
3.2 Running Gel And staining with Commasie Blue on SDS-PAGE
The gel was run and then transferred to a small container, containing 20ml commasie blue. The gel was
distained later using commasie distainer, after the bands were visible. Gels were preserved for
molecular weight determination.
MK UI UP FT WS ET
15kDa
10kDa
Figure 1 showing the results of SDS-PAGE, MK is the marker, UI, uninduced sample, UP, unpurified protein, FT, flow through,
WS, wash, ET, elute sample.
3.3 Confirming protein Expression by western-blot and immunodetection.
12% SDS-PAGE Gel was run, and the protein bands transferred from the gel to nitrocellulose membrane
using 35ml transfer buffer.
6. The membrane was transferred to a smaller container, with 8ml 3% PBST buffer. And membrane
blocked using 15ml 1% milk solution to prevent unspecific binding.
Antibodies were poured into the solution of buffer containing the membrane, the membrane washed
with TBS 3 times, and second Antibody which had a HRP conjugate was added and washed again before
adding the developing reagent for HRP and observed for 30minute. The membrane was washed , dried
and scanned.
MK UI UP FT WS ET
15kDa
10kDa
Figure 2, The results of Western blotting after immune-blotting. MK is the marker, UI, uninduced sample, UP, unpurified
protein, WS, wash, ET, elute.
3.3 Calculations for protein molecular weight determination from SDS-PAGE:
The molecular weight of an unknown protein was estimated by comparing its distance of migration
in a gel with that of the standards. A plot of log of the molecular weight (in kDa) of each band of
standard (Y) was done against relative distance traveled from the well (X). A line of best fit was
drawn connecting the points and molecular weight of protein was determined.
Relative distance travelled = Distance of Protein migration from the origin
Distance of migration of dye from the origin
7. Table 1, Showing values of migration fronts (Rf) and log of molecular weight
Relative Molecular Weight of
Migration of standards(cm) distances (X) standard. Log molecular weight (Y)
1.9 0.18 170 2.23
2.1 0.2 130 2.11
2.4 0.23 100 2
2.9 0.28 70 1.85
3.4 0.32 55 1.74
4 0.38 40 1.6
4.7 0.45 35 1.54
5.5 0.52 25 1.4
7.2 0.69 15 1.18
9.1 0.87 10 1
Sample Migration=7.6cm 0.72
Dye migration distance=10.5cm
Protein Molecular Weight determination
2.5
Log of Molecular Weight
2
1.5
1
y = -1.7178x + 2.3727
0.5
0
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1
Relative distances
Figure 3 Graph showing the relationship between molecular weight and distance travelled by the protein
8. From the equation of the line;
Y=-1.7178*0.72 + 2.3727
Y=1.135884. Protein molecular weight was obtained by calculating the antilog of 1.135884.
Protein Molecular weight =13.67kDa
4: DISCUSSION.
SDS-PAGE is a technique widely used in molecular biology, biochemistry, forensics, and genetics to
separate proteins according to their electrophoretic mobility, function of length of polypeptide chain or
molecular weight.
We determined the protein molecular weight in this experiment using a plot of log of molecular weight
(kDa) against relative distances on SDS-PAGE using a protein ladder as a standard .The molecular weight
of unknown protein was calculated as shown above indicating that the molecular weight of the protein
is 13.67 kDa .The calculation support with more confidence the prediction of the protein size .SDS PAGE
result shows that the protein was not well purified due to presence of extra small bands together with
the large and thick band of the protein of interest in elute sample.
In this experiment we successfully demonstrated the use of SDS-PAGE for protein purification and
determination of the molecular size of unknown protein sample using the empirical relationship
observed between log of molecular weight (kDa) and relative mobility on SDS-PAGE
Western blot analysis can detect protein of interest from a mixture of a great number of proteins.
Western blotting is useful to give information about the size of your protein with comparison to a size
marker or ladder in kDa, and also on protein expression. In our experiment we used Western blot
technique to confirm expression in WK6 E. coli. The Nanobody expressed contained 6x histidine tags
attached to the C-terminal end. His-tagged Nanobody was identified by probing with anti-his antibody.
The results (Fig 2) above show the Western blot results of different protein samples in different lanes.
On the second lane (UI) from the ladder, (pre induction with IPTG) there were nothing detected which
indicated there were no protein expressed. After induction with IPTG, we had a small band on UP lane
indicating that, there was protein expression. On the ET lane, a heavy band of our protein of interest
was detected and the size, determined using SDS-PAGE was approximately 13.67kDa).The sensitivity of
the assay was high and there were no contaminating bands in our results.
5: CONCLUSION.
In this experiment we were able to employ the use of Western blot and SDS-PAGE to demonstrate
expression of Nanobody containing 6xhistine tail and approximately estimate visually the size by
comparing with the ladder and by mathematical comparison of unknown protein distance of migration
in a gel with that of the standards.
9. 6: ACKNOWLEGMENT.
We acknowledge our practical instructor Steven Odongo for being helpful to us and for his technical
guidance and support during practical training. Not forgetting all the IPMB lecturers who provided us
with the necessary information on Molecular biology.
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plasmids in vitro". Proceedings of the National Academy of Sciences of the United States of America 70
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Odongo, S. IPMB General Practical Course Manual