Site-directed mutagenesis is a technique used to generate specific mutations in DNA at predetermined locations. It involves using a synthetic oligonucleotide primer containing the desired mutation to introduce changes into the DNA sequence during in vitro DNA replication or PCR. This allows researchers to study the effects of mutations and engineer proteins with improved or customized properties. Common methods for site-directed mutagenesis include using single or double primers, cassette mutagenesis by replacing DNA fragments, and PCR-based mutagenesis. The technique has various applications in investigating protein function and developing proteins for commercial uses.
The document discusses molecular diagnostics and genetic testing techniques. It provides an overview of molecular diagnostics, their significance in medicine, and how they are transforming fields like prenatal testing, disease detection, and drug selection. It then covers various immunological diagnostic methods like ELISA, radioimmunoassay, western blotting, and their characteristics. The document also discusses molecular genetic tests, genetic alterations detected, and techniques for DNA-based diagnosis of diseases. It focuses on the principles and procedures of molecular diagnostic methods like hybridization assays and PCR and their applications in detecting pathogens and genetic disorders.
Zinc finger nucleases (ZFNs) allow for highly targeted editing of the genome. ZFNs consist of a DNA-binding domain made of zinc finger proteins and a DNA-cleaving domain. The ZFN pair binds to a target site and creates a double-strand break, which the cell repairs through non-homologous end joining or homologous recombination, enabling gene knockouts or targeted changes. ZFNs work in many cell types and animal models, providing a more efficient alternative to traditional transgenic techniques. They have applications in functional genomics, cell line engineering, and animal model generation.
Cell culture based vaccine??
Cell cultures involve growing cells in a culture dish, often with a supportive growth medium. A primary cell culture consists of cells taken directly from living tissue, and may contain multiple types of cells such as fibroblasts, epithelial, and endothelial cells.
In the United States, 10 different vaccines for chicken pox, hepatitis A, polio, rabies, and rubella are cultured on aborted tissue from two fetal cell lines known as WI-38 and MRC-5. These vaccines are chicken pox, hep-A, hep-A, hep-A/hep-B, polio, rabies, rubella, measles/rubella, mumps/rubella, and MMR II (measles/mumps/rubella).
Genome editing techniques allow DNA to be inserted, deleted, modified or replaced in the genome of a living organism. Four families of engineered nucleases have been used for genome editing: meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the CRISPR/Cas9 system. Each system uses a different mechanism for recognizing and cutting DNA at specific locations to modify genes. The CRISPR/Cas9 system has become widely used due to its ease of design and lower off-target effects compared to other techniques.
This document provides guidance on how to write and publish a scientific paper. It discusses the key components of a scientific paper, including the title, authors, abstract, introduction, materials and methods, results, and discussion sections. The introduction should state the purpose and importance of the study and review relevant literature. The materials and methods section must provide enough detail that others could replicate the experiments. The results section should present representative data without interpretation. The discussion section should show relationships among facts and generalizations, not just recapitulate results. Overall, the goal is clear, logical communication of new scientific findings and conclusions.
Here are a few things not to include in a cover letter when submitting a revised manuscript:
- Details about previous rejections from other journals
- Criticism of previous reviewers/editors' assessments
- Apologies for lack of impact or interest
- Excessive focus on the manuscript's weaknesses or limitations
- Requests for special treatment or exceptions to normal policies
The cover letter should focus on addressing issues raised in the previous review, changes made to strengthen the work, and why the revised manuscript is a good fit for the journal. It's best to maintain a positive tone that emphasizes the manuscript's strengths and significance within the journal's scope.
A probe is a short sequence of DNA or RNA that is used to detect complementary DNA or RNA sequences in samples. Probes can be labeled with radioactive isotopes or fluorescent tags to allow for their detection after hybridizing to target sequences. They are commonly used techniques like Southern blots, Northern blots, and in situ hybridization to detect specific nucleic acid sequences and identify microorganisms, viruses, or genetic mutations associated with diseases. Probes provide a sensitive method for detecting nucleic acids and have many applications in medical research and diagnosis.
Site-directed mutagenesis is a technique used to generate specific mutations in DNA at predetermined locations. It involves using a synthetic oligonucleotide primer containing the desired mutation to introduce changes into the DNA sequence during in vitro DNA replication or PCR. This allows researchers to study the effects of mutations and engineer proteins with improved or customized properties. Common methods for site-directed mutagenesis include using single or double primers, cassette mutagenesis by replacing DNA fragments, and PCR-based mutagenesis. The technique has various applications in investigating protein function and developing proteins for commercial uses.
The document discusses molecular diagnostics and genetic testing techniques. It provides an overview of molecular diagnostics, their significance in medicine, and how they are transforming fields like prenatal testing, disease detection, and drug selection. It then covers various immunological diagnostic methods like ELISA, radioimmunoassay, western blotting, and their characteristics. The document also discusses molecular genetic tests, genetic alterations detected, and techniques for DNA-based diagnosis of diseases. It focuses on the principles and procedures of molecular diagnostic methods like hybridization assays and PCR and their applications in detecting pathogens and genetic disorders.
Zinc finger nucleases (ZFNs) allow for highly targeted editing of the genome. ZFNs consist of a DNA-binding domain made of zinc finger proteins and a DNA-cleaving domain. The ZFN pair binds to a target site and creates a double-strand break, which the cell repairs through non-homologous end joining or homologous recombination, enabling gene knockouts or targeted changes. ZFNs work in many cell types and animal models, providing a more efficient alternative to traditional transgenic techniques. They have applications in functional genomics, cell line engineering, and animal model generation.
Cell culture based vaccine??
Cell cultures involve growing cells in a culture dish, often with a supportive growth medium. A primary cell culture consists of cells taken directly from living tissue, and may contain multiple types of cells such as fibroblasts, epithelial, and endothelial cells.
In the United States, 10 different vaccines for chicken pox, hepatitis A, polio, rabies, and rubella are cultured on aborted tissue from two fetal cell lines known as WI-38 and MRC-5. These vaccines are chicken pox, hep-A, hep-A, hep-A/hep-B, polio, rabies, rubella, measles/rubella, mumps/rubella, and MMR II (measles/mumps/rubella).
Genome editing techniques allow DNA to be inserted, deleted, modified or replaced in the genome of a living organism. Four families of engineered nucleases have been used for genome editing: meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the CRISPR/Cas9 system. Each system uses a different mechanism for recognizing and cutting DNA at specific locations to modify genes. The CRISPR/Cas9 system has become widely used due to its ease of design and lower off-target effects compared to other techniques.
This document provides guidance on how to write and publish a scientific paper. It discusses the key components of a scientific paper, including the title, authors, abstract, introduction, materials and methods, results, and discussion sections. The introduction should state the purpose and importance of the study and review relevant literature. The materials and methods section must provide enough detail that others could replicate the experiments. The results section should present representative data without interpretation. The discussion section should show relationships among facts and generalizations, not just recapitulate results. Overall, the goal is clear, logical communication of new scientific findings and conclusions.
Here are a few things not to include in a cover letter when submitting a revised manuscript:
- Details about previous rejections from other journals
- Criticism of previous reviewers/editors' assessments
- Apologies for lack of impact or interest
- Excessive focus on the manuscript's weaknesses or limitations
- Requests for special treatment or exceptions to normal policies
The cover letter should focus on addressing issues raised in the previous review, changes made to strengthen the work, and why the revised manuscript is a good fit for the journal. It's best to maintain a positive tone that emphasizes the manuscript's strengths and significance within the journal's scope.
A probe is a short sequence of DNA or RNA that is used to detect complementary DNA or RNA sequences in samples. Probes can be labeled with radioactive isotopes or fluorescent tags to allow for their detection after hybridizing to target sequences. They are commonly used techniques like Southern blots, Northern blots, and in situ hybridization to detect specific nucleic acid sequences and identify microorganisms, viruses, or genetic mutations associated with diseases. Probes provide a sensitive method for detecting nucleic acids and have many applications in medical research and diagnosis.
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.
Gene knockout is a technique used to study gene function by inactivating genes in living organisms. It involves using gene targeting to disrupt a gene, preventing it from functioning normally. Researchers developed methods for knocking out genes in mice using embryonic stem cells, which won them the 2007 Nobel Prize in Physiology or Medicine. The basic process involves engineering a construct to disrupt a target gene, introducing it into embryonic stem cells, generating a knockout mouse, and studying the effects of the disrupted gene. Gene knockout is a valuable tool for biomedical research and understanding disease mechanisms.
This document provides an overview of gene silencing. It begins with definitions of gene silencing and discusses how it differs from gene knockout. The document then covers the short history of gene silencing research from the 1990s onwards. It describes different methods of gene silencing including transcriptional gene silencing and post-transcriptional gene silencing. Specific gene silencing techniques like RNA interference are explained in more detail. The document also includes a case study on gene silencing in petunias and discusses applications of RNAi.
Gene editing using CRISPR was originally discovered as a bacterial immune system that provides resistance to viruses. CRISPR uses specialized DNA sequences and associated Cas proteins to create targeted double-strand breaks in DNA, allowing modification of genomes. The technique has rapidly advanced due to its simplicity and versatility compared to prior tools. CRISPR holds promise for treating genetic diseases, transplantation, biotechnology, disease models, and more. It has become a widely used research tool with many companies and publications emerging around its applications.
Scientific writing is not just writing about science; it is the technical writing that scientists do to communicate their research to others. Scientific writing is predicated on the rigors of scientific inquiry, so it must reflect the same precision as that demanded in the research process.
This document discusses molecular probes, including their definition, types, preparation, and labeling. It describes the three main types of probes - oligonucleotide probes, DNA probes, and RNA probes. It explains how to prepare probes from genomic DNA, cDNA, synthetic oligonucleotides, and RNA. Methods of radioactive labeling including nick translation and oligonucleotide labeling are covered. Non-radioactive labeling using biotin and digoxigenin is also discussed. Finally, applications of molecular probes in identification of recombinant clones, fingerprinting, in situ hybridization, and medical research are summarized.
Whole genome sequencing is a technique to sequence the entire genome of an organism. It involves breaking the genome into small fragments, copying the fragments, sequencing the fragments, and reassembling the sequence data into the full genome. Key steps include isolating DNA, fragmenting it, ligating fragments into plasmids, amplifying the plasmids, sequencing the fragments using Sanger sequencing, and assembling the sequence reads into the complete genome. Whole genome sequencing allows researchers to discover coding and non-coding regions, predict disease susceptibility, and perform evolutionary studies by comparing species.
Physical methods can be used to transfer genes into cells by transiently permeabilizing cell membranes. This allows naked DNA to enter cells. Key physical methods include electroporation, gene guns, ultrasound, and hydrodynamic delivery.
Electroporation uses short electrical pulses to create temporary pores in the cell membrane through which DNA can enter. Gene guns use compressed gas to accelerate DNA-coated gold or tungsten particles into cells. Ultrasound combines microbubbles and acoustic cavitation to permeabilize membranes for DNA uptake. Hydrodynamic delivery involves rapid injection of a large DNA solution volume to generate pressure forcing DNA into organ cells like hepatocytes. These methods show promise but also have limitations like tissue damage, shallow
A bacterial plasmid is a short, usually circular, and double-stranded segment of DNA that is found in the cytoplasm separate from the main bacterial chromosome. This presentation contains plasmid features, replication, classification and its uses.
The document discusses the process of publishing scientific papers, including tips for writing and submitting research. It notes that scientific papers are intended to share original research with other scientists and involve peer review. The five steps of publication are outlined as preparing the paper, identifying the appropriate journal, rechecking the paper, submitting it, and addressing feedback. Key tips include focusing on innovative aspects, clearly structuring the paper, using appropriate methodologies, and being patient during the writing and review process.
E. Coli is a common bacterium found in the intestines of humans and other warm-blooded organisms. It can exist harmlessly or cause food poisoning. E. Coli reproduces through cell division and genetic transfer between F+ and F- cells. The life cycle involves conjugation where the F plasmid transfers DNA between cells. E. Coli is widely studied due to its rapid reproduction, hardiness, and ability to accept foreign DNA, making it useful for biotechnology and protein production.
TA cloning is a cloning technique that avoids restriction enzymes. It relies on the ability of adenine and thymine base pairs on different DNA fragments to hybridize when ligated together. The technique involves using a PCR product amplified with Taq polymerase, which leaves 3' adenine overhangs, and a linearized vector with 3' thymine overhangs. The complementary overhangs allow the PCR product to ligate directly into the vector. Examples of vectors designed for TA cloning, such as pLUG vectors, were also discussed. TOPO TA cloning uses topoisomerase enzyme to ligate PCR products to vectors. Ligation independent cloning uses annealing of single-stranded overhangs of at least 12 bases
To modifying the structure of a specific gene.
Gene targeting vector introduced into the cell.
Vector modifies the normal chromosomal gene through homologous recombination.
Useful in treating some human genetic disorders – Hemophilia, Duchenne Muscular Dystrophy.
Treating human diseases by genetic approaches – Gene Therapy.
Gene Therapy – Replacing the defective gene by normal copy of the gene.
Expressed sequence tag/EST is a short partial sequence, typically 200-400 bp long, of a complimentary DNA/Cdna.
EST is a short sub-sequence of a cDNA sequence.
Used to identify gene transcripts, and are instrumental in gene discovery and in gene-sequence determination.
Approximately 74.2 million ESTs are available in public databases.
EST results from one-short sequencing of a cloned cDNA.
Low-quality fragments.
Length is approximately 500 to 800 nucleotides.
pET vector. Plasmid for Expression by T7 RNA Polymerase.MuhammadMujahid58
The pET vector system is a powerful tool for expressing cloned genes in E. coli. It utilizes the strong T7 promoter to drive high-level expression of the gene when induced. The T7 promoter is tightly regulated by the Lac repressor protein so expression is low without induction. This prevents toxicity from overexpression. Key features include the T7 promoter, Lac repressor binding site, ribosome binding site, and antibiotic resistance gene. Expression is induced by adding IPTG which binds Lac repressor and allows transcription by T7 RNA polymerase. This results in high protein yields while avoiding metabolic burden on the host cell.
Genetic Engineering: Chapter 1- History of Genetic EngineeringHikmet Geckil
This document provides a history of genetic engineering. It discusses how genetic engineering began with selective breeding of plants and animals in prehistoric times. The modern field of genetic engineering began in 1973 when Herbert Boyer and Stanley Cohen accomplished the direct transfer of DNA between organisms. Since then, major breakthroughs included the discovery of restriction enzymes in 1969, the first recombinant DNA molecule in 1972, DNA sequencing in 1977, and PCR in 1983. Genetically modified foods and medicines have been commercialized since 1976. The latest developments include gene editing technologies and clinical applications of modified cells.
Bacteriophage vectors
Bacteriophage
WHY BACTERIOPHAGE AS A VECTOR?
M13 phage
Genome of m13 phage
Life cycle and dna replication of m13
CONSTRUCTION M13 AS PHAGE VECTOR
M13 MP 2 vector
M13MP7 VECTOR
Selection of recombinants
Lambda replacement vectors
LAMBDA EMBL 4 VECTOR
P1 PHAGE
GENOME OF P1 PHAGE
P1 PHAGE AS VECTOR
P1 phage vector system
This document provides guidance on writing scientific manuscripts. It discusses key sections of a manuscript such as the title, abstract, introduction, methods, results, discussion and references. It emphasizes logical organization, clear communication of methods and results, interpreting findings, and comparing results to prior literature. The document also offers tips for the writing process such as not procrastinating, having others review the work, and utilizing background from funded grants.
A transgenic animal is one that has had foreign DNA inserted into its genome. The first transgenic animal was a mouse created in 1982 by inserting a human growth hormone gene. Transgenic animals are created through pronuclear microinjection or stem cell methods. They have applications in medicine, agriculture, and industry. However, some argue that transgenic technology raises ethical issues.
Expression of recombinant proteins in mammalian cell linesSandeep Kumar
The speaker discusses mammalian cell-based recombinant protein production. Mammalian cells like CHO cells are commonly used as they can properly fold and modify proteins, similar to human cells. Issues include mammalian cells being fragile, slow-growing, and techniques being expensive. Benefits are low immunogenicity and high safety due to not being susceptible to human pathogens.
Pharmacogenetics is the study of genetic variations that influence individual responses to drugs. It aims to provide information to help doctors prescribe better and safer drugs at appropriate doses tailored to a patient's genetics. The study examines how genetic variations can affect drug metabolism and efficacy. Understanding a patient's genetic profile could help predict drug responses and prevent adverse reactions.
Biopharmaceuticals are large molecule drugs made using cells or enzymes, often similar to natural biological compounds. Examples include proteins, peptides, nucleic acids, and gene therapy. The first approved biopharmaceuticals were recombinant human insulin in 1982 and recombinant tissue plasminogen activator in 1986. Today, biopharmaceutical sales reach over $200 billion annually and there are over 300 approved biopharmaceutical drugs on the market. Biopharmaceuticals are mainly produced using bacterial, mammalian, yeast, plant, or animal cells transfected with plasmids containing the gene for the target protein. Purification then isolates the target protein for clinical use.
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.
Gene knockout is a technique used to study gene function by inactivating genes in living organisms. It involves using gene targeting to disrupt a gene, preventing it from functioning normally. Researchers developed methods for knocking out genes in mice using embryonic stem cells, which won them the 2007 Nobel Prize in Physiology or Medicine. The basic process involves engineering a construct to disrupt a target gene, introducing it into embryonic stem cells, generating a knockout mouse, and studying the effects of the disrupted gene. Gene knockout is a valuable tool for biomedical research and understanding disease mechanisms.
This document provides an overview of gene silencing. It begins with definitions of gene silencing and discusses how it differs from gene knockout. The document then covers the short history of gene silencing research from the 1990s onwards. It describes different methods of gene silencing including transcriptional gene silencing and post-transcriptional gene silencing. Specific gene silencing techniques like RNA interference are explained in more detail. The document also includes a case study on gene silencing in petunias and discusses applications of RNAi.
Gene editing using CRISPR was originally discovered as a bacterial immune system that provides resistance to viruses. CRISPR uses specialized DNA sequences and associated Cas proteins to create targeted double-strand breaks in DNA, allowing modification of genomes. The technique has rapidly advanced due to its simplicity and versatility compared to prior tools. CRISPR holds promise for treating genetic diseases, transplantation, biotechnology, disease models, and more. It has become a widely used research tool with many companies and publications emerging around its applications.
Scientific writing is not just writing about science; it is the technical writing that scientists do to communicate their research to others. Scientific writing is predicated on the rigors of scientific inquiry, so it must reflect the same precision as that demanded in the research process.
This document discusses molecular probes, including their definition, types, preparation, and labeling. It describes the three main types of probes - oligonucleotide probes, DNA probes, and RNA probes. It explains how to prepare probes from genomic DNA, cDNA, synthetic oligonucleotides, and RNA. Methods of radioactive labeling including nick translation and oligonucleotide labeling are covered. Non-radioactive labeling using biotin and digoxigenin is also discussed. Finally, applications of molecular probes in identification of recombinant clones, fingerprinting, in situ hybridization, and medical research are summarized.
Whole genome sequencing is a technique to sequence the entire genome of an organism. It involves breaking the genome into small fragments, copying the fragments, sequencing the fragments, and reassembling the sequence data into the full genome. Key steps include isolating DNA, fragmenting it, ligating fragments into plasmids, amplifying the plasmids, sequencing the fragments using Sanger sequencing, and assembling the sequence reads into the complete genome. Whole genome sequencing allows researchers to discover coding and non-coding regions, predict disease susceptibility, and perform evolutionary studies by comparing species.
Physical methods can be used to transfer genes into cells by transiently permeabilizing cell membranes. This allows naked DNA to enter cells. Key physical methods include electroporation, gene guns, ultrasound, and hydrodynamic delivery.
Electroporation uses short electrical pulses to create temporary pores in the cell membrane through which DNA can enter. Gene guns use compressed gas to accelerate DNA-coated gold or tungsten particles into cells. Ultrasound combines microbubbles and acoustic cavitation to permeabilize membranes for DNA uptake. Hydrodynamic delivery involves rapid injection of a large DNA solution volume to generate pressure forcing DNA into organ cells like hepatocytes. These methods show promise but also have limitations like tissue damage, shallow
A bacterial plasmid is a short, usually circular, and double-stranded segment of DNA that is found in the cytoplasm separate from the main bacterial chromosome. This presentation contains plasmid features, replication, classification and its uses.
The document discusses the process of publishing scientific papers, including tips for writing and submitting research. It notes that scientific papers are intended to share original research with other scientists and involve peer review. The five steps of publication are outlined as preparing the paper, identifying the appropriate journal, rechecking the paper, submitting it, and addressing feedback. Key tips include focusing on innovative aspects, clearly structuring the paper, using appropriate methodologies, and being patient during the writing and review process.
E. Coli is a common bacterium found in the intestines of humans and other warm-blooded organisms. It can exist harmlessly or cause food poisoning. E. Coli reproduces through cell division and genetic transfer between F+ and F- cells. The life cycle involves conjugation where the F plasmid transfers DNA between cells. E. Coli is widely studied due to its rapid reproduction, hardiness, and ability to accept foreign DNA, making it useful for biotechnology and protein production.
TA cloning is a cloning technique that avoids restriction enzymes. It relies on the ability of adenine and thymine base pairs on different DNA fragments to hybridize when ligated together. The technique involves using a PCR product amplified with Taq polymerase, which leaves 3' adenine overhangs, and a linearized vector with 3' thymine overhangs. The complementary overhangs allow the PCR product to ligate directly into the vector. Examples of vectors designed for TA cloning, such as pLUG vectors, were also discussed. TOPO TA cloning uses topoisomerase enzyme to ligate PCR products to vectors. Ligation independent cloning uses annealing of single-stranded overhangs of at least 12 bases
To modifying the structure of a specific gene.
Gene targeting vector introduced into the cell.
Vector modifies the normal chromosomal gene through homologous recombination.
Useful in treating some human genetic disorders – Hemophilia, Duchenne Muscular Dystrophy.
Treating human diseases by genetic approaches – Gene Therapy.
Gene Therapy – Replacing the defective gene by normal copy of the gene.
Expressed sequence tag/EST is a short partial sequence, typically 200-400 bp long, of a complimentary DNA/Cdna.
EST is a short sub-sequence of a cDNA sequence.
Used to identify gene transcripts, and are instrumental in gene discovery and in gene-sequence determination.
Approximately 74.2 million ESTs are available in public databases.
EST results from one-short sequencing of a cloned cDNA.
Low-quality fragments.
Length is approximately 500 to 800 nucleotides.
pET vector. Plasmid for Expression by T7 RNA Polymerase.MuhammadMujahid58
The pET vector system is a powerful tool for expressing cloned genes in E. coli. It utilizes the strong T7 promoter to drive high-level expression of the gene when induced. The T7 promoter is tightly regulated by the Lac repressor protein so expression is low without induction. This prevents toxicity from overexpression. Key features include the T7 promoter, Lac repressor binding site, ribosome binding site, and antibiotic resistance gene. Expression is induced by adding IPTG which binds Lac repressor and allows transcription by T7 RNA polymerase. This results in high protein yields while avoiding metabolic burden on the host cell.
Genetic Engineering: Chapter 1- History of Genetic EngineeringHikmet Geckil
This document provides a history of genetic engineering. It discusses how genetic engineering began with selective breeding of plants and animals in prehistoric times. The modern field of genetic engineering began in 1973 when Herbert Boyer and Stanley Cohen accomplished the direct transfer of DNA between organisms. Since then, major breakthroughs included the discovery of restriction enzymes in 1969, the first recombinant DNA molecule in 1972, DNA sequencing in 1977, and PCR in 1983. Genetically modified foods and medicines have been commercialized since 1976. The latest developments include gene editing technologies and clinical applications of modified cells.
Bacteriophage vectors
Bacteriophage
WHY BACTERIOPHAGE AS A VECTOR?
M13 phage
Genome of m13 phage
Life cycle and dna replication of m13
CONSTRUCTION M13 AS PHAGE VECTOR
M13 MP 2 vector
M13MP7 VECTOR
Selection of recombinants
Lambda replacement vectors
LAMBDA EMBL 4 VECTOR
P1 PHAGE
GENOME OF P1 PHAGE
P1 PHAGE AS VECTOR
P1 phage vector system
This document provides guidance on writing scientific manuscripts. It discusses key sections of a manuscript such as the title, abstract, introduction, methods, results, discussion and references. It emphasizes logical organization, clear communication of methods and results, interpreting findings, and comparing results to prior literature. The document also offers tips for the writing process such as not procrastinating, having others review the work, and utilizing background from funded grants.
A transgenic animal is one that has had foreign DNA inserted into its genome. The first transgenic animal was a mouse created in 1982 by inserting a human growth hormone gene. Transgenic animals are created through pronuclear microinjection or stem cell methods. They have applications in medicine, agriculture, and industry. However, some argue that transgenic technology raises ethical issues.
Expression of recombinant proteins in mammalian cell linesSandeep Kumar
The speaker discusses mammalian cell-based recombinant protein production. Mammalian cells like CHO cells are commonly used as they can properly fold and modify proteins, similar to human cells. Issues include mammalian cells being fragile, slow-growing, and techniques being expensive. Benefits are low immunogenicity and high safety due to not being susceptible to human pathogens.
Pharmacogenetics is the study of genetic variations that influence individual responses to drugs. It aims to provide information to help doctors prescribe better and safer drugs at appropriate doses tailored to a patient's genetics. The study examines how genetic variations can affect drug metabolism and efficacy. Understanding a patient's genetic profile could help predict drug responses and prevent adverse reactions.
Biopharmaceuticals are large molecule drugs made using cells or enzymes, often similar to natural biological compounds. Examples include proteins, peptides, nucleic acids, and gene therapy. The first approved biopharmaceuticals were recombinant human insulin in 1982 and recombinant tissue plasminogen activator in 1986. Today, biopharmaceutical sales reach over $200 billion annually and there are over 300 approved biopharmaceutical drugs on the market. Biopharmaceuticals are mainly produced using bacterial, mammalian, yeast, plant, or animal cells transfected with plasmids containing the gene for the target protein. Purification then isolates the target protein for clinical use.
Genetics and molecular aspects of epilepsyLarry Baum
This document summarizes genetics and molecular aspects of epilepsy. It discusses classification of epilepsy, ways to classify epilepsy genes, and functions of known epilepsy genes. The main classifications of epilepsy genes are by gene function (e.g. channel, synapse, brain organization), variant type (rare mutation, common polymorphism, copy number variant), seizure type (generalized, partial), syndrome, and cause (idiopathic, symptomatic). Many epilepsy genes have been identified, most commonly SCN1A, KCNQ2, GABAA receptor subunits, and nicotinic acetylcholine receptor subunits. Epilepsy can be caused by changes in genes involved in ion channels, synapses, or brain development/organization
This document provides an overview of biostatistics. It defines biostatistics and discusses variables that can be studied, including discrete and continuous variables. It describes common software used for analysis and summarizes typical descriptive measures like mean, median, standard deviation, etc. The document outlines common types of comparisons between continuous and categorical variables, including t-tests, ANOVA, and chi-square tests. It also discusses concepts like alpha, beta, power, and cautions around hypothesis testing and interpreting statistical significance.
Common genetic variants that alter the risk of developing epilepsyLarry Baum
This document summarizes a genome-wide association study of common genetic variants associated with non-idiopathic, or symptomatic, epilepsy. The study used a two-stage design, genotyping over 500,000 SNPs in stage 1 in over 500 epilepsy patients and nearly 3,000 controls, and following up the top hits in stage 2 with over 500 additional patients and 500 controls. Several genes were identified as associated with epilepsy risk, including CAMSAP1L1, SNAR-H, KDM3A, ERBB4, SPEF2, KCND2, and DSCAM, which have functions related to neuronal development, histone modification, and ion channel activity. This study provides new insights into the
Pharmacogenetics and therapeutic drug monitoring practiceLarry Baum
This document discusses pharmacogenetics and therapeutic drug monitoring. It describes how pharmacogenetics can help determine the best drug and dose for individuals by examining genetic factors that influence drug metabolism and response. Two key types of pharmacogenetic effects - analog and digital - are described. Several examples of pharmacogenetic testing for drugs like carbamazepine, allopurinol and cancer therapies are provided. The role of genes like HLA-B*1502, HLA-B*5801, CYP2C9 and VKORC1 in drug responses are outlined. Therapeutic drug monitoring is also discussed as a way to track drug levels in patients' blood to ensure efficacy and safety. Several classes of drugs that are commonly monitored
This document discusses the genetics of various forms of dementia. It begins by providing background on genes, DNA mutations, and genetic inheritance. It then examines specific genes linked to early-onset Alzheimer's disease like APP, PSEN1, and PSEN2. It also discusses the ApoE4 gene variant as a risk factor for late-onset Alzheimer's. Other dementias covered include vascular dementia, dementia with Lewy bodies, and genetic factors involved in each. The goal of genetic studies of dementia is to better understand disease development and inheritance to enable earlier diagnosis, prevention and treatment.
Unlocking the mysteries of reproduction: Exploring fecundity and gonadosomati...AbdullaAlAsif1
The pygmy halfbeak Dermogenys colletei, is known for its viviparous nature, this presents an intriguing case of relatively low fecundity, raising questions about potential compensatory reproductive strategies employed by this species. Our study delves into the examination of fecundity and the Gonadosomatic Index (GSI) in the Pygmy Halfbeak, D. colletei (Meisner, 2001), an intriguing viviparous fish indigenous to Sarawak, Borneo. We hypothesize that the Pygmy halfbeak, D. colletei, may exhibit unique reproductive adaptations to offset its low fecundity, thus enhancing its survival and fitness. To address this, we conducted a comprehensive study utilizing 28 mature female specimens of D. colletei, carefully measuring fecundity and GSI to shed light on the reproductive adaptations of this species. Our findings reveal that D. colletei indeed exhibits low fecundity, with a mean of 16.76 ± 2.01, and a mean GSI of 12.83 ± 1.27, providing crucial insights into the reproductive mechanisms at play in this species. These results underscore the existence of unique reproductive strategies in D. colletei, enabling its adaptation and persistence in Borneo's diverse aquatic ecosystems, and call for further ecological research to elucidate these mechanisms. This study lends to a better understanding of viviparous fish in Borneo and contributes to the broader field of aquatic ecology, enhancing our knowledge of species adaptations to unique ecological challenges.
EWOCS-I: The catalog of X-ray sources in Westerlund 1 from the Extended Weste...Sérgio Sacani
Context. With a mass exceeding several 104 M⊙ and a rich and dense population of massive stars, supermassive young star clusters
represent the most massive star-forming environment that is dominated by the feedback from massive stars and gravitational interactions
among stars.
Aims. In this paper we present the Extended Westerlund 1 and 2 Open Clusters Survey (EWOCS) project, which aims to investigate
the influence of the starburst environment on the formation of stars and planets, and on the evolution of both low and high mass stars.
The primary targets of this project are Westerlund 1 and 2, the closest supermassive star clusters to the Sun.
Methods. The project is based primarily on recent observations conducted with the Chandra and JWST observatories. Specifically,
the Chandra survey of Westerlund 1 consists of 36 new ACIS-I observations, nearly co-pointed, for a total exposure time of 1 Msec.
Additionally, we included 8 archival Chandra/ACIS-S observations. This paper presents the resulting catalog of X-ray sources within
and around Westerlund 1. Sources were detected by combining various existing methods, and photon extraction and source validation
were carried out using the ACIS-Extract software.
Results. The EWOCS X-ray catalog comprises 5963 validated sources out of the 9420 initially provided to ACIS-Extract, reaching a
photon flux threshold of approximately 2 × 10−8 photons cm−2
s
−1
. The X-ray sources exhibit a highly concentrated spatial distribution,
with 1075 sources located within the central 1 arcmin. We have successfully detected X-ray emissions from 126 out of the 166 known
massive stars of the cluster, and we have collected over 71 000 photons from the magnetar CXO J164710.20-455217.
Authoring a personal GPT for your research and practice: How we created the Q...Leonel Morgado
Thematic analysis in qualitative research is a time-consuming and systematic task, typically done using teams. Team members must ground their activities on common understandings of the major concepts underlying the thematic analysis, and define criteria for its development. However, conceptual misunderstandings, equivocations, and lack of adherence to criteria are challenges to the quality and speed of this process. Given the distributed and uncertain nature of this process, we wondered if the tasks in thematic analysis could be supported by readily available artificial intelligence chatbots. Our early efforts point to potential benefits: not just saving time in the coding process but better adherence to criteria and grounding, by increasing triangulation between humans and artificial intelligence. This tutorial will provide a description and demonstration of the process we followed, as two academic researchers, to develop a custom ChatGPT to assist with qualitative coding in the thematic data analysis process of immersive learning accounts in a survey of the academic literature: QUAL-E Immersive Learning Thematic Analysis Helper. In the hands-on time, participants will try out QUAL-E and develop their ideas for their own qualitative coding ChatGPT. Participants that have the paid ChatGPT Plus subscription can create a draft of their assistants. The organizers will provide course materials and slide deck that participants will be able to utilize to continue development of their custom GPT. The paid subscription to ChatGPT Plus is not required to participate in this workshop, just for trying out personal GPTs during it.
The debris of the ‘last major merger’ is dynamically youngSérgio Sacani
The Milky Way’s (MW) inner stellar halo contains an [Fe/H]-rich component with highly eccentric orbits, often referred to as the
‘last major merger.’ Hypotheses for the origin of this component include Gaia-Sausage/Enceladus (GSE), where the progenitor
collided with the MW proto-disc 8–11 Gyr ago, and the Virgo Radial Merger (VRM), where the progenitor collided with the
MW disc within the last 3 Gyr. These two scenarios make different predictions about observable structure in local phase space,
because the morphology of debris depends on how long it has had to phase mix. The recently identified phase-space folds in Gaia
DR3 have positive caustic velocities, making them fundamentally different than the phase-mixed chevrons found in simulations
at late times. Roughly 20 per cent of the stars in the prograde local stellar halo are associated with the observed caustics. Based
on a simple phase-mixing model, the observed number of caustics are consistent with a merger that occurred 1–2 Gyr ago.
We also compare the observed phase-space distribution to FIRE-2 Latte simulations of GSE-like mergers, using a quantitative
measurement of phase mixing (2D causticality). The observed local phase-space distribution best matches the simulated data
1–2 Gyr after collision, and certainly not later than 3 Gyr. This is further evidence that the progenitor of the ‘last major merger’
did not collide with the MW proto-disc at early times, as is thought for the GSE, but instead collided with the MW disc within
the last few Gyr, consistent with the body of work surrounding the VRM.
hematic appreciation test is a psychological assessment tool used to measure an individual's appreciation and understanding of specific themes or topics. This test helps to evaluate an individual's ability to connect different ideas and concepts within a given theme, as well as their overall comprehension and interpretation skills. The results of the test can provide valuable insights into an individual's cognitive abilities, creativity, and critical thinking skills
Immersive Learning That Works: Research Grounding and Paths ForwardLeonel Morgado
We will metaverse into the essence of immersive learning, into its three dimensions and conceptual models. This approach encompasses elements from teaching methodologies to social involvement, through organizational concerns and technologies. Challenging the perception of learning as knowledge transfer, we introduce a 'Uses, Practices & Strategies' model operationalized by the 'Immersive Learning Brain' and ‘Immersion Cube’ frameworks. This approach offers a comprehensive guide through the intricacies of immersive educational experiences and spotlighting research frontiers, along the immersion dimensions of system, narrative, and agency. Our discourse extends to stakeholders beyond the academic sphere, addressing the interests of technologists, instructional designers, and policymakers. We span various contexts, from formal education to organizational transformation to the new horizon of an AI-pervasive society. This keynote aims to unite the iLRN community in a collaborative journey towards a future where immersive learning research and practice coalesce, paving the way for innovative educational research and practice landscapes.
Or: Beyond linear.
Abstract: Equivariant neural networks are neural networks that incorporate symmetries. The nonlinear activation functions in these networks result in interesting nonlinear equivariant maps between simple representations, and motivate the key player of this talk: piecewise linear representation theory.
Disclaimer: No one is perfect, so please mind that there might be mistakes and typos.
dtubbenhauer@gmail.com
Corrected slides: dtubbenhauer.com/talks.html
Remote Sensing and Computational, Evolutionary, Supercomputing, and Intellige...University of Maribor
Slides from talk:
Aleš Zamuda: Remote Sensing and Computational, Evolutionary, Supercomputing, and Intelligent Systems.
11th International Conference on Electrical, Electronics and Computer Engineering (IcETRAN), Niš, 3-6 June 2024
Inter-Society Networking Panel GRSS/MTT-S/CIS Panel Session: Promoting Connection and Cooperation
https://www.etran.rs/2024/en/home-english/
Current Ms word generated power point presentation covers major details about the micronuclei test. It's significance and assays to conduct it. It is used to detect the micronuclei formation inside the cells of nearly every multicellular organism. It's formation takes place during chromosomal sepration at metaphase.
ESR spectroscopy in liquid food and beverages.pptxPRIYANKA PATEL
With increasing population, people need to rely on packaged food stuffs. Packaging of food materials requires the preservation of food. There are various methods for the treatment of food to preserve them and irradiation treatment of food is one of them. It is the most common and the most harmless method for the food preservation as it does not alter the necessary micronutrients of food materials. Although irradiated food doesn’t cause any harm to the human health but still the quality assessment of food is required to provide consumers with necessary information about the food. ESR spectroscopy is the most sophisticated way to investigate the quality of the food and the free radicals induced during the processing of the food. ESR spin trapping technique is useful for the detection of highly unstable radicals in the food. The antioxidant capability of liquid food and beverages in mainly performed by spin trapping technique.
4. Publishing in journals with few or bad papers
Publishing in journals that university library
doesn’t carry. If you can’t access the journal,
others in your field also can’t, so your work will
be unread and thus wasted, like Mendel.
Problem 1: Junk journals
5. Ignoring the journal formatting requirements
Paper could be rejected.
Paper could be delayed.
Copyeditor could introduce errors when formatting the paper.
Check format requirements when choosing a journal.
Find them in the journal’s Instructions for Authors. Examples:
http://eneuro.org/information-for-authors
http://www.springer.com/medicine/neurology/journal/415?
detailsPage=pltci_1753915
Length (maximum number of words)
Figures and tables (maximum number)
References
Use journal’s format.
Use RefWorks or other software to save time to reformat if a
journal rejects your paper and you thus need to submit to
another journal.
Problem 2: Journal format
6. Unlike a short story, writing a paper from start to
finish may not best help it flow smoothly.
Why?
Different types of sections
Analogy with a painting
Outline first
Figures: skeleton to hang the meat of the paper
Results
Order
1. Conclusions
2. Introduction
3. Methods
4. Abstract
Problem 3: One direction
7. Authors may emphasize their favorite
experiments.
Those that took the most time and effort
Those that were successful
But these may not be the most important for the
reader. They don’t care how many hours you
put into it.
Focus on what the reader needs to see in order
to understand the main point.
Problem 4: Effort bias
8. The point is to help the reader understand.
Tell a story.
Make it clear and simple.
Use active voice.
Use few and short words.
As if you’re explaining to a friend: a general
scientist or even a lay person, not a specialist
You do need details.
But details shouldn’t derail the train of the story.
Problem 5: Shoveling details
9. Problem 6: No paragraphs
Not using paragraphs
Use paragraphs
To focus readers on one
point at a time to get
your message across
Left indent (Enter, Tab)
or Blank line (Enter twice)
10. Problem 7: Hidden paragraphs
Using Enter key to separate paragraphs
Show paragraphs correctly
Left indent (Enter, tab), or
Blank line (Enter twice)
←¶?
13. Sentences stuck together
Examples:
“A significance threshold of 0.05 was applied for all linear
regression analyses, for all voxel based analyses and ANOVA a
threshold of 0.05 corrected for multiple comparisons was applied.”
“The EOAD and LOAD groups consisted of 24 and 36 patients,
respectively, and did not significantly differ in clinical severity and
APOE ε4 genotype but the MMSE score was significantly lower in
the LOAD group.”
Make two sentences or a compound sentence
Examples:
“A significance threshold of 0.05 was applied for all linear
regression analyses; for all voxel based analyses and ANOVA a
threshold of 0.05 corrected for multiple comparisons was applied.”
“The EOAD and LOAD groups consisted of 24 and 36 patients,
respectively, and did not significantly differ in clinical severity and
APOE ε4 genotype, but the MMSE score was significantly lower in
the LOAD group.”
Problem 9: Run-on sentences
14. Many papers have many language errors.
Ways to reduce language errors:
Spell-checker
Helps, but misses correctly-spelled wrong words.
Example: “The statistical significances of differences in frequencies
of variants between the groups were tested using the personal chi-
square test.”
Co-authors
All co-authors should read the paper before agreeing to have their
name on it.
In reading it, they should proofread it.
If they’re too busy to even read the paper, they don’t deserve to be
a co-author.
You need someone other than the author to proofread because the
author is too familiar with the paper and won’t recognize errors.
Proofreading services
There are many.
Fee may be fixed or depend on length or time.
Problem 10: Poor proofreading
15. Problem 11: Too many digits
How old are you?
Why didn’t you tell me the exact answer?
Use a similarly practical expectation in papers.
Significant digits
2 for standard deviation
1 more than might be relevant
16. Problem 12: Nonsense numbers
Not checking numbers for obvious errors
Greek letters deleted or converted (µl to ml or l)
Wrong units
Examples:
“The cells were incubated for 8 minutes in 25 ml lysis solution
containing DNase I at room temperature. The incubation
was subsequently quenched with 2.5 μL of stop solutions”
“neurons were seeded in a 96-well culture plate.... the left
cells were incubated with 100 mL (0.5 mg/mL) of 3-(4,5-
Dimethyl-2-Thiazolyl)-2,5-diphenyl tetrazolium bromide
(MTT....”
Proofread. Do numbers and units make sense?
17. Problem 13: Fonts
Using inconsistent or wrong fonts
Greek letters deleted or converted (µl to ml or l)
Chinese font for degrees C (℃).
Superscript or subscript converted to normal font (H2O, cm2).
Different size fonts for equivalent text (It can look messy.)
Check each time you copy and paste from other files.
Txt
Good: Can paste into, then copy from, plain text files to erase unwanted formatting
(as when pasting text from a pdf into a Word file)
Bad: But also erases any wanted formatting (superscript, subscript)
Pdf: need to delete Enter characters and correct other changes
Web page: need to change font and correct formatting
Proofread to find and correct
If you want to only use one font, you can Select All and choose the font and
size. But check carefully in case you forgot that you wanted a different font
buried somewhere.
Can magnify when you proofread to see easier.
18. Should support each claim with at least one reference.
Problem 14: Bad references
“Alzheimer’s disease is epidemic with an incidence that increases with age
such that its onset is nearly assured for those who live long enough. For
example, it is estimated that about 40% of those aged 85 or older have AD.
It is predicted that by 2050, over 100 million people will have AD. Those with
a history of head injury are at increased risk as may be those with obesity,
diabetes mellitus, hypertension, and renal disease. Clearly, interventions that
prevent, stabilize, remediate, or cure AD are desperately needed.”
Check the studies you cite.
Are they the papers you intended?
Do they actually prove your claim?
Cite in or near the sentence with the claim.
Use the journal’s citation format consistently.
Original paper vs. review or confirmation
Prefer original
But can cite both
20. Shoveling details on a poster is an even
bigger problem for posters than papers.
That’s because people view a poster for
only a few moments.
Think of billboards:
Problem: Busy poster
21.
22. How many words on each billboard?
Do they get their message across?
You don’t need to flood the viewer with
information to make your point.
Make posters easy and fast to absorb.
Aim for a few seconds for the key point and a
few minutes for all points.
Cut, cut, cut until only essentials remain.
Graphs & photos can be better than text.
Introduction, methods, results, conclusion
Readable from 1 m away
Problem: Busy poster