The document discusses prokaryotic expression of proteins in E. coli. It describes how a cloned gene is inserted into an expression vector which is then introduced into E. coli for large-scale protein expression. Typical expression vectors contain a coding sequence, promoter, transcriptional regulatory elements, and sequences for autonomous replication in the host. The general procedure involves obtaining the gene of interest, preparing the expression vector, inserting the gene, transforming the host strain, inducing expression, and analyzing the expressed protein.
Strain improvement Part II, Generation of mutants producing high level of pri...Renu Jaisinghani
This presentation, describes about various mutants that can be generated by carrying out process of mutation, so that high yielding mutants can be obtained that can be used for industrial production of primary metabolite.
Metabolic engineering is a branch of bioengineering where the use of genetic engineering and recombinant technology to modify the metabolism of an organism.
Strain improvement Part II, Generation of mutants producing high level of pri...Renu Jaisinghani
This presentation, describes about various mutants that can be generated by carrying out process of mutation, so that high yielding mutants can be obtained that can be used for industrial production of primary metabolite.
Metabolic engineering is a branch of bioengineering where the use of genetic engineering and recombinant technology to modify the metabolism of an organism.
Serum-free Media for Therapeutic Cell Manufacturing – Challenges and InnovationsMerck Life Sciences
The need for high quality materials that are animal origin-free and compatible with a limited number of downstream processing steps will increase as cell therapies achieve clinical success. Large scale manufacturing necessitates transition from planar culture to technologies such as stirred tank bioreactors where culture of suspension cells or adherent-dependent cells on microcarriers is enabled.
This webinar will discuss challenges and solutions to the elimination of animal-derived components from cell culture processes, with focus on human mesenchymal stromal/stem cells (hMSCs). Fetal bovine serum in particular is associated with regulatory, supply, and consistency challenges, yet a wide range of performance has been observed between different serum-free media formulations for expansion of hMSCs in planar formats. Moreover, a positive performance in static culture is not necessarily predictive of that under agitated conditions with microcarriers, highlighting ongoing challenges to the generation of a fully chemically-defined and scalable cell culture medium. Through use of pharma-grade basal media manufactured with advanced milling technology and EMPROVE® raw materials, as well as transition to serum-free supplementation and process development activities, the robust expansion of hMSCs across platforms has been achieved.
Presented by Aletta Schnitzler, Senior Scientist on 5/5/16
Cloning and extracellular expression of recombinant tissue plasminogen activa...bioejjournal
Tissue plasminogen activator (tPA) has noteworthy application in treatment of acute myocardial
infarctions. This study focuses on expression of rt-PA using microbial systems in order to reduce cost
without compromising on quality as an alternative to commercial (rt-PA)produced by using mammalian
host systems. In the present study, Pichia pastoris X-33strain was used as a host with pICZA expression
vector to obtain extracellular expression of full length tPA gene. Specific primers were designed in such a
way to get native tPA protein sequence in subsequent purification steps. Further, construct pICZA-tPA
was developed and electroporated into host to achieve stablert-PA gene by achieving genome integration.
The transformants were screened for phenotypic characters.Mut+phenotypic colony named Pichia tPA-3
showed expression of rt-PA at 66 kDa on SDS PAGE. Size Exclusion Chromatography (SEC) was
performed, resulting in product peak at same RT as reference standard. (alteplase).Cloning and expression
of rt-PA was successfully achieved in microbial system. Further process optimization at larger scales will
surely provide cost effective alternative to mammalian system forrt-PA production.
Protein expression and purification services from creative biomartAnne Ehlert
Creative BioMart is committed to providing advanced tools for protein expression and purification. As a leading supplier for reagents in the biotechnology field, we understand the importance of convenient and easy-to-use systems for high level expression and sample purification. We invite you to review our growing range of expression systems resulting from our experience in cloning, overexpression and purification.
Serum-free Media for Therapeutic Cell Manufacturing – Challenges and InnovationsMerck Life Sciences
The need for high quality materials that are animal origin-free and compatible with a limited number of downstream processing steps will increase as cell therapies achieve clinical success. Large scale manufacturing necessitates transition from planar culture to technologies such as stirred tank bioreactors where culture of suspension cells or adherent-dependent cells on microcarriers is enabled.
This webinar will discuss challenges and solutions to the elimination of animal-derived components from cell culture processes, with focus on human mesenchymal stromal/stem cells (hMSCs). Fetal bovine serum in particular is associated with regulatory, supply, and consistency challenges, yet a wide range of performance has been observed between different serum-free media formulations for expansion of hMSCs in planar formats. Moreover, a positive performance in static culture is not necessarily predictive of that under agitated conditions with microcarriers, highlighting ongoing challenges to the generation of a fully chemically-defined and scalable cell culture medium. Through use of pharma-grade basal media manufactured with advanced milling technology and EMPROVE® raw materials, as well as transition to serum-free supplementation and process development activities, the robust expansion of hMSCs across platforms has been achieved.
Presented by Aletta Schnitzler, Senior Scientist on 5/5/16
Cloning and extracellular expression of recombinant tissue plasminogen activa...bioejjournal
Tissue plasminogen activator (tPA) has noteworthy application in treatment of acute myocardial
infarctions. This study focuses on expression of rt-PA using microbial systems in order to reduce cost
without compromising on quality as an alternative to commercial (rt-PA)produced by using mammalian
host systems. In the present study, Pichia pastoris X-33strain was used as a host with pICZA expression
vector to obtain extracellular expression of full length tPA gene. Specific primers were designed in such a
way to get native tPA protein sequence in subsequent purification steps. Further, construct pICZA-tPA
was developed and electroporated into host to achieve stablert-PA gene by achieving genome integration.
The transformants were screened for phenotypic characters.Mut+phenotypic colony named Pichia tPA-3
showed expression of rt-PA at 66 kDa on SDS PAGE. Size Exclusion Chromatography (SEC) was
performed, resulting in product peak at same RT as reference standard. (alteplase).Cloning and expression
of rt-PA was successfully achieved in microbial system. Further process optimization at larger scales will
surely provide cost effective alternative to mammalian system forrt-PA production.
Protein expression and purification services from creative biomartAnne Ehlert
Creative BioMart is committed to providing advanced tools for protein expression and purification. As a leading supplier for reagents in the biotechnology field, we understand the importance of convenient and easy-to-use systems for high level expression and sample purification. We invite you to review our growing range of expression systems resulting from our experience in cloning, overexpression and purification.
this is a presentation on gene expression vector that includes what is expression vector, how many types of expression vector and difference between cloning and expression vector
Expression and purification of recombinant proteins in Bacterial and yeast sy...Shreya Feliz
This presentation gives the information about bacterial and yeast system as host for expressing recombinant proteins, suitable vectors, strains of host, Pros and cons of this system, different purification techniques and commercially available proteins produced so far by this system.
Genetically modified microbes.
its an informative presentation about the Geneticatly modified microbs .how genetically microbes developed and what mechanism they are made and how the will be beneficial for the enviroment .mechanism ,advantages disadvantages etc
Screening of metabolite and various rDNA technology for strain improvement.pptxAindrila
This slide is about the different types and importance of metabolites. How we can do screening of strains and use different techniques, especially rDNA technology for strain improvement.
Purification of protein involved in Mycobacterium tuberculosis as a potential...AmitSingh65788
Antibiotic resistance poses a significant challenge in the treatment of tuberculosis, requiring the development of new antibiotics with unique molecular mechanisms. In our research, we have identified a specific protein in Mycobacterium tuberculosis as a potential drug target. Let's explore our journey to synthesize and purify this protein.
In order to produce the protein of interest, we employ recombinant DNA technology. This involves isolating and cloning the coding sequence of the protein into an expression plasmid vector. Bacteria such as E. coli are commonly used for this purpose due to their simple manipulation and cost-effectiveness
This presentation content information about Protein Purification Steps .What is Protein purification, why their is need of purification, what are biopharmaceuticals, Methods of identification of protein etc.
Potential therapy derived from banana protein is effective against SARS-CoV-2Creative BioMart
On January 13, 2020, a paper was published online touting the creation of a possible therapy that could be used against all known strains of the flu. A week later, the first laboratory-confirmed case of SARS-CoV-2 sparked the two-and-a-half-year COVID-19 pandemic in the United States. Interestingly, before the virus temporarily halted their work, the international team of researchers for this flu paper also investigated treatments for the coronavirus.
CAR-T Cell Anti-Cancer Therapy Ushered in A Revolutionary Breakthrough!Creative BioMart
Chimeric antigen receptor T cells (CAR-T) and T cell antigen receptor chimeric T cells (TCR-T) are currently the "top stream" in adoptive T cell tumor immunotherapy. In particular, CAR-T therapy, which has been approved by the FDA, is rewriting the treatment paradigm of some hematological tumors.
In recent years, the increase in the number of multi-drug resistant pathogens and food safety have become serious global problems, and it is increasingly important to find or develop a new generation of antibacterial drugs or preservatives. Scientists have discovered that bacteria-produced bacteriocins can control clinically relevant susceptible and resistant bacteria, and purified bacteriocins can be added to foods as natural preservatives. Bacteriocins can be added to animal feeds as anti-pathogen additives to protect livestock from pathogen damage. In medicine, bacteriocin has the potential to replace antibiotics as antibacterial drugs and is a new type of anticancer drug.
Creative BioMart to Attend BIO International Convention June 13-16, 2022Creative BioMart
Creative BioMart, a leading biotechnology company supplying comprehensive protein products and services for the science community, recently announced that it will be attending the BIO International Convention in San Diego, California, from June 13-16, 2022.
Third Natural Eukaryotic Epigenetic Mark Found.pdfCreative BioMart
A group at the Marine Biology Laboratory (MBL) now found a third epigenetic mark in this freshwater invertebrate, Adineta vaga, which has previously been found only in bacteria. For the first time, a horizontally transferred gene has been shown to remodel gene regulatory systems in eukaryotes.
ELife: T Interferon Suppresses Inflammatory Diseases by Balancing the MicrobiomeCreative BioMart
The authors report that STAT1KO mice spontaneously develop inflammatory diseases characterized by bone marrow hyperplasia and splenic accumulation of hematopoietic stem cells.
Chemokines are small proteins, usually ~70–80 amino acid residues, with conserved sequence and structural features and expressed in tissues during normal immune surveillance or in response to injury or infection.
Creative BioMart has more than 20 years of experience in protein characterization. We have extensive experience in developing and establishing protein identification methods to help our customers generate the data they need to obtain the level of product identification required for regulatory submission. These characterization services are also part of our stability and product launch support services, where we provide critical identity, purity, and performance measurements.
The latest research has identified FGL1 as an immune inhibitory ligand of LAG-3, an immune inhibitory receptor that mediates T-cell suppression. Blocking FGL1/LAG-3 interaction improves anti-tumor immunity.
Creative BioMart has currently developed FGL1 proteins from multiple species. Their binding activity with LAG-3 protein has been verified by functional ELISA.
https://www.creativebiomart.net/resource/pdfdownload/dbd8e3ddfc158995b7e56f7277140c50.pdf
Protein quantification is divided into "total quantification method" of whole protein and "individual quantification method" of specific protein according to its purpose. It is an indispensable part of biological experiments.
To master the basic operation process of primary culture and subculture of mammalian cells, lay a foundation for the application of bioengineering in medicine.
Electrophoresis technology is the basic technology of molecular biology. This experiment helps better understanding of DNA electrophoresis tank and gel electrophoresis technology.
Observation of Io’s Resurfacing via Plume Deposition Using Ground-based Adapt...Sérgio Sacani
Since volcanic activity was first discovered on Io from Voyager images in 1979, changes
on Io’s surface have been monitored from both spacecraft and ground-based telescopes.
Here, we present the highest spatial resolution images of Io ever obtained from a groundbased telescope. These images, acquired by the SHARK-VIS instrument on the Large
Binocular Telescope, show evidence of a major resurfacing event on Io’s trailing hemisphere. When compared to the most recent spacecraft images, the SHARK-VIS images
show that a plume deposit from a powerful eruption at Pillan Patera has covered part
of the long-lived Pele plume deposit. Although this type of resurfacing event may be common on Io, few have been detected due to the rarity of spacecraft visits and the previously low spatial resolution available from Earth-based telescopes. The SHARK-VIS instrument ushers in a new era of high resolution imaging of Io’s surface using adaptive
optics at visible wavelengths.
Slide 1: Title Slide
Extrachromosomal Inheritance
Slide 2: Introduction to Extrachromosomal Inheritance
Definition: Extrachromosomal inheritance refers to the transmission of genetic material that is not found within the nucleus.
Key Components: Involves genes located in mitochondria, chloroplasts, and plasmids.
Slide 3: Mitochondrial Inheritance
Mitochondria: Organelles responsible for energy production.
Mitochondrial DNA (mtDNA): Circular DNA molecule found in mitochondria.
Inheritance Pattern: Maternally inherited, meaning it is passed from mothers to all their offspring.
Diseases: Examples include Leber’s hereditary optic neuropathy (LHON) and mitochondrial myopathy.
Slide 4: Chloroplast Inheritance
Chloroplasts: Organelles responsible for photosynthesis in plants.
Chloroplast DNA (cpDNA): Circular DNA molecule found in chloroplasts.
Inheritance Pattern: Often maternally inherited in most plants, but can vary in some species.
Examples: Variegation in plants, where leaf color patterns are determined by chloroplast DNA.
Slide 5: Plasmid Inheritance
Plasmids: Small, circular DNA molecules found in bacteria and some eukaryotes.
Features: Can carry antibiotic resistance genes and can be transferred between cells through processes like conjugation.
Significance: Important in biotechnology for gene cloning and genetic engineering.
Slide 6: Mechanisms of Extrachromosomal Inheritance
Non-Mendelian Patterns: Do not follow Mendel’s laws of inheritance.
Cytoplasmic Segregation: During cell division, organelles like mitochondria and chloroplasts are randomly distributed to daughter cells.
Heteroplasmy: Presence of more than one type of organellar genome within a cell, leading to variation in expression.
Slide 7: Examples of Extrachromosomal Inheritance
Four O’clock Plant (Mirabilis jalapa): Shows variegated leaves due to different cpDNA in leaf cells.
Petite Mutants in Yeast: Result from mutations in mitochondrial DNA affecting respiration.
Slide 8: Importance of Extrachromosomal Inheritance
Evolution: Provides insight into the evolution of eukaryotic cells.
Medicine: Understanding mitochondrial inheritance helps in diagnosing and treating mitochondrial diseases.
Agriculture: Chloroplast inheritance can be used in plant breeding and genetic modification.
Slide 9: Recent Research and Advances
Gene Editing: Techniques like CRISPR-Cas9 are being used to edit mitochondrial and chloroplast DNA.
Therapies: Development of mitochondrial replacement therapy (MRT) for preventing mitochondrial diseases.
Slide 10: Conclusion
Summary: Extrachromosomal inheritance involves the transmission of genetic material outside the nucleus and plays a crucial role in genetics, medicine, and biotechnology.
Future Directions: Continued research and technological advancements hold promise for new treatments and applications.
Slide 11: Questions and Discussion
Invite Audience: Open the floor for any questions or further discussion on the topic.
This presentation explores a brief idea about the structural and functional attributes of nucleotides, the structure and function of genetic materials along with the impact of UV rays and pH upon them.
Richard's entangled aventures in wonderlandRichard Gill
Since the loophole-free Bell experiments of 2020 and the Nobel prizes in physics of 2022, critics of Bell's work have retreated to the fortress of super-determinism. Now, super-determinism is a derogatory word - it just means "determinism". Palmer, Hance and Hossenfelder argue that quantum mechanics and determinism are not incompatible, using a sophisticated mathematical construction based on a subtle thinning of allowed states and measurements in quantum mechanics, such that what is left appears to make Bell's argument fail, without altering the empirical predictions of quantum mechanics. I think however that it is a smoke screen, and the slogan "lost in math" comes to my mind. I will discuss some other recent disproofs of Bell's theorem using the language of causality based on causal graphs. Causal thinking is also central to law and justice. I will mention surprising connections to my work on serial killer nurse cases, in particular the Dutch case of Lucia de Berk and the current UK case of Lucy Letby.
Richard's aventures in two entangled wonderlandsRichard Gill
Since the loophole-free Bell experiments of 2020 and the Nobel prizes in physics of 2022, critics of Bell's work have retreated to the fortress of super-determinism. Now, super-determinism is a derogatory word - it just means "determinism". Palmer, Hance and Hossenfelder argue that quantum mechanics and determinism are not incompatible, using a sophisticated mathematical construction based on a subtle thinning of allowed states and measurements in quantum mechanics, such that what is left appears to make Bell's argument fail, without altering the empirical predictions of quantum mechanics. I think however that it is a smoke screen, and the slogan "lost in math" comes to my mind. I will discuss some other recent disproofs of Bell's theorem using the language of causality based on causal graphs. Causal thinking is also central to law and justice. I will mention surprising connections to my work on serial killer nurse cases, in particular the Dutch case of Lucia de Berk and the current UK case of Lucy Letby.
Deep Behavioral Phenotyping in Systems Neuroscience for Functional Atlasing a...Ana Luísa Pinho
Functional Magnetic Resonance Imaging (fMRI) provides means to characterize brain activations in response to behavior. However, cognitive neuroscience has been limited to group-level effects referring to the performance of specific tasks. To obtain the functional profile of elementary cognitive mechanisms, the combination of brain responses to many tasks is required. Yet, to date, both structural atlases and parcellation-based activations do not fully account for cognitive function and still present several limitations. Further, they do not adapt overall to individual characteristics. In this talk, I will give an account of deep-behavioral phenotyping strategies, namely data-driven methods in large task-fMRI datasets, to optimize functional brain-data collection and improve inference of effects-of-interest related to mental processes. Key to this approach is the employment of fast multi-functional paradigms rich on features that can be well parametrized and, consequently, facilitate the creation of psycho-physiological constructs to be modelled with imaging data. Particular emphasis will be given to music stimuli when studying high-order cognitive mechanisms, due to their ecological nature and quality to enable complex behavior compounded by discrete entities. I will also discuss how deep-behavioral phenotyping and individualized models applied to neuroimaging data can better account for the subject-specific organization of domain-general cognitive systems in the human brain. Finally, the accumulation of functional brain signatures brings the possibility to clarify relationships among tasks and create a univocal link between brain systems and mental functions through: (1) the development of ontologies proposing an organization of cognitive processes; and (2) brain-network taxonomies describing functional specialization. To this end, tools to improve commensurability in cognitive science are necessary, such as public repositories, ontology-based platforms and automated meta-analysis tools. I will thus discuss some brain-atlasing resources currently under development, and their applicability in cognitive as well as clinical neuroscience.
2. Introduction
A method in which a cloned gene is inserted into a suitable vector and introduced into
Escherichia coli for expression of a large amount of protein is generally referred to as
prokaryotic expression. This method has applications in protein purification,
localization and functional analysis. E. coli used to express recombinant proteins has
the following characteristics: easy to grow and control; materials used for bacterial
culture are less expensive than mammalian cell systems; A wide variety of E. coli strains
and matching plasmids with various properties are available. However, proteins
expressed in E. coli often form inclusion bodies to affect the biological activity and
conformation of the expressed protein due to lack of modification and post-
translational processing such as glycosylation and phosphorylation.
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3. Introduction
Expression vectors play an important role in genetic engineering. Prokaryotic
expression vectors are usually plasmids. Typical expression vectors should have the
following components:
(1) Selecting a coding sequence of the marker;
(2) A promoter capable of transcribed;
(3) Transcriptional regulatory sequences (transcriptional terminator, ribosome binding
site);
(4) A multi-restriction cleavage site linker;
(5) Sequences autonomously replicated in the host.
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4. Introduction
The general procedure for prokaryotic expression is as follows: obtaining the gene of
interest - preparing the expression vector - inserting the gene of interest into the
expression vector (sequencing verification) - transforming the expression host strain -
inducing expression of the target protein - analysis of the expressed protein -
amplification, purification, further detection.
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5. Primary reagent
1. LB medium.
2. 100 mM IPTG (isopropylthio-β-D-galactoside): 2.38 g of IPTG was dissolved in 100 ml
of ddH2O, filtered through a 0.22 μm filter, and stored at -20 °C.
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6. Experimental procedure
(1) Obtaining the gene of interest
(2) Construction of recombinant expression vector
(3) Obtaining the expression strain containing the recombinant expression
plasmid
(4) Induced expression
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7. Precautions
1. When selecting an expression vector, it should be considered according to the final
application of the expressed protein. For ease of purification, fusion expression can be
selected; if a native protein is obtained, non-fusion expression can be selected.
2. In the fusion expression, when the foreign DNA is selected and linked to the carrier
molecule, the reading of the cryptographic structure during transcription and
translation cannot be interfered.
Collected by Creative BioMart.
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