Plasmids are small, circular DNA molecules that can replicate independently of the host chromosome. They are commonly used in molecular biology to clone and amplify genes. The summarized document describes the key functional elements found in yeast overexpression plasmids, including selection markers like URA3, origins of replication, promoters, and tags. It also summarizes how plasmids are constructed through molecular cloning and purified from bacteria using their unique physical properties like supercoiling and renaturation compared to host chromosomal DNA and proteins. Purification involves alkaline lysis to break open cells, neutralization to allow plasmid renaturation, and multiple wash steps on a silica resin column to isolate pure plasmid DNA.
description of plasmids and types and importance of plasmids and artificial plasmids(PBR322,cosmids,phagemids) and selection of the recombinants and uses and advantages and disadvantages of the plasmids
description of plasmids and types and importance of plasmids and artificial plasmids(PBR322,cosmids,phagemids) and selection of the recombinants and uses and advantages and disadvantages of the plasmids
Plasmid is a double stranded, circular extra chromosomal DNA of bacterium. It is used in recombinant DNA experiments to clone genes from other organisms and make large quantities of their DNA. Plasmid can be transferred between same species or between different species. Size of plasmids range from 1-1000 kilo base pairs. Plasmids are part of mobilomes (total of all mobile genetic elements in a genome) like transposons or prophages and are associated with conjugation. Even the largest plasmids are considerably smaller than the chromosomal DNA of the bacterium, which can contain several million base pairs.
A recombinant DNA molecule is produced by joining together two or more DNA segments usually originating from two different organisms.
More Specifically, a recombinant DNA molecule is a vector into which desired DNA fragment has been inserted to enable its cloning in an appropriate host.
Recombinant DNA molecules are produced with one of the following objectives:
1. To obtain large number of copies of specific DNA fragments.
2. Large scale production of the protein encoded by the gene.
3. Integration of the desired DNA fragment into target organism where it expresses itself.
Drought tolerant-genetically modified plants:
Present abiotic stress is a major challenge in our quest for sustainable food production as these may reduce the potential yields by 70% in crop plants
Of all abiotic stress, drought is regarded as the most damaging
Transgenic plants carrying genes for abiotic stress tolerance are being developed for water stress management
Conventional breeding approaches, involving inter specific and inter generic hybridizations and mutagenesis have been limited success.
Major problems have been the complexity of drought tolerance & low genetic yield components under drought conditions.
Unlike conventional plant breeding there is no need of repeated back crossing
Gene pyramiding or gene stacking through co-transformation of different genes with similar effects can be achieved.
The content provided about "plasmid and its types with current research applications. The content presented with best of my knowledge and reference with several articles from appropriate source. I hope this will be useful.
Explore natural remedies for syphilis treatment in Singapore. Discover alternative therapies, herbal remedies, and lifestyle changes that may complement conventional treatments. Learn about holistic approaches to managing syphilis symptoms and supporting overall health.
Plasmid is a double stranded, circular extra chromosomal DNA of bacterium. It is used in recombinant DNA experiments to clone genes from other organisms and make large quantities of their DNA. Plasmid can be transferred between same species or between different species. Size of plasmids range from 1-1000 kilo base pairs. Plasmids are part of mobilomes (total of all mobile genetic elements in a genome) like transposons or prophages and are associated with conjugation. Even the largest plasmids are considerably smaller than the chromosomal DNA of the bacterium, which can contain several million base pairs.
A recombinant DNA molecule is produced by joining together two or more DNA segments usually originating from two different organisms.
More Specifically, a recombinant DNA molecule is a vector into which desired DNA fragment has been inserted to enable its cloning in an appropriate host.
Recombinant DNA molecules are produced with one of the following objectives:
1. To obtain large number of copies of specific DNA fragments.
2. Large scale production of the protein encoded by the gene.
3. Integration of the desired DNA fragment into target organism where it expresses itself.
Drought tolerant-genetically modified plants:
Present abiotic stress is a major challenge in our quest for sustainable food production as these may reduce the potential yields by 70% in crop plants
Of all abiotic stress, drought is regarded as the most damaging
Transgenic plants carrying genes for abiotic stress tolerance are being developed for water stress management
Conventional breeding approaches, involving inter specific and inter generic hybridizations and mutagenesis have been limited success.
Major problems have been the complexity of drought tolerance & low genetic yield components under drought conditions.
Unlike conventional plant breeding there is no need of repeated back crossing
Gene pyramiding or gene stacking through co-transformation of different genes with similar effects can be achieved.
The content provided about "plasmid and its types with current research applications. The content presented with best of my knowledge and reference with several articles from appropriate source. I hope this will be useful.
Explore natural remedies for syphilis treatment in Singapore. Discover alternative therapies, herbal remedies, and lifestyle changes that may complement conventional treatments. Learn about holistic approaches to managing syphilis symptoms and supporting overall health.
New Drug Discovery and Development .....NEHA GUPTA
The "New Drug Discovery and Development" process involves the identification, design, testing, and manufacturing of novel pharmaceutical compounds with the aim of introducing new and improved treatments for various medical conditions. This comprehensive endeavor encompasses various stages, including target identification, preclinical studies, clinical trials, regulatory approval, and post-market surveillance. It involves multidisciplinary collaboration among scientists, researchers, clinicians, regulatory experts, and pharmaceutical companies to bring innovative therapies to market and address unmet medical needs.
- Video recording of this lecture in English language: https://youtu.be/lK81BzxMqdo
- Video recording of this lecture in Arabic language: https://youtu.be/Ve4P0COk9OI
- Link to download the book free: https://nephrotube.blogspot.com/p/nephrotube-nephrology-books.html
- Link to NephroTube website: www.NephroTube.com
- Link to NephroTube social media accounts: https://nephrotube.blogspot.com/p/join-nephrotube-on-social-media.html
Flu Vaccine Alert in Bangalore Karnatakaaddon Scans
As flu season approaches, health officials in Bangalore, Karnataka, are urging residents to get their flu vaccinations. The seasonal flu, while common, can lead to severe health complications, particularly for vulnerable populations such as young children, the elderly, and those with underlying health conditions.
Dr. Vidisha Kumari, a leading epidemiologist in Bangalore, emphasizes the importance of getting vaccinated. "The flu vaccine is our best defense against the influenza virus. It not only protects individuals but also helps prevent the spread of the virus in our communities," he says.
This year, the flu season is expected to coincide with a potential increase in other respiratory illnesses. The Karnataka Health Department has launched an awareness campaign highlighting the significance of flu vaccinations. They have set up multiple vaccination centers across Bangalore, making it convenient for residents to receive their shots.
To encourage widespread vaccination, the government is also collaborating with local schools, workplaces, and community centers to facilitate vaccination drives. Special attention is being given to ensuring that the vaccine is accessible to all, including marginalized communities who may have limited access to healthcare.
Residents are reminded that the flu vaccine is safe and effective. Common side effects are mild and may include soreness at the injection site, mild fever, or muscle aches. These side effects are generally short-lived and far less severe than the flu itself.
Healthcare providers are also stressing the importance of continuing COVID-19 precautions. Wearing masks, practicing good hand hygiene, and maintaining social distancing are still crucial, especially in crowded places.
Protect yourself and your loved ones by getting vaccinated. Together, we can help keep Bangalore healthy and safe this flu season. For more information on vaccination centers and schedules, residents can visit the Karnataka Health Department’s official website or follow their social media pages.
Stay informed, stay safe, and get your flu shot today!
Ozempic: Preoperative Management of Patients on GLP-1 Receptor Agonists Saeid Safari
Preoperative Management of Patients on GLP-1 Receptor Agonists like Ozempic and Semiglutide
ASA GUIDELINE
NYSORA Guideline
2 Case Reports of Gastric Ultrasound
ARTIFICIAL INTELLIGENCE IN HEALTHCARE.pdfAnujkumaranit
Artificial intelligence (AI) refers to the simulation of human intelligence processes by machines, especially computer systems. It encompasses tasks such as learning, reasoning, problem-solving, perception, and language understanding. AI technologies are revolutionizing various fields, from healthcare to finance, by enabling machines to perform tasks that typically require human intelligence.
Prix Galien International 2024 Forum ProgramLevi Shapiro
June 20, 2024, Prix Galien International and Jerusalem Ethics Forum in ROME. Detailed agenda including panels:
- ADVANCES IN CARDIOLOGY: A NEW PARADIGM IS COMING
- WOMEN’S HEALTH: FERTILITY PRESERVATION
- WHAT’S NEW IN THE TREATMENT OF INFECTIOUS,
ONCOLOGICAL AND INFLAMMATORY SKIN DISEASES?
- ARTIFICIAL INTELLIGENCE AND ETHICS
- GENE THERAPY
- BEYOND BORDERS: GLOBAL INITIATIVES FOR DEMOCRATIZING LIFE SCIENCE TECHNOLOGIES AND PROMOTING ACCESS TO HEALTHCARE
- ETHICAL CHALLENGES IN LIFE SCIENCES
- Prix Galien International Awards Ceremony
New Directions in Targeted Therapeutic Approaches for Older Adults With Mantl...i3 Health
i3 Health is pleased to make the speaker slides from this activity available for use as a non-accredited self-study or teaching resource.
This slide deck presented by Dr. Kami Maddocks, Professor-Clinical in the Division of Hematology and
Associate Division Director for Ambulatory Operations
The Ohio State University Comprehensive Cancer Center, will provide insight into new directions in targeted therapeutic approaches for older adults with mantle cell lymphoma.
STATEMENT OF NEED
Mantle cell lymphoma (MCL) is a rare, aggressive B-cell non-Hodgkin lymphoma (NHL) accounting for 5% to 7% of all lymphomas. Its prognosis ranges from indolent disease that does not require treatment for years to very aggressive disease, which is associated with poor survival (Silkenstedt et al, 2021). Typically, MCL is diagnosed at advanced stage and in older patients who cannot tolerate intensive therapy (NCCN, 2022). Although recent advances have slightly increased remission rates, recurrence and relapse remain very common, leading to a median overall survival between 3 and 6 years (LLS, 2021). Though there are several effective options, progress is still needed towards establishing an accepted frontline approach for MCL (Castellino et al, 2022). Treatment selection and management of MCL are complicated by the heterogeneity of prognosis, advanced age and comorbidities of patients, and lack of an established standard approach for treatment, making it vital that clinicians be familiar with the latest research and advances in this area. In this activity chaired by Michael Wang, MD, Professor in the Department of Lymphoma & Myeloma at MD Anderson Cancer Center, expert faculty will discuss prognostic factors informing treatment, the promising results of recent trials in new therapeutic approaches, and the implications of treatment resistance in therapeutic selection for MCL.
Target Audience
Hematology/oncology fellows, attending faculty, and other health care professionals involved in the treatment of patients with mantle cell lymphoma (MCL).
Learning Objectives
1.) Identify clinical and biological prognostic factors that can guide treatment decision making for older adults with MCL
2.) Evaluate emerging data on targeted therapeutic approaches for treatment-naive and relapsed/refractory MCL and their applicability to older adults
3.) Assess mechanisms of resistance to targeted therapies for MCL and their implications for treatment selection
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These simplified slides by Dr. Sidra Arshad present an overview of the non-respiratory functions of the respiratory tract.
Learning objectives:
1. Enlist the non-respiratory functions of the respiratory tract
2. Briefly explain how these functions are carried out
3. Discuss the significance of dead space
4. Differentiate between minute ventilation and alveolar ventilation
5. Describe the cough and sneeze reflexes
Study Resources:
1. Chapter 39, Guyton and Hall Textbook of Medical Physiology, 14th edition
2. Chapter 34, Ganong’s Review of Medical Physiology, 26th edition
3. Chapter 17, Human Physiology by Lauralee Sherwood, 9th edition
4. Non-respiratory functions of the lungs https://academic.oup.com/bjaed/article/13/3/98/278874
NVBDCP.pptx Nation vector borne disease control programSapna Thakur
NVBDCP was launched in 2003-2004 . Vector-Borne Disease: Disease that results from an infection transmitted to humans and other animals by blood-feeding arthropods, such as mosquitoes, ticks, and fleas. Examples of vector-borne diseases include Dengue fever, West Nile Virus, Lyme disease, and malaria.
Title: Sense of Smell
Presenter: Dr. Faiza, Assistant Professor of Physiology
Qualifications:
MBBS (Best Graduate, AIMC Lahore)
FCPS Physiology
ICMT, CHPE, DHPE (STMU)
MPH (GC University, Faisalabad)
MBA (Virtual University of Pakistan)
Learning Objectives:
Describe the primary categories of smells and the concept of odor blindness.
Explain the structure and location of the olfactory membrane and mucosa, including the types and roles of cells involved in olfaction.
Describe the pathway and mechanisms of olfactory signal transmission from the olfactory receptors to the brain.
Illustrate the biochemical cascade triggered by odorant binding to olfactory receptors, including the role of G-proteins and second messengers in generating an action potential.
Identify different types of olfactory disorders such as anosmia, hyposmia, hyperosmia, and dysosmia, including their potential causes.
Key Topics:
Olfactory Genes:
3% of the human genome accounts for olfactory genes.
400 genes for odorant receptors.
Olfactory Membrane:
Located in the superior part of the nasal cavity.
Medially: Folds downward along the superior septum.
Laterally: Folds over the superior turbinate and upper surface of the middle turbinate.
Total surface area: 5-10 square centimeters.
Olfactory Mucosa:
Olfactory Cells: Bipolar nerve cells derived from the CNS (100 million), with 4-25 olfactory cilia per cell.
Sustentacular Cells: Produce mucus and maintain ionic and molecular environment.
Basal Cells: Replace worn-out olfactory cells with an average lifespan of 1-2 months.
Bowman’s Gland: Secretes mucus.
Stimulation of Olfactory Cells:
Odorant dissolves in mucus and attaches to receptors on olfactory cilia.
Involves a cascade effect through G-proteins and second messengers, leading to depolarization and action potential generation in the olfactory nerve.
Quality of a Good Odorant:
Small (3-20 Carbon atoms), volatile, water-soluble, and lipid-soluble.
Facilitated by odorant-binding proteins in mucus.
Membrane Potential and Action Potential:
Resting membrane potential: -55mV.
Action potential frequency in the olfactory nerve increases with odorant strength.
Adaptation Towards the Sense of Smell:
Rapid adaptation within the first second, with further slow adaptation.
Psychological adaptation greater than receptor adaptation, involving feedback inhibition from the central nervous system.
Primary Sensations of Smell:
Camphoraceous, Musky, Floral, Pepperminty, Ethereal, Pungent, Putrid.
Odor Detection Threshold:
Examples: Hydrogen sulfide (0.0005 ppm), Methyl-mercaptan (0.002 ppm).
Some toxic substances are odorless at lethal concentrations.
Characteristics of Smell:
Odor blindness for single substances due to lack of appropriate receptor protein.
Behavioral and emotional influences of smell.
Transmission of Olfactory Signals:
From olfactory cells to glomeruli in the olfactory bulb, involving lateral inhibition.
Primitive, less old, and new olfactory systems with different path
1. Plasmids
Indispensable tools that allow molecular biologists to obtain
essentially unlimited amounts of a DNA sequence
Definition: Small circular extrachromosomal DNA molecules, that
replicate independently of the host chromosomes
2. How are plasmids constructed?
What functional elements are found in our yeast
overexpression plasmids?
How are plasmids purified?
3. GFP gene
This gene encodes green
fluorescent protein, which
glows in UV light
Kanamycin
resistance
gene
the enzyme
encoded by
this gene
stops the
antibiotic
kanamycin
from working
Origin of
replication
this is needed so
that the plasmid
can be copied,
using the
bacterium’s DNA
copying machinery
The plasmid
4. A Brief History of Plasmids
• Genetic evidence for the existence of plasmids initially
came from studies carried out in the laboratories of J.
Lederberg and W. Hayes in the early 1950s
• 1952:It was Joshua Lederberg who proposed the term
plasmid in 1952 for extranuclear structures that are able
to reproduce in an autonomous state.
• he word 'plasmid' was first coined by Joshua Lederberg
in 1952. He used it to describe 'any extrachromosomal
hereditary element'. Lederberg first used the term in a
paper he published describing some experiments he and
his graduate student Norton Zinder conducted on
Salmonella bacteria and its virus P22.
J. Lederberg, 1952, Physiol. Rev. 32, 403-430
5. • In the late 1950s, a number of laboratories took up the
study of plasmids once the discovery was made that
extrachromosomal antibiotic resistance (R) factors are the
responsible agents for the transmissibility of multiple
antibiotic resistance among the enterobacterial
• The earliest studies on R plasmids, carried out mainly by
scientists in Japan (8), and the pioneering work on
colicinogenic (Col) plasmids by P. Fredericq of Belgium in
the mid-1950s (9) resulted in the identification of a variety
of naturally occurring plasmids that, subsequently, were
analyzed in the 1960s for the plasmid properties of
autonomous replication, mobility, incompatibility, and host
range.
6. • The list of plasmids available for study during this
period was augmented significantly by the
isolation by several laboratories of F-prime
factors from E. coli strains that carried a
chromosomally integrated form of the F factor
(10).
• The detailed genetic analysis of these F-prime
plasmids contributed greatly to our
understanding of the chromosomal state of a
plasmid and the mechanics of chromosomal gene
transfer from donor to recipient bacterium
7. • have no distinct 5’ or 3’ beginning or end.
• double-stranded DNA molecules
• ranging from a few to several hundred
kilobases
• provide one or more benefits to the host
such as resistance to antibiotics,
degradative functions, and virulence.
9. The structure of a plasmid
• Origin of replication (OR),
antibiotic resistance gene
(ARG) and multiple cloning
site (MCS).
• between 1 to more than
1,000 kbp
10. • The final breakthrough came in 1973, when the
first artificial plasmid was constructed by Boyer
and Cohen. By then, scientists had discovered
that special proteins, known as restriction
enzymes, could “cut” DNA by cleaving its
intramolecular bonds. Particularly, Boyer has
isolated a restriction enzyme, EcoRI 1, which
could recognize and cut a particular sequence
of DNA to produce unpaired DNA bases on one
strand, known as “cohesive end” or “sticky end”
(Figure 1) [3].
11. Plasmids used in molecular biology have been constructed in the lab
Molecular cloning
Enzymes are used to insert desired
pieces of foreign DNA into plasmids
Bacterial cells are transformed with
the plasmids. Copies of the
plasmids are purified from bacteria.
12. Shuttle vectors have origins of replication and selectable markers
for propagation in both bacteria and yeast
We are using plasmids that have been termed shuttle vectors,
because they can be propagated in either bacteria or yeast
Plasmids are propagated in
bacteria, which grow quickly
and maintain multiple copies
of the plasmids
non-pathogenic strain
of Escherichia coli
Saccharomyces cerevisiae
deletion strains
Plasmid-encoded genes
are expressed in yeast,
and phenotypes are
analyzed
13. How are plasmids constructed?
What functional elements are found in our yeast
overexpression plasmids?
How are plasmids purified?
14. 1 URA3
2 β-lactamase gene
3 pBR322 ori
4 yeast 2 µm origin
5 GAL1 promoter
6 C-terminal tags
1
2
3
4
5 6
ORF goes
here
*Plasmid names begin with a lower case “p”
The pBG1805*-derived plasmids are complex vectors designed
to express S. cerevisiae ORFs
15. pYES2.1-based plasmids used for S. pombe ORFs have many
elements in common with pBG1805-based plasmids
pYES2.1 (5886 bp)
used for S. pombe ORFs
1
2
3
4
5
7
2
1 3
4
5
6
URA3
β-lactamase gene
pBR322 ori
yeast 2 µm origin
GAL1 promoter
C-terminal tags
7
pBG1805 (6573 bp)
used for S. cerevisiae ORFs
1
2
3
4
5 6
ORF goes
here ORF goes
here
16. How are plasmids constructed?
What functional elements are found in our yeast
overexpression plasmids?
How are plasmids purified?
17. Plasmids are much smaller than bacterial
chromosomes
Plasmids are supercoiled in their native form
Supercoiling allows plasmids to renature quickly
after they are denatured
Plasmid purification is based on their distinctive
physical properties
Plasmids used in molecular biology are highly engineered and
contain elements of use to researchers
18. Plasmid purification from bacteria relies on their unique
physical properties
Bacterial cell with
plasmids
contains
MANY different,
well-folded proteins
1-2 copies of large
(>Mbp) , circular
bacterial DNA
complexed with
proteins
Multiple copies of small
(5-15 kbp) plasmids
Purification involves sequential denaturation and renaturation steps
19. Cells are first treated with base and a detergent
breaks open membrane
and denatures both DNA
and proteins
Proteins denature
irreversibly
Chromosomal DNA
denatures—will have
difficulty renaturing
because of its length
and many proteins
complexed to it
Plasmids denature, but
strands stay together
because of supercoiling
20. Extract is neutralized to allow DNA molecules to renature
Plasmids
renature and are
suspended in the
SUPERNATANT
following
centrifugation
Proteins and
chromosomal DNA form
aggregate irreversibly,
forming a PRECIPITATE
that can be collected by
centrifugation
When purifying plasmids, use a micropipette to remove the
supernatant for further processing steps
21. Zyppy purification kit use multiple steps to purify plasmids
Alkaline lysis
Neutralization
Purification of plasmid DNA on a silica resin
Elution of purified DNA from he silica resin
Let's look at the individual steps……………..
22. 1 Transformed E. coli cultures are
concentrated by centrifugation
2. The cell pellet is resuspended
in 600 µL TE buffer by vortexing
3. 100 µL of 7X Blue Zyppy lysis buffer
is added
0.1 N NaOH in buffer lyses the cells
GENTLY mix the contents by inverting the tube 4-6 times
Solution changes from cloudy to clear when cells are lysed
Warning: too much mechanical agitation can shear chromosomal DNA
Alkaline lysis
23. Neutralization
4. Add 350 µL yellow Zyppy
Neutralization buffer
Mix by inverting several times
A heavy precipitate will begin to form immediately!
The initial “glop” will become more granular when
neutralization is complete—but don’t overdo it!
The precipitate contains denatured proteins and
the denatured chromosomal DNA.
5. Spin down the denatured molecules
for 3 minutes at top speed. CAREFULLY
remove the supernatant containing the
plasmid – Don't be greedy! Purity is
preferred to yield!
24. Purification on
Zyppy silica resin
6. Apply the supernatant to the spin
column. Place the column in the
collection tube. Centrifuge the column
for ~15 seconds at top speed.
7. Discard the flow through in the
collection tube. Add 200 µL Zyppy
EndoWash. Centrifuge ~15 sec.
EndoWash contains guanidine
hydrochloride and
isopropanol. It removes
contaminating proteins that
are bound to the resin.
8. Discard the flow through in the
collection tube. Add 400 µL Column
Wash. Centrifuge 1 min.
25. Plasmid Elution
9. Transfer the column to a clean,
LABELED microcentrifuge tube
10. Add 50 µL TE buffer directly on
top of the column. Allow the column to
stand upright in the test tube for ~10
min. (Plasmid is being eluted.)
11. Spin the column for 30 seconds.
Plasmid DNA will be collected in the
microcentrifuge tube. Pure plasmid DNA
collects here!