It is defined simply as a technique to efficiently and stably introduce foreign genes into the genome of target cells.
The insertion of unrelated, therapeutic genetic information in the form of DNA into target cells
Definition - Rolling circle replication is a process of unidirectional nucleic acid replication.
* can rapidly synthesize multiple copies of circular molecules of DNA or RNA, such as plasmids.
* Eucaryotic also replicate.
* widely used in molecular biology & biomedical
nanotechnology, especially in the field of
biosensing (as a method of signal Amplification).
Steps:
Circular ds DNA will be “nicked”
3` end is elongated →Leading strand
5` end displaced → Lagging strand
made up of double stranded by OKAZAKI fragments.
4) Replication of both “ unnicked” and displaced ss DNA
5) Displaced DNA circulates and synthesis its own complementary strand.
Initation-- phosphate ends, by the action of:
a) Helicase
b) Topoisomerases
c) Single stranded binding proteins(SSBPs)
Elongation-OH group of broken strand, using the unbroken strand as a template. The polymerase will start to move in a circle for elongation, due to which it is named as Rolling Circle Model.
end will be displaced and will grow out like a waving thread.
Termination-* At the point of termination, the linear DNA molecule is cleaved from the circle resulting in a double stranded circular DNA molecule and a single- stranded linear DNA molecule.
* The linear single stranded molecule is circularized by the action of ligase and then replication to double stranded circular plasmid molecule.
Example- Conjugation of F+ and F- bacteria
Diagrammatic representation of Rolling circle
some Examples-Viral DNA
* Human herpes virus
* Human papilloma virus
* Geminivirus
Viral RNA
* pospiviridiae
* Avsunviridiae
Reference:- https://en. m. wikipedia.org
what- when- how.com
https//www.sciencedirect.com
www.slideshare.com
Genetics-notes.wikispace.com
you tube
Prescott 5th edition page.no: 236, 237
Brock biology of microorganism , page.no: 253,616
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
Each cell in the human contains all the genetic material for the growth and development of a human
Some of these genes will be need to be expressed all the time
These are the genes that are involved in of vital biochemical processes such as respiration
Other genes are not expressed all the time
They are switched on an off at need
GENE CLONING,ITS HISTORY, NEW ADVENT IN GENE CLONING, PCR IMPORTANCE ,APPLICATION OF GENE CLONING,STEPS OF GENE CLONING,Antisense technology,Gene cloning in agriculture,Somatic cell therapy,Role of gene cloning in identification of genes responsible for human diseases,Synthesis of other recombinant human proteins and recombinant vaccines
Gene cloning in medicine,Recombinant protein from yeast,Problems with the production of recombinant protein in E.coli ,Expression of foreign genes in E.coli,Production of recombinant protein ,PCR can also be used to purify a gene,Obtaining a pure sample of a gene by cloning,Why gene cloning and PCR are so important,The advent of gene cloning and the polymerase
chain reaction.
Autonomously replicating circular fragment present in DNA is called plasmids.
The term plasmid was first introduced by American molecular biologist Joshua Lederberg in1952.
An episome is a plasmid capable of inserting DNA into the host chromosome.
Because of their ability to transfer DNA from one bacterium to another, plasmids are extensively used in recombinant DNA technology or genetic engineering.
Definition - Rolling circle replication is a process of unidirectional nucleic acid replication.
* can rapidly synthesize multiple copies of circular molecules of DNA or RNA, such as plasmids.
* Eucaryotic also replicate.
* widely used in molecular biology & biomedical
nanotechnology, especially in the field of
biosensing (as a method of signal Amplification).
Steps:
Circular ds DNA will be “nicked”
3` end is elongated →Leading strand
5` end displaced → Lagging strand
made up of double stranded by OKAZAKI fragments.
4) Replication of both “ unnicked” and displaced ss DNA
5) Displaced DNA circulates and synthesis its own complementary strand.
Initation-- phosphate ends, by the action of:
a) Helicase
b) Topoisomerases
c) Single stranded binding proteins(SSBPs)
Elongation-OH group of broken strand, using the unbroken strand as a template. The polymerase will start to move in a circle for elongation, due to which it is named as Rolling Circle Model.
end will be displaced and will grow out like a waving thread.
Termination-* At the point of termination, the linear DNA molecule is cleaved from the circle resulting in a double stranded circular DNA molecule and a single- stranded linear DNA molecule.
* The linear single stranded molecule is circularized by the action of ligase and then replication to double stranded circular plasmid molecule.
Example- Conjugation of F+ and F- bacteria
Diagrammatic representation of Rolling circle
some Examples-Viral DNA
* Human herpes virus
* Human papilloma virus
* Geminivirus
Viral RNA
* pospiviridiae
* Avsunviridiae
Reference:- https://en. m. wikipedia.org
what- when- how.com
https//www.sciencedirect.com
www.slideshare.com
Genetics-notes.wikispace.com
you tube
Prescott 5th edition page.no: 236, 237
Brock biology of microorganism , page.no: 253,616
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
Each cell in the human contains all the genetic material for the growth and development of a human
Some of these genes will be need to be expressed all the time
These are the genes that are involved in of vital biochemical processes such as respiration
Other genes are not expressed all the time
They are switched on an off at need
GENE CLONING,ITS HISTORY, NEW ADVENT IN GENE CLONING, PCR IMPORTANCE ,APPLICATION OF GENE CLONING,STEPS OF GENE CLONING,Antisense technology,Gene cloning in agriculture,Somatic cell therapy,Role of gene cloning in identification of genes responsible for human diseases,Synthesis of other recombinant human proteins and recombinant vaccines
Gene cloning in medicine,Recombinant protein from yeast,Problems with the production of recombinant protein in E.coli ,Expression of foreign genes in E.coli,Production of recombinant protein ,PCR can also be used to purify a gene,Obtaining a pure sample of a gene by cloning,Why gene cloning and PCR are so important,The advent of gene cloning and the polymerase
chain reaction.
Autonomously replicating circular fragment present in DNA is called plasmids.
The term plasmid was first introduced by American molecular biologist Joshua Lederberg in1952.
An episome is a plasmid capable of inserting DNA into the host chromosome.
Because of their ability to transfer DNA from one bacterium to another, plasmids are extensively used in recombinant DNA technology or genetic engineering.
BACTERIAL RECOMBINATION,PLASMIDS AND EPISOMESsushma93
Genetic recombination - transfer of DNA from one organism (donor) to another organism (recipient). The transferred donor DNA may then be integrated into the recipient's genetic material by various mechanisms
Bacterial recombination occurs in three ways
Transformation
Transduction
Conjugation
1a- What are three ways that bacteria can exchange genetic informati.pdfeyelineoptics
1a- What are three ways that bacteria can exchange genetic information? How are these similar
and how are they different? How can they be used to map bacterial genomes?
b) Explain how genes in phages are mapped. (This is different from the above question)
Solution
Ques-1: What are three ways that bacteria can exchange genetic information? How are these
similar and how are they different? How can they be used to map bacterial genomes?
Answer:
Horizontal gene transfer (HGT) or lateral gene transfer is the transfer of genes between two
organisms that are phylogenetically unrelated but can readily exchange genes by conjugation or
transduction or transformation. HGT commonly occurs in prokaryotes and it plays a key role in
their evolution. HGT accelerates evolution.
Horizontal gene transfer: 1. conjugation, 2. transformation; 3. transduction
Similarities & differences:
Horizontal gene transfer is the transfer of genomes by between bacteria by the routes that does
not involve parent-offspring transmission. Conjugation, transduction and transformation are the
three mechanism of horizontal gene transfer.
In the above case, it has clearly illustrated that the conditions, which can successfully prevents
transduction and abolish transfer of chromosomal DNA from one bacterial cell to another by
generalized transduction with P22 bacteriophage. Initially recipient cell- surface receptors are
important for the attachment of the bacteriophage (P22) and finally spikes of phage enable
bacterial membrane to lyse finally incorporate phage genome into the bacterial cell for
integration. Therefore, for efficient transduction, presence of recipient cell- surface receptors is
essential in the culture otherwise, bacteria exert resistant effects to the phages.
Transformation type of gene transfer occurs between bacteria. The genes released by dead
bacteria can be transferred to the live bacteria, and get integrated into its genome, which results
in genetic recombination of the host.
Transduction is also involving the genetic material transfer, but this needs bacteriophage as
media. The bacteriophage infects the bacteria in lytic cycle and releases the new virions, the
capsids of some of these virions carry the bacterial DNA fragments into the new host bacteria,
followed by genetic recombination of the host bacteria.
Conjugation: It is the transfer of genetic material from cell to cell by means of direct contact or a
bridge like structure. Bacterial conjugation needs the conjugation tube for genome transfer, both
transduction and transformation does not need this.
Mapping the genome -mechanisms
F factor is mainly pertaining to a class of conjugate plasmids, which meticulously originated to
control sexual functions in prokaryotes via fertility inhibition. The origin of F-factor is mainly
with most common functional segments such as gene traJ, Ori, OriC. The structure of this F
factor contains OriT for origin of transfer and acts as a starting point for gene transmission.
Leptospirosis: Its Epidemiology, Diagnosis and Control Chandrani Goswami
Leptospirosis is a zoonosis caused by pathogenic spirochetes of the genus Leptospira.
Disease was first described by Adolf Weil in 1886
In 1908, a Japanese research group led by Ryokichi Inada and Yutaka to first identified the bacterium as the causative agent of leptospirosis and noted its presence in rats in 1916
Generally it is transmitted by the infected urine of rodents.
Leptospirosis is in the group of 17 neglected tropical diseases, categorized by WHO.
Leptospirosis is an underreported disease, and there are no reliable global incidence figures (WHO, 2015)
Synonyms: Weil's Syndrome, Weil-Vasiliev disease, Swineherd's disease, Rice-field fever, Waterborne fever, Nanukayami fever, Cane-cutter fever, Swamp fever, Mud fever, Stuttgart disease, and Canicola fever.
Global travel and spread of COVID 19: Current epidemiological statusChandrani Goswami
International departures and arrivals across the world have increased significantly in just 10 years, contributing to the rapid spread of coronavirus globally
OVERVIEW OF coronavirus
Epidemiology CoVs:
COVID-19 Transmission
COVID-19 Distribution
SARS-CoV-2 Strain
Heart failure (HF), often used to mean chronic heart failure (CHF), occurs when the heart is unable to pump sufficiently to maintain blood flow to meet the needs of the body.
Somatic cells are mainly epithelial cells that has been shed from the epithelial lining of the gland and White blood cells (leukocyte) has entered the mammary gland in response to injury or infection.
Refers to inflammation of the mammary gland, which is characterized by physical, chemical as well as bacteriological changes in the milk and pathological changes in the udder tissues.
Major agents and their characteristics which has beenChandrani Goswami
Biological weapon, also called germ weapon
“Any of a number of disease-producing agents such as bacteria, viruses, rickettsia, fungi, toxins, or other biological agents that may be utilized as weapons against humans, animals, or plants”.
Nutraceutical market, scope and growth: Herbal drug technologyLokesh Patil
As consumer awareness of health and wellness rises, the nutraceutical market—which includes goods like functional meals, drinks, and dietary supplements that provide health advantages beyond basic nutrition—is growing significantly. As healthcare expenses rise, the population ages, and people want natural and preventative health solutions more and more, this industry is increasing quickly. Further driving market expansion are product formulation innovations and the use of cutting-edge technology for customized nutrition. With its worldwide reach, the nutraceutical industry is expected to keep growing and provide significant chances for research and investment in a number of categories, including vitamins, minerals, probiotics, and herbal supplements.
Multi-source connectivity as the driver of solar wind variability in the heli...Sérgio Sacani
The ambient solar wind that flls the heliosphere originates from multiple
sources in the solar corona and is highly structured. It is often described
as high-speed, relatively homogeneous, plasma streams from coronal
holes and slow-speed, highly variable, streams whose source regions are
under debate. A key goal of ESA/NASA’s Solar Orbiter mission is to identify
solar wind sources and understand what drives the complexity seen in the
heliosphere. By combining magnetic feld modelling and spectroscopic
techniques with high-resolution observations and measurements, we show
that the solar wind variability detected in situ by Solar Orbiter in March
2022 is driven by spatio-temporal changes in the magnetic connectivity to
multiple sources in the solar atmosphere. The magnetic feld footpoints
connected to the spacecraft moved from the boundaries of a coronal hole
to one active region (12961) and then across to another region (12957). This
is refected in the in situ measurements, which show the transition from fast
to highly Alfvénic then to slow solar wind that is disrupted by the arrival of
a coronal mass ejection. Our results describe solar wind variability at 0.5 au
but are applicable to near-Earth observatories.
This pdf is about the Schizophrenia.
For more details visit on YouTube; @SELF-EXPLANATORY;
https://www.youtube.com/channel/UCAiarMZDNhe1A3Rnpr_WkzA/videos
Thanks...!
THE IMPORTANCE OF MARTIAN ATMOSPHERE SAMPLE RETURN.Sérgio Sacani
The return of a sample of near-surface atmosphere from Mars would facilitate answers to several first-order science questions surrounding the formation and evolution of the planet. One of the important aspects of terrestrial planet formation in general is the role that primary atmospheres played in influencing the chemistry and structure of the planets and their antecedents. Studies of the martian atmosphere can be used to investigate the role of a primary atmosphere in its history. Atmosphere samples would also inform our understanding of the near-surface chemistry of the planet, and ultimately the prospects for life. High-precision isotopic analyses of constituent gases are needed to address these questions, requiring that the analyses are made on returned samples rather than in situ.
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.
(May 29th, 2024) Advancements in Intravital Microscopy- Insights for Preclini...Scintica Instrumentation
Intravital microscopy (IVM) is a powerful tool utilized to study cellular behavior over time and space in vivo. Much of our understanding of cell biology has been accomplished using various in vitro and ex vivo methods; however, these studies do not necessarily reflect the natural dynamics of biological processes. Unlike traditional cell culture or fixed tissue imaging, IVM allows for the ultra-fast high-resolution imaging of cellular processes over time and space and were studied in its natural environment. Real-time visualization of biological processes in the context of an intact organism helps maintain physiological relevance and provide insights into the progression of disease, response to treatments or developmental processes.
In this webinar we give an overview of advanced applications of the IVM system in preclinical research. IVIM technology is a provider of all-in-one intravital microscopy systems and solutions optimized for in vivo imaging of live animal models at sub-micron resolution. The system’s unique features and user-friendly software enables researchers to probe fast dynamic biological processes such as immune cell tracking, cell-cell interaction as well as vascularization and tumor metastasis with exceptional detail. This webinar will also give an overview of IVM being utilized in drug development, offering a view into the intricate interaction between drugs/nanoparticles and tissues in vivo and allows for the evaluation of therapeutic intervention in a variety of tissues and organs. This interdisciplinary collaboration continues to drive the advancements of novel therapeutic strategies.
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.
2. GENE TRANSFER
• It is defined simply as a technique to efficiently
and stably introduce foreign genes into the
genome of target cells.
• The insertion of unrelated, therapeutic genetic
information in the form of DNA into target cells.
3. INTRODUCTION
Gene transfer in which bacteria genotype can be
changed by transfer of genetic material from
one bacterium to another.
In such instances, the transferred DNA either:
a) Recombine with genome of the recipient
bacterium or
b) Is on the plasmid capable of replication in the
recipient bacterium without recombination.
4. Genetic recombination it occurs when sequence
of DNA from two separate sources are
integrated.
The directed desirable gene transfer from one
organism to another and the subsequent stable
integration & expression of foreign gene into the
genome is referred as genetic transformation.
The transferred gene is known as transgene and
the organism that develop after a successful gene
transfer is known as transgenic.
5. MECHANISM OF GENE TRANSFER
There are four mechanisms of gene transfer and
recombination between bacteria cells.
1. Conjugation
2. Transformation
3. Transduction
4. Cell- cell fusion
6. DNA TRANSFER BY ARTIFICIAL
METHODS
Physical methods
1. Microinjection
2. Biolistics transformation
Chemical methods
1. DNA transfer by calcium phosphate method
2. Liposome mediated transfer
Electrical methods
1. Electroporation
7. CONJUGATION
Bacteria conjugation in E.coli was discovered by
Lederberg and Tatum in 1946, when they
observed sex like exchange between two mutants
strains of E.coli called K12.
Conjugation involves the transfer of plasmid
from a donor( bacteria contain F plasmid) to the
recipient(bacteria lacking F plasmid).
8. Conjugation is more common in gram negative
between strain of same species or closely related
species.
Some plasmid like F plasmid(fertility factor or
sex factor) they carry tra gene that mediate their
own transfer through sex pilus.
Bacteria that have a F plasmid are referred to as
as F+ or male. Those that do not have an F
plasmid are F- of female.
The F plasmid consists of 25 genes that mostly
code for production of sex pilli.
9. Conjugation are of different types:
1) F+ conjugation- During conjugation the F+ bacteria
synthesise a modified pilus (sex pilus) through which
genetic material transfer, the result is two male cells.
• This attached pilus is a temporary cytoplasmic bridge
through which a replicating F plasmid is transferred
from the male to the female.
• Conjugation in gram positive does not depend on sex
pili, the donor cells form a protein adhesin on the
surface that causes donor and recipient cells to
aggregate. Eg. Bacillus subtillis, Streptocoocus lactis,
Enterococcus fecaelis
10.
11. 2) Resistant plasmid conjugation- Some gram negative
bacteria(eg. E.coli, shigella) contain plasmids that
contain antibiotic resistance gene called R factor. The R
factor has two component-
i) RTF( resistance transfer factor) that codes for sex
transfer like F factor.
ii) r determinant that codes for antibiotic resistance.
Sometimes RTF may dissociated from the r
determinants and the two component may exist as
separate entities. Under such condition, although the
host cell remains drug resistant, the drug resistance is
not transferable.
12. 3) High frequency recombinant (Hfr) conjugation-
When the F+ plasmid is integrated within the bacterial
chromosome, the cell is called an Hfr cell
Plasmid that are capable of integrating into the
chromosome is called episome.
HFR cells are able to transfer chromosomal gene to
recipient with high frequency.
The recipient F- cell usually remain F- after conjugation
because only part of the F plasmid from the donor Hfr
cell to the recipient have been transferred.
13. 4) Sexduction- When F- factor reverts to free state
from the integrated state, it may sometimes carry with
it some genetic material from the host cell adjacent to
the integration site of the F plasmid. Such type of
hybrid molecule and F factor plasmid incorporating
some host chromosomal gene is called F prime (F’)
factor. When F’ cell mates with a recipient(F-) cell the
recipient bacterium become F’. This process of gene
transfer is termed as sexduction.
14. TRANSFORMATION• Involves the uptake of free or naked DNA via a
recipient released by the donor.
• Transformation, originally discovered in
pneumococcus by Griffith(1928) known as Griffith’s
experiment, has also observed in Bacillus, Neisseria,
Haemophilus bacteria.
• Griffith’s experiment in Pneumococcus
Type I strain(smooth strain) injected to mice it dies
Type II strain (rough strain) injected to mice it survives
15. • Heat killed capsulated type I injected to mice it
survives.
• Mixed culture(heat killed capsulated type I and type II
strain) injected to mice it dies,he discovered life
smooth strain from the blood collected from mice.
• In 1944 Avery, Mcleod and Mc Carty found that the
factor that transform other strain was found to be
DNA.
• Transformation occurs naturally in some species of
bacteria , but it can also be effected by artificial
treatment in other species.
16.
17. TRANSDUCTION
Gene transfer from a donor to a recipient by way of a
bacteriophage.
If the lysogenic cycle is adopted, the phage
chromosome is integrated (by covalent bonds) into the
bacterial chromosome, this is called prophage.this
process- lysogeny or lysogenic conversion,bacteria-
lysogenic bacteria.
The lytic cycle leads to the production of new phage
particles which are released by lysis of the host.
18.
19. Transduction has been observed im a diverse group of
bacteria- Escherichia, Salmonella, Shigella, klebsiella,
proteus, psedomonas, bacillus and streptococcus.
Two ways in which transduction occurs:
1) Generalised transduction eg. Phages P22 of
Salmonella typhimurium, phage P1 of E.coli
2) Restricted or specialised transduction eg. Lamda
phage of E.coli transfers gal gene.
20. Generalised transduction Restricted transduction
Transfer Any gene Genes immediately adjacent to the
site of prophage attachment
Induction Transducing phage is
produced upon induction of
prophage and in infection with
virulent phages.
Transducing phage is produced
upon induction of only prophage
Content in
transducing
phage
Only bacterial DNA Hybrid- contains both bacterial
and phage DNA
Transducing
paricles
Replace recipient gene when
integrated in chromosome
Added to genome of recipient cell
21. Cell to cell fusion(protoplast fusion)
• In gram positive bacteria???
• Cell membrane of participating cells are joined
together in protoplast fusion which inturn generates
cytoplasmic bridge between the cells resulting in
mixing of their cytoplasm and exchange of genetic
material.
• Protoplast fusion is an artificial method of gene
transfer.
22. SCREENING OF TRANSGENE
• The presence of transgene or gene of interest is
detected by several methods:
• A selectable marker gene
• Southern blot techniques
• Northern bolt technique
• Western blot technique
23. APPLICATION
• Clinical gene transfer applications
• Vaccine Development
• Production of transgenic animals
• Treatment of Cancer, AIDS
• Gene Discovery
• Gene Therapy
• Enhancing the resistance of plants
• GMO