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
• Plasmids are extra-chromosomal genetic elements that replicate independently of the host chromosome.
• They are small, circular (some are linear), double-stranded DNA molecules that exist in bacterial cells and in some eukaryotes.
There is the fifth video by Miss Aymen Arif Sindh Biotechnologist Association has taken initiative for all young scientists, researchers, and students to have the platform to show their talent and interest in different activities.
Topic: Plasmids and its types
Presentation by: Aymen Arif
Research Officer at Halal Food and testing Laboratory,
Industrial Analytical Center, H.E.J (ICCBS).
Youtube: https://www.youtube.com/watch?v=-spdnc-2z6Q
• Plasmids are extra-chromosomal genetic elements that replicate independently of the host chromosome.
• They are small, circular (some are linear), double-stranded DNA molecules that exist in bacterial cells and in some eukaryotes.
There is the fifth video by Miss Aymen Arif Sindh Biotechnologist Association has taken initiative for all young scientists, researchers, and students to have the platform to show their talent and interest in different activities.
Topic: Plasmids and its types
Presentation by: Aymen Arif
Research Officer at Halal Food and testing Laboratory,
Industrial Analytical Center, H.E.J (ICCBS).
Youtube: https://www.youtube.com/watch?v=-spdnc-2z6Q
On the basis of need of specific content of any topic, i prepared a slides of plasmid for needy students. I'm also a student that's why i know how useful a proper presentation for us.
In this presentation, i try to cover some basic knowledge regarding to plasmid. If you like this ppt than please let me know, it gives me a motivation. If you need other topics ppt then write a topic name on comment section. THANK YOU
Recombinant baculoviruses are widely used to
express heterologous genes in cultured insect cells
and insect larvae. For large-scale applications, the
baculovirus expression vector system (BEVS) is particularly
advantageous.
Along the prokaryotic vectors some Eukaryotic vectors are also present. These are basically used for the expression of eukaryotic DNA of interest in the Eukaryotes
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
This lecture is intended as an introduction to the fundamental concepts associated with plasmid DNA. Plasmids can be applied as vectors in Genetic Engineering for the production of recombinant proteins as well as the construction of genomic libraries for DNA sequencing projects.
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
On the basis of need of specific content of any topic, i prepared a slides of plasmid for needy students. I'm also a student that's why i know how useful a proper presentation for us.
In this presentation, i try to cover some basic knowledge regarding to plasmid. If you like this ppt than please let me know, it gives me a motivation. If you need other topics ppt then write a topic name on comment section. THANK YOU
Recombinant baculoviruses are widely used to
express heterologous genes in cultured insect cells
and insect larvae. For large-scale applications, the
baculovirus expression vector system (BEVS) is particularly
advantageous.
Along the prokaryotic vectors some Eukaryotic vectors are also present. These are basically used for the expression of eukaryotic DNA of interest in the Eukaryotes
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
This lecture is intended as an introduction to the fundamental concepts associated with plasmid DNA. Plasmids can be applied as vectors in Genetic Engineering for the production of recombinant proteins as well as the construction of genomic libraries for DNA sequencing projects.
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
A genetically engineered DNA molecule from bacteria , phage or yeast to carry foreign DNA for the purpose of cloning and expression of the inserted DNA of interest in RDT
The main difference between plasmid and vectors is that plasmid is an extra-chromosomal element of mainly bacterial cells whereas vector is a vehicle that carries foreign DNA molecules into another cell. Vectors are mainly used in the recombinant DNA technology to introduce foreign DNA molecules into cells.
After the end of the presentation we’ll know -
What is cloning vector?
Why cloning vector?
History
Features of a cloning vector
Types of cloning vector
Plasmid
Bacteriophage
Cosmid
Bacterial Artificial Chromosome (BAC)
Yeast Artificial Chromosome (BAC)
Human Artificial Chromosome (HAC)
Retroviral Vectors
What determines choice of vector?
Vector in molecular gene cloning
Objectives:
After the end of the presentation we’ll know -
What is cloning vector?
Why cloning vector?
History
Features of a cloning vector
Types of cloning vector
Plasmid
Bacteriophage
Cosmid
Bacterial Artificial Chromosome (BAC)
Yeast Artificial Chromosome (BAC)
Human Artificial Chromosome (HAC)
Retroviral Vectors
What determines choice of vector?
Vector in molecular gene cloning
Cloning vector - The molecular analysis of DNA has been made possible by the cloning of DNA. The two molecules that are required for cloning are the DNA to be cloned and a cloning vector.
A cloning vector is a small piece of DNA taken from a virus, a plasmid or the cell of a higher organism, that can be stably maintained in an organism and into which a foreign DNA fragment can be inserted for cloning purposes.
Most vectors are genetically engineered.
The cloning vector is chosen according to the size and type of DNA to be cloned.
The vector therefore contains features that allow for the convenient insertion or removal of DNA fragment in or out of the vector, for example by treating the vector and the foreign DNA with a restriction enzyme and then ligating the fragments together.
After a DNA fragment has been cloned into a cloning vector, it may be further subcloned into another vector designed for more specific use.
A detailed explanation of cloning strategies which involves isolation of DNA fragments from the sample and introduction in to a vector with restriction enzymes and introduced in to host by different methods and finally screening of the host cells with the recombinants based on protein,nucleicacid and antibiotic assays
control of gene expression by sigma factor and post transcriptional controlIndrajaDoradla
explanation of control of gene expression by sigma factor and decription of sigma factor and detailed explation of post transcriptional control by antisense technology and rna i
description of transgenic animals and production with desired traits using different methods and their applications and their advantages and disadvantages
Professional air quality monitoring systems provide immediate, on-site data for analysis, compliance, and decision-making.
Monitor common gases, weather parameters, particulates.
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.
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.
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.
Cancer cell metabolism: special Reference to Lactate PathwayAADYARAJPANDEY1
Normal Cell Metabolism:
Cellular respiration describes the series of steps that cells use to break down sugar and other chemicals to get the energy we need to function.
Energy is stored in the bonds of glucose and when glucose is broken down, much of that energy is released.
Cell utilize energy in the form of ATP.
The first step of respiration is called glycolysis. In a series of steps, glycolysis breaks glucose into two smaller molecules - a chemical called pyruvate. A small amount of ATP is formed during this process.
Most healthy cells continue the breakdown in a second process, called the Kreb's cycle. The Kreb's cycle allows cells to “burn” the pyruvates made in glycolysis to get more ATP.
The last step in the breakdown of glucose is called oxidative phosphorylation (Ox-Phos).
It takes place in specialized cell structures called mitochondria. This process produces a large amount of ATP. Importantly, cells need oxygen to complete oxidative phosphorylation.
If a cell completes only glycolysis, only 2 molecules of ATP are made per glucose. However, if the cell completes the entire respiration process (glycolysis - Kreb's - oxidative phosphorylation), about 36 molecules of ATP are created, giving it much more energy to use.
IN CANCER CELL:
Unlike healthy cells that "burn" the entire molecule of sugar to capture a large amount of energy as ATP, cancer cells are wasteful.
Cancer cells only partially break down sugar molecules. They overuse the first step of respiration, glycolysis. They frequently do not complete the second step, oxidative phosphorylation.
This results in only 2 molecules of ATP per each glucose molecule instead of the 36 or so ATPs healthy cells gain. As a result, cancer cells need to use a lot more sugar molecules to get enough energy to survive.
Unlike healthy cells that "burn" the entire molecule of sugar to capture a large amount of energy as ATP, cancer cells are wasteful.
Cancer cells only partially break down sugar molecules. They overuse the first step of respiration, glycolysis. They frequently do not complete the second step, oxidative phosphorylation.
This results in only 2 molecules of ATP per each glucose molecule instead of the 36 or so ATPs healthy cells gain. As a result, cancer cells need to use a lot more sugar molecules to get enough energy to survive.
introduction to WARBERG PHENOMENA:
WARBURG EFFECT Usually, cancer cells are highly glycolytic (glucose addiction) and take up more glucose than do normal cells from outside.
Otto Heinrich Warburg (; 8 October 1883 – 1 August 1970) In 1931 was awarded the Nobel Prize in Physiology for his "discovery of the nature and mode of action of the respiratory enzyme.
WARNBURG EFFECT : cancer cells under aerobic (well-oxygenated) conditions to metabolize glucose to lactate (aerobic glycolysis) is known as the Warburg effect. Warburg made the observation that tumor slices consume glucose and secrete lactate at a higher rate than normal tissues.
What is greenhouse gasses and how many gasses are there to affect the Earth.moosaasad1975
What are greenhouse gasses how they affect the earth and its environment what is the future of the environment and earth how the weather and the climate effects.
Comparing Evolved Extractive Text Summary Scores of Bidirectional Encoder Rep...University of Maribor
Slides from:
11th International Conference on Electrical, Electronics and Computer Engineering (IcETRAN), Niš, 3-6 June 2024
Track: Artificial Intelligence
https://www.etran.rs/2024/en/home-english/
This pdf is about the Schizophrenia.
For more details visit on YouTube; @SELF-EXPLANATORY;
https://www.youtube.com/channel/UCAiarMZDNhe1A3Rnpr_WkzA/videos
Thanks...!
2. What is?
• A plasmid is a small, circular, double-stranded DNA molecule that is distinct
from a cell's chromosomal DNA It replicates independently of
chromosomal DNA Plasmids naturally exist in bacterial cells, and they also
occur in some eukaryotes.
• plasmids carry genes that are beneficial to the host organisms. Often, the
genes carried in plasmids provide bacteria with genetic advantages, such as
antibiotic resistance. Plasmids have a wide range of lengths, from roughly one
thousand DNA base pairs to hundreds of thousands of base pairs.
• When a bacterium divides, all of the plasmids contained within the cell are
copied such that each daughter cell receives a copy of each plasmid. Bacteria
can also transfer plasmids to one another through a process called conjugation
5. ADVANTAGE
• Scientists have taken advantage of plasmids to use them as tools to
clone, transfer, and manipulate genes. Plasmids that are used
experimentally for these purposes are called vectors. Researchers
can insert DNA fragments or genes into a plasmid vector, creating a
so-called recombinant plasmid. Then, because bacteria divide
rapidly, they can be used as factories to copy DNA fragments in
large quantities. Artificial plasmids are widely used
as vectors in molecular cloning, serving to drive the replication
of recombinant DNA sequences within host organisms. In the
laboratory, plasmids may be introduced into a cell
via transformation.
• Characteristics such as flexibility, versatility, safety, and cost-
effectiveness enable molecular biologists to broadly utilize
plasmids across a wide range of applications. Some common
plasmid types include cloning plasmids, expression plasmids, gene
knock-down plasmids, reporter plasmids, viral plasmids,
and genome engineering plasmids.
6.
7. Types of Plasmids
There are five main types of plasmids:
•fertility F-plasmids
• resistance plasmid
• virulence plasmids
• degradative plasmids
•Col plasmids.
8.
9.
10.
11. ARTIFICIAL PLASMID
• pBR322 is a purpose built plasmid vector and was one of the first
widely used cloning vector it has a relatively small size of 4361
bp. This is important because transformation efficiency is inversly
proportional to size.
Nomenclature of pBR322
• Created in 1977 in the laboratory of Herbert Boyer at the
university of california, san Francisco
‘p’ plasmid
‘BR’ Boliver and Rodriguez (researchers who developed
it)
‘322’ distinguishes those plasmids from others (like
pBR325,pBR327 etc) developed in the same laboratory
Features of pBR322
• pBR322 has 4361 base pairs in length
• It has two antibiotic resistance genes they are:
a)Ampicillin resistance gene(AmpR)
R
12. • Origin of replication(ori)
• It has unique restriction sites for more than 40 restriction enzymes.11
of these 40 sites lie with in the TetR gene. There are 2 sites for
restriction enzymes HindIII and claI with in the promoter of TetR
gene. They are 6 restriction sites inside the AmpR gene.
Construction of pBR322
• The ampR gene originally resided on the plasmid RSF2124(a
naturally occurring antibiotic resistance plasmid in E.coli)
• The tetR is derived from Psc101 (a second antibiotic resistant
13. • The origin of replication is derived from pMB1, which is closely
related to the colicin producing plasmid colE1
INSERTIONAL INACTIVATION
• It is a technique used in recombinant DNA technology to select
recombinant colonies
• It involves the inactivation of a gene up on insertion of another
gene inside in its place or with in its coding sequence
For example-pBR322 is opened by using a restriction enzyme
BamHI where restriction site lies with in the tetracycline resistant
gene. The foreign DNA also isolated with the same type of
restriction enzyme and DNA ligase is added to the mixture
containing linearized pBR322 and the foreign gene two types of
vectors are formed
Recombinant vector foreign gene
Un altered vector foreign gene
15. ampR tetR
Foreign gene
ampR ampRtetR
Insertion
inactivation
Unaltered vector Altered vector
E.coli
Non transformed cell Transformed cells with and with
Out recombinant vector
INTERPRETATION
Non transformed ampS,tetS
T.With nonrecombinant ampR,tetR
T,with recombinant ampR,tetS
Transformation
16. Non transformed cell Transformed cells with and with
Out recombinant vector
Amp
Non transformed cells dead
Transformed cells live
Amp
Transformed cells are transferred
Master plate
tet
Replica plate
Transformed with recombinant dead
Transformed with unaltered live
Compared with the master plate and recombinants are picked
INTERPRETATION
Non transformed ampS,tetS
T.With nonrecombinant ampR,tetR
T,with recombinant ampR,tetS
17. USES
• It is widely used as a cloning vector
• It is used as a model system for study of prokaryotic transcription
and translation
ADVANTAGES
• Due to small size enables easy purification and manipulation
• As due to the presence of 2 selectable markers (amp,tet) allow
easy selection of recombinant DNA
• It can be amplified up to 1000-3000 copies
DISADVANTAGES
• It has very high mobility that is it can move to another cell in the
presence of conjugative plasmid like F-cell.due to this the vector
may lost in the mixed host of cells
• There is a limitation in the size of gene of interest that it can
accommodate
• Screening process is time consuming and laborious
• No high copy number
19. • These are artificially constructed
• Phagemids are the vectors that are made up of a plasmid and phage DNA
Nomenclature of phagemid
Phage part of DNA obtained from bacteriophage
Mid part of DNA obtained from plasmid
Features of phagemid
• It contains 2961bp
• It has ampicillin resistant containing gene
• It has 2 ori genes
• It has Mcs
• It has lacZ gene
• Example – most commonly used phagemids are PBluescript series
Construction of PBluescript
• marker genes plasmid
• Ori plasmid produce dsDNA
F1/M13 phage produce ss DNA
• Mcs plasmid (21 unique restriction sites) inserted in lacZ
• T7 &T3 promoters produce RNA polymerase
20. Some imp characters
• Similarly to a plasmid, a phagemid can be used to clone DNA
fragments and be introduced into a bacterial host by a range of
techniques (transformation, electroporation).
Production of dsDNA
• When a phagemid is introduced in to a host it normally replicates
and produce dsDNA
E.coli
pBluescript II SK (+/-) phagemids
21. Production of ssDNA
• However, infection of a bacterial host containing a phagemid with a
'helper' phage. e.g. VCSM13 or M13K07, provides the necessary viral
components to enable single stranded DNA replication and packaging
of the phagemid DNA into phage particles.
•These are secreted through the cell wall and released into the medium.
Filamentous phage (M13)retard bacterial growth but, in contrast to
lambda and T7 phage, are not generally lytic.
•Helper phage are usually engineered to package less efficiently than the
phagemid so that the resultant phage particles contain predominantly
phagemid DNA. F1 Filamentous phage infection requires the presence of
a pilus so only bacterial hosts containing the F-plasmid or its derivatives
can be used to generate phage particles.
22. Production of 2 diff series (depend on sense + & anti sense –
strand)
• The pBluescript II SK and KS vector series represent two
orientations of the MCS within the lacZ gene encoding the N-
terminal fragment of beta-galactosidase (KS represents the
orientation of the MCS in which lacZ transcription proceeds from
KpnI to SacI, while SK - from SacI to KpnI).
Production of RNA transcripts
• In vitro transcription of T3 and T7 RNA polymerase
pBluescript II SK (+/-) phagemids
23. Screening of recombinants
Interpretation
• The recombinants are screened by growing the cells in a medium
containing IPTG(iso propyl β D1 thio galactopyranoside)and x-gal
• When a foreign gene is inserted in the LacZ the E.coli cells fails to
produce β galactosidase so no hydrolysis of x-gal so the
bacterial colony remains white
LacZ gene produces β galactosidase it hydrolysis the x-gal and IPTG containing medium
and produce blue colour
White recombinants
Blue Non recombinants
24. Uses
pBluescript II phagemids are designed for DNA cloning, dideoxy
DNA sequencing, in vitro mutagenesis and in vitro transcription
in a single system.
Advantages
• It can be used to provide single strand or double stranded material
with out recloning
• Carrying capacity is higher than phage vectors
• Higher efficiency in transformation than phage vectors
• Phagemids are generally more stable than the recombinant phage
vectors
• Screening and storage is easier
26. • These are artificially constructed (hybrid)
• Cosmids are the vectors that are made up of a plasmid and phage DNA
Nomenclature of cosmid
Cos part of DNA containing cohesive sites
Mid part of DNA obtained from plasmid
Construction
Cosmids were first developed by collins and
Hohn in 1978
Features of cosmid
• It can carry about 44 kbp
• It has antibiotic resistant containing gene(selectable marker)
• It has ori gene
• It has Mcs(poly linker)
• One or two cos sites (require for packaging in to phage particle)
• The cos sites allow the vector to be packaged and transmitted to bacteria
like a phage vector
27. • Cloning Steps :-
• Preparation . - Target DNA: the genomic DNA to be cloned has to be cut into the
appropriate size range of restriction fragments , By using Restriction Enzymes , And
Cut the Cosmid Cloning site ( poly linker ) by the same Restriction enzyme , Then
Ligation of physically unlinked fragments.it results in the formation of long
concatamers and these are used efficiently for packing
• Packaging - the total DNA is transferred into an appropriate E. coli host via a
technique called in vitro packaging (recombinant plasmids contained in phage shells
) . - they can also be packaged in phage capsids, which allows the foreign genes to
be transferred into or between cells by transduction (a process resembling viral
infection )
28. • In general when a phage infects a bacteria it infects with high efficiency
and when it enters it gets circularized due to the presence of cos sites and
undergo recombination with the bacterial genome with site directed
integrated manner
• So during cloning of cosmid vector when the difference between cos sites is
about at a distance of 50kb away from each other they could be efficiently
packaged in to the head if the distance is less than <50kb or <30kbp then no
packaging in to the head takes place
29. More distance between cos sites Less distance between cos sites
Transduction : The phage particle with cosmid vector is now inserted in to
the host bacteria that process similar to the infection of viral particle and
when the DNA enters in to the host it gets circularized
30. • Plating & Growing & Selection & Isolation - Colonies are formed in
selective media , just as with a plasmid vector . - The media is : Liquid
media . - So that the transformed cells can be identified by plating on a
medium containing the antibiotic ( Ampicillin Agar plate ). - Those cells
which did not take up the cosmid would be unable to grow.
Uses of Cosmid Vectors:
• Cosmids are used for construction of genomic libraries of eukaryotes since
these can be used for cloning large fragments of DNA.
31. Advantages of cosmids
• Can be used as cloning vectors
• Cosmids are high insert capacity vectors
• Cosmids are used for the analysis of complex genomes
• Large genes can be studied intact and genetic linkage studies can be carried
out at molecular level
• Background molecules which do not have the insert DNA or have the
smaller inserts are eliminated during packaging
• Transformation efficiency of cosmids is high when compared to plasmids
• Easy screening method is found
Disadvantages of cosmids
• Not easy to handle these are large plasmids
• Not stable can acquire mutations
Examples of cosmid vectors
• Commonly used vectors include "SuperCos 1"