BAC & YAC are artificially prepared chromosomes to clone DNA sequences.yeast artificial chromosome is capable of carrying upto 1000 kbp of inserted DNA sequence
BAC & YAC are artificially prepared chromosomes to clone DNA sequences.yeast artificial chromosome is capable of carrying upto 1000 kbp of inserted DNA sequence
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
What are an expression vector? Detailed description of plant gene structure. Plant expression vector systems are generally consists of Ri and Ti plasmids.
The other vectors which are generally used are DNA and RNA viruses.
This presentation covers a general introduction to expression vector, its components, types, and its application. Then it covers some of the expression system with examples.
Introduction
Ti plasmid
Agrobacterium tumefaciens
Ti plasmid structure
Overview of infection process
Ti plasmid derived vector systems
Cointegrate vectors
Binary vectors
Agrobacterium mediated transformation of explants
Conclusions
References
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
What are an expression vector? Detailed description of plant gene structure. Plant expression vector systems are generally consists of Ri and Ti plasmids.
The other vectors which are generally used are DNA and RNA viruses.
This presentation covers a general introduction to expression vector, its components, types, and its application. Then it covers some of the expression system with examples.
Introduction
Ti plasmid
Agrobacterium tumefaciens
Ti plasmid structure
Overview of infection process
Ti plasmid derived vector systems
Cointegrate vectors
Binary vectors
Agrobacterium mediated transformation of explants
Conclusions
References
Artificial chromosome I Bacterial Artificial Chromosome I Yeast Artificial C...DevikaPatel12
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#BAC #YAC #bacterialartificialchromosome #yeastartificialchromosome #artificialchromosome #genetics #geneticengineering #DNA #dna #gene #genetherapy
Yeast Artificial Chromosome also known as YAC is an artificial vector used in molecular biology for genetic recombination, gene cloning and biotechnology.
It is the basics of vector cloning which necessary for every and each student who is intrested in biotechnology. It is only starting, if you want to more than this then please comment on it.
Gene Cloning Vectors - Plasmids, Bacteriophages and Phagemids.Ambika Prajapati
A cloning vector is a small piece of DNA that can be stably maintained in an organism, and into which a foreign DNA fragment can be inserted for cloning purposes. The cloning vector may be DNA taken from a virus, the cell of a higher organism, or it may be the plasmid of a bacterium.
They allow the exogenous DNA to be inserted, stored, and manipulated mainly at DNA level.
Types -
1.Plasmid vectors.
2.Bacteriophage vectors .
3.Phagemids.
A brief information about the SCOP protein database used in bioinformatics.
The Structural Classification of Proteins (SCOP) database is a comprehensive and authoritative resource for the structural and evolutionary relationships of proteins. It provides a detailed and curated classification of protein structures, grouping them into families, superfamilies, and folds based on their structural and sequence similarities.
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.
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.
(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.
The increased availability of biomedical data, particularly in the public domain, offers the opportunity to better understand human health and to develop effective therapeutics for a wide range of unmet medical needs. However, data scientists remain stymied by the fact that data remain hard to find and to productively reuse because data and their metadata i) are wholly inaccessible, ii) are in non-standard or incompatible representations, iii) do not conform to community standards, and iv) have unclear or highly restricted terms and conditions that preclude legitimate reuse. These limitations require a rethink on data can be made machine and AI-ready - the key motivation behind the FAIR Guiding Principles. Concurrently, while recent efforts have explored the use of deep learning to fuse disparate data into predictive models for a wide range of biomedical applications, these models often fail even when the correct answer is already known, and fail to explain individual predictions in terms that data scientists can appreciate. These limitations suggest that new methods to produce practical artificial intelligence are still needed.
In this talk, I will discuss our work in (1) building an integrative knowledge infrastructure to prepare FAIR and "AI-ready" data and services along with (2) neurosymbolic AI methods to improve the quality of predictions and to generate plausible explanations. Attention is given to standards, platforms, and methods to wrangle knowledge into simple, but effective semantic and latent representations, and to make these available into standards-compliant and discoverable interfaces that can be used in model building, validation, and explanation. Our work, and those of others in the field, creates a baseline for building trustworthy and easy to deploy AI models in biomedicine.
Bio
Dr. Michel Dumontier is the Distinguished Professor of Data Science at Maastricht University, founder and executive director of the Institute of Data Science, and co-founder of the FAIR (Findable, Accessible, Interoperable and Reusable) data principles. His research explores socio-technological approaches for responsible discovery science, which includes collaborative multi-modal knowledge graphs, privacy-preserving distributed data mining, and AI methods for drug discovery and personalized medicine. His work is supported through the Dutch National Research Agenda, the Netherlands Organisation for Scientific Research, Horizon Europe, the European Open Science Cloud, the US National Institutes of Health, and a Marie-Curie Innovative Training Network. He is the editor-in-chief for the journal Data Science and is internationally recognized for his contributions in bioinformatics, biomedical informatics, and semantic technologies including ontologies and linked data.
1. Bacterial
and yeast artificial chromosomes
PRESENTATION BY
N.CHITRA.
M.Sc.,Microbiology ( 2nd year )
ST.PETER’S INSTITUTE OF
HIGHER EDUCATION AND
RESEARCH,
AVADI,
CHENNAI – 54.
2. Yeast Artificial Chromosomes (YACs)
Yeast artificial chromosomes (YACs) are genetically engineered
chromosomes derived from the DNA of the yeast.
It is a human-engineered DNA molecule used to clone DNA
sequences in yeast cells.
They are the products of a recombinant DNA cloning
methodology to isolate and propagate very large segments
of DNA in a yeast host.
By inserting large fragments of DNA, the inserted sequences can
be cloned and physically mapped using a process called
chromosome walking.
The amount of DNA that can be cloned into a YAC is, on average,
from 200 to 500 kb.
However, as much as 1 Mb (mega, 106) can be cloned into a YAC.
3.
4. Structure
A yeast artificial
chromosome cloning
vector consists of two
copies of a yeast
telomeric sequence
(telomeres are the
sequences at the ends
of chromosomes), a
yeast centromere, a
yeast ars (an
autonomously
replicating sequence
where DNA replication
begins),and
appropriate selectable
markers.
5.
6. Working Principle of Yeast Artificial
Chromosomes
The yeast artificial chromosome, which is often shortened to YAC, is an
artificially constructed system that can undergo replication. The design
of a YAC allows extremely large segments of genetic material to be
inserted. Subsequent rounds of replication produce many copies of the
inserted sequence, in a genetic procedure known as cloning.
The principle is similar to that for plasmids or cosmids.
The experimenter introduces some typical elements that are necessary
for correct replication.
In the case of YACs, the replication origins are the centromeres and
telomeres of the yeast chromosomes, which must be inserted into the
DNA being cloned.
The constructs can be transformed in yeast Spheroplast and are then
replicated there.
In contrast to the vectors, YACs are not circular; they are made of linear
DNA.
7.
8.
9. Process
YAC vector is initially propagated as circular plasmid
inside bacterial host utilizing bacterial ori sequence.
The circular plasmid is cut at a specific site using
restriction enzymes to generate a linear chromosome
with two telomere sites at terminals.
The linear chromosome is again digested at a specific
site with two arms with different selection marker.
The genomic insert is then ligated into YAC vector using
DNA ligase enzyme.
The recombinant vectors are transformed into yeast
cells and screened for the selection markers to obtain
recombinant colonies.
10.
11. Advantages of Yeast Artificial
Chromosomes
Yeast artificial chromosomes (YACs) provide the largest insert
capacity of any cloning system.
Yeast expression vectors, such as YACs, YIPs (yeast integrating
plasmids), and YEPs (yeast episomal plasmids), have
advantageous over bacterial artificial chromosomes (BACs).
They can be used to express eukaryotic proteins that require
post-translational modification.
A major advantage of cloning in yeast, a eukaryote, is that many
sequences that are unstable, underrepresented, or absent when
cloned into prokaryotic systems, remain stable and intact in YAC
clones.
It is possible to reintroduce YACs intact into mammalian cells
where the introduced mammalian genes are expressed and used
to study the functions of genes in the context of flanking
sequences.
12. Uses of Yeast Artificial Chromosomes
Yeast artificial chromosomes (YACs) were originally
constructed in order to study chromosome behavior in
mitosis and meiosis without the complications of
manipulating and destabilizing native chromosomes.
YACs representing contiguous stretches of genomic
DNA (YAC contigs) have provided a physical map
framework for the human, mouse, and
even Arabidopsis genomes.
YACs are extremely popular for those trying to analyze
entire genomes.
13. Limitations of Yeast Artificial
Chromosomes
A problem encountered in constructing and using YAC libraries is that
they typically contain clones that are chimeric, i.e., contain DNA in a
single clone from different locations in the genome.
YAC clones frequently contain deletions, rearrangements, or
noncontiguous pieces of the cloned DNA. As a result, each YAC clone
must be carefully analyzed to be sure that no rearrangements of the
DNA have occurred.
The efficiency of cloning is low (about 1000 clones are obtained per
microgram of vector and insert DNA).
YACs have been found to be less stable than BACs.
The yield of YAC DNA isolated from a yeast clone containing a YAC is
quite low.
The YAC DNA is only a few percents of the total DNA in the
recombinant yeast cell. It is difficult to obtain even 1 μg of YAC DNA.
The cloning of YACs is too complicated to be carried out by a lone
researcher.
15. Bacterial Artificial Chromosomes
BAC vectors are plasmids
constructed with the
replication origin of E. coli F
factor, and so can be
maintained in a single copy
per cell. These vectors can
hold DNA fragments of up to
300 kb
Recombinant BACs are
introduced into E. coli by
electroportation
Once in the cell, the rBAC
replicates like an F factor
16.
17. Characteristic features of BAC vectors
The original BAC vector, pBAC108L, is based on a mini-F plasmid, pMBO131
(Figure 1) which encodes genes essential for self-replication and regulates its
copy number inside a cell. The unidirectional self-replicating genes
are oriS and repE while parA and parB maintain copy number to one or two for
each E. coli genome.
Multiple cloning sites is present, flanked by “universal promoters” T7 and SP6,
all flanked by GC-rich restriction enzyme sites for insert excision.
Presence of cosN and loxP sites(cloned in by bacteriophage l terminase and P1
Cre recombinase, respectively) permits linearization of the plasmid for
convenient restriction mapping.
There is a chloramphenicol resistance gene for negative selection of non-
transformed bacteria.
Vector is 6900 bp in length and is capable of maintaining insert DNA in excess
of 300 kilobases (kb)
18. Other BAC Vectors
There have been many modifications done to
increase the ease-of-use as well as for use in
specific systems and situations.
pBeloBAC11 2 and pBACe3.6 modified BAC
vectors based on pBAC108L
They are commonly used as a basis for further
modification.
19. pBAC108L
The first BAC
vector.
Has a set of
regulatory genes,
OriS, and repE
which control F-
factor replication,
and parA and parB
which limit the
number of copies to
one or two.
pBAC108L lacked
a selectable marker
for recombinants.
Thus clones with
inserts had to be
identified by colony
hybridization
20. pBeloBAC11
The primary characteristic of this vector is the
addition of a lacZ gene into the multiple cloning site
2 of pBAC108L.
Plates supplemented with X-gal/IPTG, an intact lacZ
gene encodes b-galactosidase which catalyses the
supplemented substrate into a blue substance.
Successful ligation of insert DNA into the vector
inactivates lacZ, generating white colonies, indicating
the presence of a successful vector-insert ligation.
It is still a low-copy number plasmid due to presence
of parA and parB.
Size of vector is 7507 bp in length.
21.
22. pBACe3.6
This vector is based on pBAC108L but is more highly modified than
pBeloBAC11.
In order to overcome the issue of low plasmid copy numbers, the P1
replicon in F’ was deleted and a removable high copy number replicon
originating from an inserted pUC19 was introduced.
This vector contains 2.7 kb pUClink stuffer fragment which is flanked
by two sets of six restriction sites within a sacB region.
Levansucrase, a product of sacB gene, which converts sucrose (sup-
plemented in the media) to levan, which is toxic to E. coli host cells.
Hence, if the vector is re-ligated without an insert, the
functional sacB produces levansucrase and the cells die before forming
colonies. Successful ligation of an insert into the vector increases the
distance from the promoter to the coding region of sacB, disrupting
toxic gene expression in the presence of sucrose.
23.
24.
25. In addition to this vectors, there are many
specialized BAC vectors carrying a variety of
different combinations of drug resistance genes.
Besides, many different selection mechanisms and
markers are available. Modifications of cloning
sites (unique restriction endonuclease sites) are
also common as per the addition of genes and
promoters specific to different strains of bacteria
26. Development of
BAC vector
Bacterial plasmid is cut within
lacZ gene with restriction
enzymes.
Gene of Interest is cut out
using restriction enzyme.
F+ Plasmid and gene of
interest ligate.
BAC is electroporated into
E.coli cells.
Cells are plated on medium.
White colonies are composed
of transformed lacZ cells.
27.
28. Advantages of BAC Vectors
The large size of BACs help to minimize site of integration
effects, a phenomenon which has been defined as endogenous
sequences (such as gene coding regions and distal regulatory
elements) to be disrupted, and to produce potentially
undesirable phenotypes in gene cloning technology.
Endogenous gene expression more accurately than other cloning
systems.
The human genome BACs consist of the full gene
structure(which play very important role in gene regulation).
Therefore the human genome BACs will ensure full mRNA
processing and splicing when genes are transcribed, and
produce the full complement of protein isoforms once mRNAs
are translated.
It can be transfected and expressed in mammalian cell lines even
if transfection efficiency and copy numbers are low.
29. Disadvantages of BAC vectors
A construct containing a large genomic fragment
is likely to contain non-related genes which may
lead to indirect, non-specific gene expression and
unanticipated changes in the cell phenotype.
Recombinant BAC constructs can be time-
consuming and labor-intensive.
The large size BAC DNA constructs are more
easily degraded and sheard during manipulation
before transfection.
30.
31. Applications of BAC vectors
BACs are useful for the construction of genomic libraries
but their range of use is vast. It spans from basic science
to economically rewarding industrial research, and fields
as prosaic as animal husbandry.
In genomic analyses, it helps in determining
phylogenetic lineage det between species.
Helps in study of horizontal gene transfer and since
bacterial genes are usually clustered, the ability of BAC
vectors to accommodate large inserts has allowed the
study of entire bacterial pathways.
By isolating DNA directly from soil or from marine
environments, the “metagenomes” of those organisms
which are either uncultureable or are termed viable but
uncultureable can be cloned into BAC vectors and
indirectly studied.
32. In industrial research fields where BAC vectors are invaluable
tools in cataloguing novel genomes is in the discovery of
novel enzymes. Work has been done on identifying enzymes
that are involved in biopolymer hydrolysis or even
radioactive waste management.
BAC vectors have been instrumental in studying large double
stranded DNA viruses both from an academic point of view
and as a tool to develop improved vaccines.
In genomic research, high throughput determination of gains
and losses of genetic material using high resolution BAC
arrays and comparative genomic hybridization (CGH) have
been developed into the new tools for translational research
in solid tumors and neurodegenerative disorders.
34. YAC is a genetically engineered
chromosome with the use of yeast DNA for
the purpose of cloning.
Gender YACs were designed to clone large
fragments of genomic DNA into yeast.
Insert Length YACs can contain
megabase-sized genomic inserts.(1000 kb –
2000 kb).
Construction YAC DNA is difficult to
purify intact and requires high concentration
for generating YAC vector system.
Chimerism YACs are often chimeric.
Stability YAC is unstable.
Modifications Yeast recombination is very
feasible and always remains active. Hence it
can generate deletions and other
rearrangements in a YAC.
Maintenance Manipulating recombinant
YACs usually requires YAC to be transferred
into E. coli for subsequent manipulation.
Hence, it is a laborious process.
BAC is a genetically engineered DNA
molecule using E. coli DNA for the purpose of
cloning.
Gender BACs were developed for cloning
large genomic fragments into Escherichia coli.
Insert Length BACs can carry inserts of
200–300 kb or less.
Construction BAC is easy to purify intact
and can be easily constructed.
Chimerism BACs are rarely chimeric.
Stability BAC is stable.
Modifications E. coli recombination is
prevented and is turned on when required.
Hence, it reduces the unwanted
rearrangements in BACs.
Maintenance BAC modification occurs
directly in E. coli. So there is no need for DNA
transfer. Hence, the process is not laborious.
DIFFERENCE BETWEEN YAC AND BAC VECTORS
