- Lambda bacteriophage cloning vectors were developed to overcome limitations in the size of DNA that could be inserted into unmodified lambda vectors.
- Segments of the non-essential lambda genome could be deleted to allow insertion of up to 18kb of new DNA while still allowing packaging.
- Natural selection was used to generate lambda strains lacking restriction sites, allowing restriction-based cloning.
- The first lambda vectors were insertion and replacement vectors, while later cosmids allowed cloning of fragments up to 52kb.
BAC & YAC are artificially prepared chromosomes to clone DNA sequences.yeast artificial chromosome is capable of carrying upto 1000 kbp of inserted DNA sequence
molecular biology phage vector, full lifecycle and all necessary information regarding lambda phage, it contain 2 types that is insertion and replacement.
BAC & YAC are artificially prepared chromosomes to clone DNA sequences.yeast artificial chromosome is capable of carrying upto 1000 kbp of inserted DNA sequence
molecular biology phage vector, full lifecycle and all necessary information regarding lambda phage, it contain 2 types that is insertion and replacement.
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
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.
MBB 501 PLANT BIOTECHNOLOGY
INFORMATION ABOUT DIFFERENT DNA MODIFYING ENZYMES
WHAT IS AN ENZYME?
Alkaline Phosphatase
Polynucleotide kinase
Terminal deoxyneucleotidyl transferase
Nucleases
Exonuclease
Bal31 Exonuclease III
Endonuclease
S1 endonulease
Deoxyribonuclease 1 (Dnase 1)
RNase A
RNase H
Restriction Endonuclease
PvuI
PvuII
Different types of endonuclease enzymes
The recognition sequences for some of the most frequently used restriction endonucleases.
Categorization of enzymes
Isoschizomers
Neoschizomers
Isocaudomers
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
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.
MBB 501 PLANT BIOTECHNOLOGY
INFORMATION ABOUT DIFFERENT DNA MODIFYING ENZYMES
WHAT IS AN ENZYME?
Alkaline Phosphatase
Polynucleotide kinase
Terminal deoxyneucleotidyl transferase
Nucleases
Exonuclease
Bal31 Exonuclease III
Endonuclease
S1 endonulease
Deoxyribonuclease 1 (Dnase 1)
RNase A
RNase H
Restriction Endonuclease
PvuI
PvuII
Different types of endonuclease enzymes
The recognition sequences for some of the most frequently used restriction endonucleases.
Categorization of enzymes
Isoschizomers
Neoschizomers
Isocaudomers
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.
(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 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 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.
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.
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.
Seminar of U.V. Spectroscopy by SAMIR PANDASAMIR PANDA
Spectroscopy is a branch of science dealing the study of interaction of electromagnetic radiation with matter.
Ultraviolet-visible spectroscopy refers to absorption spectroscopy or reflect spectroscopy in the UV-VIS spectral region.
Ultraviolet-visible spectroscopy is an analytical method that can measure the amount of light received by the analyte.
Introduction:
RNA interference (RNAi) or Post-Transcriptional Gene Silencing (PTGS) is an important biological process for modulating eukaryotic gene expression.
It is highly conserved process of posttranscriptional gene silencing by which double stranded RNA (dsRNA) causes sequence-specific degradation of mRNA sequences.
dsRNA-induced gene silencing (RNAi) is reported in a wide range of eukaryotes ranging from worms, insects, mammals and plants.
This process mediates resistance to both endogenous parasitic and exogenous pathogenic nucleic acids, and regulates the expression of protein-coding genes.
What are small ncRNAs?
micro RNA (miRNA)
short interfering RNA (siRNA)
Properties of small non-coding RNA:
Involved in silencing mRNA transcripts.
Called “small” because they are usually only about 21-24 nucleotides long.
Synthesized by first cutting up longer precursor sequences (like the 61nt one that Lee discovered).
Silence an mRNA by base pairing with some sequence on the mRNA.
Discovery of siRNA?
The first small RNA:
In 1993 Rosalind Lee (Victor Ambros lab) was studying a non- coding gene in C. elegans, lin-4, that was involved in silencing of another gene, lin-14, at the appropriate time in the
development of the worm C. elegans.
Two small transcripts of lin-4 (22nt and 61nt) were found to be complementary to a sequence in the 3' UTR of lin-14.
Because lin-4 encoded no protein, she deduced that it must be these transcripts that are causing the silencing by RNA-RNA interactions.
Types of RNAi ( non coding RNA)
MiRNA
Length (23-25 nt)
Trans acting
Binds with target MRNA in mismatch
Translation inhibition
Si RNA
Length 21 nt.
Cis acting
Bind with target Mrna in perfect complementary sequence
Piwi-RNA
Length ; 25 to 36 nt.
Expressed in Germ Cells
Regulates trnasposomes activity
MECHANISM OF RNAI:
First the double-stranded RNA teams up with a protein complex named Dicer, which cuts the long RNA into short pieces.
Then another protein complex called RISC (RNA-induced silencing complex) discards one of the two RNA strands.
The RISC-docked, single-stranded RNA then pairs with the homologous mRNA and destroys it.
THE RISC COMPLEX:
RISC is large(>500kD) RNA multi- protein Binding complex which triggers MRNA degradation in response to MRNA
Unwinding of double stranded Si RNA by ATP independent Helicase
Active component of RISC is Ago proteins( ENDONUCLEASE) which cleave target MRNA.
DICER: endonuclease (RNase Family III)
Argonaute: Central Component of the RNA-Induced Silencing Complex (RISC)
One strand of the dsRNA produced by Dicer is retained in the RISC complex in association with Argonaute
ARGONAUTE PROTEIN :
1.PAZ(PIWI/Argonaute/ Zwille)- Recognition of target MRNA
2.PIWI (p-element induced wimpy Testis)- breaks Phosphodiester bond of mRNA.)RNAse H activity.
MiRNA:
The Double-stranded RNAs are naturally produced in eukaryotic cells during development, and they have a key role in regulating gene expression .
1. Cloning vectors based on lambda bacteriophage
• Two problems had to be solved before lambda based cloning vectors could be
developed:
• (1)The DNA molecule can be increased in size by only about 5%, representing the
addition of only 3 kb of new DNA.
• If the total size of the molecule is more than52 kb, then it cannot be packaged
into the head structure and infective phage particles are not formed.
• This severely limits the size of a DNA fragment that can be inserted into an un
modified vector
2. • (2)The lambda genome is so large that it has more than one recognition
sequence for virtually every restriction endonuclease.
• Restriction cannot be used to cleave the normal lambda molecule in a way that
will allow insertion of new DNA,
• Because the molecule would be cut into several small fragments that would be
very unlikely to re-form a viable lambda genome on re ligation
3. Segments of the lambda genome can be deleted
without impairing viability
• Large segment in the central region of the lambda DNA molecule can be removed
without affecting the ability of the phage to infect E. coli cells.
• Removal of all or part of this non-essential region, between positions 20 and 35
on the map decreases the size of the resulting lambda molecule by up to 15 kb.
• This means that as much as 18 kb of new DNA can now be added before the cut-
off point for packaging is reached
4. • This “non-essential” region in fact contains most of the genes involved in
integration and excision of the eprophage from the E. coli chromosome.
• A deleted lambda genome is therefore non-lysogenic and can follow only the lytic
infection cycle.
5. Natural selection can be used to isolate modified
lambda that lack certain restriction sites
• Even a deleted lambda genome, with the non-essential region removed, has
multiple recognition sites for most restriction endonucleases.
• If just one or two sites need to be removed, then the technique of in vitro
mutagenesis can be used.
• For example, an EcoRI site, GAATTC, could be changed to GGATTC, which is not
recognized by the enzyme.
• However, in vitro mutagenesis was in its early stage. when the first lambda
vectors were under development, and even today would not be an efficient
means of changing more than a few sites in a single molecule.
6. • Instead, natural selection was used to provide strains of lambda that lack the
unwanted restriction sites.
• Natural selection can be brought into play by using as a host an E. coli strain that
produces EcoRI.
• Most lambda DNA molecules that invade the cell are destroyed by this restriction
endonuclease, but a few survive and produce plaques.
• These are mutant phages, from which one or more EcoRI sites have been lost
spontaneously.
• Several cycles of infection will eventually result in lambda molecules that lack all
or most of the EcoRI sites.
7.
8. Insertion and replacement vectors
• The first two classes of vector to be produced were lambda insertion and lambda
replacement(or substitution) vectors.
•Insertion vectors
• With an insertion vector a large segment of the non-essential region has been
deleted, and the two arms ligated together.
• An insertion vector possesses at least one unique restriction site into which new
DNA can be inserted.
• The size of the DNA fragment that an individual vector can carry depends, of
course, on the extent to which the non-essential region has been deleted.
• Two popular insertion vectors are:
9.
10. •Lambda gt10
• which can carry up to 8 kb of new DNA, inserted into a unique EcoRI site located
in the cI gene.
• Insertional inactivation of this gene means that recombinants are distinguished
as clear rather than turbid plaques.
11. Lambda ZAPII
• In which insertion of upto 10 kb DNA into any of 6 restriction sites with in a poly
linker inactivates the lacZ′ gene carried by the vector.
• Recombinants give clear rather than blue plaques on X-gal agar.
12. Replacement vectors
• A lmbda replacement vector has two recognition sites for the restriction
endonuclease used for cloning.
• These sites flank a segment of DNA that is replaced by the DNA to be cloned
• Often the replaceable fragment (or “stuffer fragment”) carries additional
restriction sites that can be used to cut it up into small pieces so that its own
re-insertion during a cloning experiment is very unlikely.
• Replacement vectors are generally designed to carry larger pieces of DNA than
insertion vectors can handle.
• Recombinant selection is often on the basis of size, with non-recombinant
vectors being too small to be packaged into lambda phage heads .
13.
14. Lambda EMBL4
• It can carry up to 20 kb of inserted DNA by replacing a segment flanked by pairs
of EcoRI, BamHI, and SalI sites.
• Any of these three restriction endonucleases can be used to remove the stuffer
fragment, so DNA fragments with a variety of sticky ends can be cloned.
• Recombinant selection with lambda EMBL4 can be on the basis of size, or can
utilize the Spi phenotype.
15. Cloning experiments with lambda insertion or
replacement vectors
• A cloning experiment with a lambda vector can proceed along the same lines as
with a plasmid vector
• The lambda molecules are restricted, new DNA is added, the mixture is ligated,
and the resulting molecules used to transfect a competent E. coli host
• This type of experiment requires that the vector be in its circular form, with the
cos sites hydrogen bonded to each other.
• Although satisfactory for many purposes, a procedure based on transfection is
not particularly efficient. A greater number of recombinants will be obtained if
one or two refinements are introduced.
• The first is to use the linear form of the vector. When the linear form of the
vector is digested with the relevant restriction endonuclease, the left and right
arms are released as separate fragments.
16.
17. • A recombinant molecule can be constructed by mixing together the DNA to be
cloned with the vector arms
• Ligation results in several molecular arrangements, including catenanes
comprising left arm–DNA–right arm repeated many times
• If the inserted DNA is the correct size, then the cos sites that separate these
structures will be the right distance apart for in vitro packaging .
• Recombinant phage are therefore produced in the test tube and can be used to
infect an E. coli culture.
• This strategy, in particular the use of in vitro packaging, results in a large number
of recombinant plaques
18.
19. Long DNA fragments can be cloned using a cosmid
• The final and most sophisticated type of lambda-based vector is the cosmid.
• Cosmids are hybrids between a phage DNA molecule and a bacterial plasmid,
• And their design centers on the fact that the enzymes that package the lambda
DNA molecule into the phage protein coat need only the cos sites in order to
function.
• The in vitro packaging reaction works not only with lambda genomes, but also
with any molecule that carries cos sites separated by 37–52 kb of DNA.
• A cosmid is basically a plasmid that carries a cos
• It also needs a selectable marker, such as the ampicillin resistance gene, and a
plasmid origin of replication
20. • cosmids lack all the lambda genes and so do not produce plaques.
• Instead colonies are formed on selective media, just as with a plasmid vector
21. • A cloning experiment with a cosmid is carried out as follows.
• The cosmid is opened at its unique restriction site and new DNA fragments
inserted.
• These fragments are usually produced by partial digestion with a restriction
endonuclease, as total digestion almost invariably results in fragments that are
too small to be cloned with a cosmid.
• Ligation is carried out so that catenanes are formed. Providing the inserted DNA
is the right size, in vitro packaging cleaves the cossites and places the
recombinant cosmids in mature phage particles.
• These lambda phage are then used to infect an E. coli culture, though of course
plaques are not formed.
• Instead, infected cells are plated onto a selective medium and antibiotic-resistant
colonies are grown. All colonies are recombinants, as non-recombinant linear
cosmids are too small to be packaged into lambda heads
22.
23. Vectors for other bacteria
• Cloning vectors have also been developed for several other species of bacteria,
including Streptomyces, Bacillus, and Pseudomonas.
• Some of these vectors are based on plasmids specific to the host organism, and
some on broad host range plasmids able to replicate in a variety of bacterial
hosts.
• A few are derived from bacteriophages specific to these organisms.
• Antibiotic resistance genes are generally used as the selectable markers.
• Most of these vectors are very similar to E. coli vectors in terms of their general
purposes and uses.