This document discusses different types of vectors that can be used for genetic engineering, including animal viruses, plant viruses, retroviruses, shuttle vectors, and binary vectors. It provides details on the structure and use of tobacco mosaic virus (TMV) as a plant viral vector, describing how foreign genes can be stably replicated and spread systemically in plants using this system. It also summarizes key features of yeast episomal plasmids (YEps), retroviral vectors based on murine leukemia virus, and the binary vector system used for plant transformation via Agrobacterium tumefaciens.
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.
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
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.
BAC & YAC are artificially prepared chromosomes to clone DNA sequences.yeast artificial chromosome is capable of carrying upto 1000 kbp of inserted DNA sequence
RNA interference (RNAi): Cellular process by which an mRNA is targeted for degradation by a dsRNA with a strand complementary to a fragment of such mRNA.
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
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.
BAC & YAC are artificially prepared chromosomes to clone DNA sequences.yeast artificial chromosome is capable of carrying upto 1000 kbp of inserted DNA sequence
RNA interference (RNAi): Cellular process by which an mRNA is targeted for degradation by a dsRNA with a strand complementary to a fragment of such mRNA.
One of the first plasmids to be used in recombinant genetics was called pBR322. It is approximately 4300 bp in length and has two antibiotic resistance genes: Ap (Ampicillin) and Tc (Tetracycline). Bacteria cells that are successfully transformed with this plasmid are able to grow in the presence of both ampicillin and tetracycline antibiotics
Recombinant viral vectors are genetic engineering tools commonly used for gene transfer purpose with high transfection efficiency and site specific gene insertion.
A comprehensive study of shuttle vector & binary vector and its rules of in ...PRABAL SINGH
Vector: A vector is a DNA molecule that has the ability to replicate autonomously in an appropriate host cell and into which the DNA fragment to be cloned is integrated for cloning
Various virus vector related to Plant and Animal for gene cloning and transfo...PrabhatSingh628463
Various virus vector related to Plant and Animal for gene cloning and transformation and Expression vector and it’s mode of expression in plant and animal cells
Transgensis: The process of transfer of gene from one organism to another organism.
Transgene: the gene responsible for transfer
Transgenic Mice: can be done by three methods
1)Retroviral Method: by using retroviral vector transgene is inserted into the egg
2) Dna Microinjection: Direct inoculation of transgene into the male pronuclues
3) Embryonic Stem cell: transgene is inserted during embryonic stage of the embryo
There are many applications, limitations .
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.
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.
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.
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.
Earliest Galaxies in the JADES Origins Field: Luminosity Function and Cosmic ...Sérgio Sacani
We characterize the earliest galaxy population in the JADES Origins Field (JOF), the deepest
imaging field observed with JWST. We make use of the ancillary Hubble optical images (5 filters
spanning 0.4−0.9µm) and novel JWST images with 14 filters spanning 0.8−5µm, including 7 mediumband filters, and reaching total exposure times of up to 46 hours per filter. We combine all our data
at > 2.3µm to construct an ultradeep image, reaching as deep as ≈ 31.4 AB mag in the stack and
30.3-31.0 AB mag (5σ, r = 0.1” circular aperture) in individual filters. We measure photometric
redshifts and use robust selection criteria to identify a sample of eight galaxy candidates at redshifts
z = 11.5 − 15. These objects show compact half-light radii of R1/2 ∼ 50 − 200pc, stellar masses of
M⋆ ∼ 107−108M⊙, and star-formation rates of SFR ∼ 0.1−1 M⊙ yr−1
. Our search finds no candidates
at 15 < z < 20, placing upper limits at these redshifts. We develop a forward modeling approach to
infer the properties of the evolving luminosity function without binning in redshift or luminosity that
marginalizes over the photometric redshift uncertainty of our candidate galaxies and incorporates the
impact of non-detections. We find a z = 12 luminosity function in good agreement with prior results,
and that the luminosity function normalization and UV luminosity density decline by a factor of ∼ 2.5
from z = 12 to z = 14. We discuss the possible implications of our results in the context of theoretical
models for evolution of the dark matter halo mass function.
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.
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. Animal and Plant Viruses as
vector,
Binary and shuttle vector
Promila Sheoran
Ph.D. Biotechnology
GJU S&T Hisar
2. Animal viruses as vector
•The first eukaryotic DNA virus was SV40, for which a complete nucleotide sequence
and a detailed understanding of transcription were available.
•The genome of SV40 contains very little non-essential DNA so it is necessary to insert
the foreign gene in place of essential viral genes and to propagate the recombinant
genome in the presence of a helper virus.
•This virus is capable of infecting several mammalian species, following a lytic cycle in
some host and a lysogenic cycle in others.
3. The genome is 5.2kb in size and contains
two sets of genes, the early genes,
expressed early in the infection cycle
and coding for proteins involved in viral
DNA replication, and the late genes,
coding for viral capsid proteins.
However, all work using SV40 virions to propagate recombinant DNA molecules is
severely constrained by the facts that the viral genome is small, 5.24 kb, and that the
packaging limits are strict. Such systems can not, therefore, be used for the analysis of
most eukaryotic genes.
4. Adenoviruses, are a group of viruses which enable larger fragments of DNA to be
cloned than it is possible with an SV40 vector. Adenoviruses are more difficult to
handle because the genomes are bigger.
5. •Papillomaviruses, which also have a relatively high capacity for inserted DNA,
have the important advantage of enabling a stable transformed cell line to be
obtained.
•Papillomavirus transformed cells don't contain integrated viral DNA rather they
contain between 50 and 300 copies of unintegrated, circular viral DNA although
some proportion of these viral genomes exists as concatamers and/or catenates.
• Bovine papillomavirus (BPV), which causes warts on cattle, has an unusal
infection cycle in mouse cells, taking the form of a multi copy plasmid with about
100 molecules present per cell.
•It doesn't cause the death of the mouse cell, and BPV molecules are passed to
daughter cells on cell division. Shuttle vectors consisting of BPV and pBR322
sequences, and capable of replication in both mouse and bacterial cells, are
therefore of great value in animal cell biotechnology.
6.
7. Retroviruses
•Retroviruses, though have single-stranded RNA genomes but provides perhaps the
most promising vector system of all.
• During the process of reverse transcription, sequences from the termini of viral
RNA are duplicated to generate long terminal repeats(LTRs).
• These long terminal repeats contain both the promoter and the polyadenylation
signal for the transcription of viral mRNAs.
•The specificity of proviral DNA integration is also determined by the long terminal
repeats. Although retroviruses can integrate at many sites within the cellular
genome, integrative recombination always occurs at particular sites at the ends of
the LTRs.
•The sequences appropriately inserted between the two LTRs will be integrated
intact. A further great advantage of retroviruses is that they are natural transducing
viruses.
8.
9. •Retroviral vectors are most frequently based upon the Moloney murine leukaemia
virus (Mo-MLV), which is an amphotrophic virus, capable of infecting both mouse
cells, enabling vector development in mouse models, & human cells, enabling
human treatment.
• The viral genes (gag, pol & env) are replaced with the transgene of interest &
expressed on plasmids in the packaging cell line. Because the non-essential genes
lack the packaging sequence (psi) they are not included in the virion particle.
•To prevent recombination resulting in replication competent retroviruses, all
regions of homology with the vector backbone should be removed & the non-
essential genes should be expressed by at least two transcriptional units.
•The essential regions include the 5' & 3' LTRs & the packaging sequence lying
downstream of the 5' LTR. To aid identification of transformed cells selectable
markers, such as neomycin & beta galactosidase, can be included & transgenes
expression can be improved with the addition of internal ribosome sites. The
available carrying capacity for retroviral vectors is approximately 7.5 kb, which is too
small for some genes even if the cDNA is used.
10. Plant viruses as vector
•The expression of foreign genes in plants has proven advantageous to the study of
molecular biology. Stable gene transfer to whole plants can be achieved by a
combination of DNA transformation and tissue culture techniques or by
virus transfection.
• With viral-based vectors the ease of infection avoids the time-consuming
procedures, "position" effects, and "somaclonal" variation seen when foreign genes
are integrated into the plant genome
11. • Several features of tobacco mosaic virus (TMV) suggest that it might be usefully
adapted for such a purpose:
(i) tobamoviruses have a wide host range;
(ii) they can move cell-to-cell mediated by a virus-encoded peptide;
(iii) they exhibit rapid systemic spread in plants;
(iv) TMV infections are maintained for the lifetime of the plant;
(v) TMV RNA is replicated to high levels as autonomous sequences;
(vi) this replication results in rapid and productive cytoplasmic gene expression;
(vii) temperature-sensitive mutations of RNA synthesis are available to
modulate expression of foreign genes;
(viii) TMV also lacks the packaging constraints found with nonhelical viruses,
including existing DNA plant virus vectors; and
(ix) the TMV genome can now be manipulated as a DNA copy and then
transcribed in vitro to produce infectious RNA molecules.
12. •The TMV genome consists of one 6395-nucleotide molecule of messenger-sense,
single-stranded RNA, encoding at least four proteins.
•The 126- and 183-kDa replicase proteins are translated directly from the genomic
RNA, whereas the 30-kDa cell-to-cell movement protein and 17.5-kDa coat protein
are translated from two 3'-coterminal subgenomic RNAs produced during
replication.
•Because the 126- and 183-kDa proteins are required for TMV RNA replication, it
was decided to express the foreign sequences by means of a subgenomic RNA 3'
of these genes. For brome mosaic virus, subgenomic RNA synthesis has been
shown to be internally initiated on minus-sense RNA.
•Functionally similar sequences occur upstream of the highly expressed TMV coat
protein ORF and are within the 30-kDa protein-coding region. The 203 bases
upstream of the respective coat protein initiation codons of TMV-U1 and
odontoglossum ringspot virus (ORSV), a tobamovirus isolated from Cymbidium
spp., exhibit a remarkably low sequence similarity [45% compared with 65% for
the 3' 2.4 kilobases (kb) of the respective genomes].
13. •The expression vector pTB2 was designed to avoid deletion of foreign inserts due to
flanking repeated sequences by using these two heterologous promoters from TMV-
U1 and ORSV to synthesize subgenomic RNAs for the foreign gene and ORSV coat
protein gene, respectively.
•To determine whether foreign sequences inserted into such a TMV-based expression
vector could be replicated stably and whether they could be propagated systemically,
two bacterial sequences were tested in the vector.
•The DHFR sequence from plasmid R67 chosen for its small size of 238 base pairs
(bp)] and the larger (832-bp) NPTII ORF from transposon TnS (24) were cloned
into the Xho I site of pTB2, downstream of the TMV-U1 coat protein subgenomic
promoter, to produce pTBD4 and pTBN62, respectively
14. FIG. 1. Construction of expression vector pTB2 and derivatives containing dihydrofolate
reductase (DHFR; pTBD4) and neomycin phosphotransferase II (NPTII; pTBN62) genes.
Thin lines and open boxes
(ORFs) are sequences
transcribed in vitro by E. coli
RNA polymerase from its
promoter (hatched box in top
diagram).
ORFs for the 126-, 183-, 30-
kDa and coat proteins (126K,
183K, 30K, and cp,
respectively) are TMV-U1
sequences, except where
noted as being from ORSV
(o).
15. Thick lines represent bacterial
cloning vector sequences.
Narrow open box (o) delineates
203 ORSV bases upstream of
coat protein initiation codon.
Vertical lines in pTBD4 and
pTBN62 indicate extent of foreign
sequences. Selected restriction
sites are shown (B, BamHI; K, Kpn
I; N, Nsi I; Nc, Nco I; P, Pst I; S, Spi
I; and X, Xho I).
16. •Two bacterial sequences inserted independently into TB2 moved systemically in
Nicotiana benthamna, although they differed in their stability on serial passage.
•Systemic expression of the bacterial protein neomycin phosphotransferase was
demonstrated. Hybrid RNAs containing both TMV-U1 and the inserted bacterial gene
sequences were encapsidated by the odontoglossum ringspot virus coat protein,
facilitating their transmission and amplification on passaging to subsequent plants.
•The vector TB2 provides a rapid means of expressing genes and gene variants in
plants.
17. Shuttle Vector
•These vectors have been designed to replicate in cells of two different species;
therefore, they contain two origins of replication, one specific for each host species, as
well as those genes necessary for their replication and not provided by the host cells.
•These vectors are created by recombinant techniques.
•Some of them can be grown in two different prokaryotic species, usually E. coli and a
eukaryotic one, e.g. yeast, plants, animals.
•Since these vectors can be grown in one host and then moved into another without any
extra manipulation, they are called shuttle vectors.
•YEp13 is an example of shuttle vector.
18. •The yeast Saccharomyces cerevisiae is one of the most important organisms in
biotechnology. Development of cloning vectors for yeast was initially stimulated by
the discovery of a plasmid that is present in most strains of S. cerevisiae. The
2 µm plasmid, as it is called, is one of only a very limited number of plasmids found
in eukaryotic cells.
•Vectors derived from the 2 µm plasmid are called yeast episomal plasmids (YEps).
Some YEps contain the entire 2 µm plasmid, others include just the 2 µm origin of
replication. An example of the latter type is YEp13.
•The 2 µm plasmid is an excellent basis for a cloning vector. It is 6 kb in size, which is
ideal for a vector, and exists in the yeast cell at a copy number of between 70 and
200. Replication makes use of a plasmid origin, several enzymes provided by the host
cell, and the proteins coded by the REP1 and REP2 genes carried by the plasmid.
19. •However, all is not perfectly straightforward in using the 2 µm plasmid as a
cloning vector. First, there is the question of a selectable marker.
•Here gene LEU2, which codes for b-isopropyl-malate dehydrogenase, one of the
enzymes involved in the conversion of pyruvic acid to leucine is used as marker.
•In order to use LEU2 as a selectable marker, a special kind of host organism is
needed. The host must be an auxotrophic mutant that has a non-functional LEU2
gene.
•Such a leu2− yeast is unable to synthesize leucine and can survive only if this
amino acid is supplied as a nutrient in the growth medium. Selection is possible
because transformants contain a plasmid-borne copy of the LEU2 gene, and so
are able to grow in the absence of the amino acid.
• In a cloning experiment, cells are plated out onto minimal medium, which
contains no added amino acids. Only transformed cells are able to survive and
form colonies.
20.
21. •YEp13 illustrates several general features of yeast cloning vectors. First, it
is a shuttle vector.
• As well as the 2 µm origin of replication and the selectable LEU2 gene,
YEp13 also includes the entire pBR322 sequence, and can therefore
replicate and be selected for in both yeast and E. coli.
22.
23. Uses of Shuttle Vectors
•There are several lines of reasoning behind the use of shuttle vectors. One is that
it might be difficult to recover the recombinant DNA molecule from a transformed
yeast colony.
•This is not such a problem with YEps, which are present in yeast cells primarily as
plasmids, but with other yeast vectors, which may integrate into one of the yeast
chromosomes, purification might be impossible.
•This is a disadvantage because in many cloning experiments purification of
recombinant DNA is essential in order for the correct construct to be identified by,
for example, DNA sequencing.
•The standard procedure when cloning in yeast is therefore to perform the initial
cloning experiment with E. coli, and to select recombinants in this organism.
Recombinant plasmids can then be purified, characterized, and the correct
molecule introduced into yeast.
24. Binary vector
•The binary vector is a shuttle vector, so-called because it is able to replicate in
multiple hosts (E. coli and Agrobacterium tumefaciens).
•A transfer DNA (T-DNA) binary system is a pair of plasmids consisting of a binary
plasmid and a helper plasmid. The two plasmids are used together (thus binary)
to produce genetically modified plants. They are artificial vectors that have both
been created from the naturally occurring Ti plasmid found in Agrobacterium
tumefaciens.
•Systems in which T-DNA and vir genes are located on separate replicons are
called T-DNA binary systems. T-DNA is located on the binary vector (the non-T-
DNA region of this vector containing origin(s) of replication that could function
both in E. coli and in Agrobacterium tumefaciens, and antibiotic-resistance genes
used to select for the presence of the binary vector in bacteria, became known as
vector backbone sequences).
•The replicon containing the vir genes became known as the vir helper. Strains
harboring this replicon and a T-DNA are considered disarmed if they do not
contain oncogenes that could be transferred to a plant.
25. •The discovery that the vir genes do not need to be in the same plasmid with a T-DNA
region to lead its transfer and insertion into the plant genome led to the construction of
a system for plant transformation where the T-DNA region and the vir region are on
separate plasmids.
•In the binary vector system, the two different plasmids employed are:
•First is a wide-host-range small replicon, which has an origin of replication (ori) that
permits the maintenance of the plasmid in a wide range of bacteria including E. coli and
Agrobacterium. This plasmid typically contains:
1. foreign DNA in place of T-DNA,
2. the left and right T-DNA borders (or at least the right T-border),
3. markers for selection and maintenance in both E. coli and A. tumefaciens,
4. a selectable marker for plants.
•The plasmid is said to be "disarmed", since its tumor-inducing genes located in the T-
DNA have been removed.
26. Second is a helper Ti plasmid, harbored in A. tumefaciens, which lacks the entire T-
DNA region but contains an intact vir region.
27. In general, the transformation procedure is as follows:
•The recombinant small replicon is transferred via bacterial conjugation or
direct transfer to A. tumefaciens harboring a helper Ti plasmid,
•the plant cells are co-cultivated with the Agrobacterium, to allow transfer
of recombinant T-DNA into the plant genome, and
•transformed plant cells are selected under appropriate conditions.
28. Possible pitfalls
•A possible disadvantage may ensue from the fact that the stability of wide host
range replicons in E. coli and Agrobacterium varies considerably. Depending on the
orientation, plasmids with two different origins of replication may be unstable in E.
coli where both origins are active.
Advantages
•Compared with co-integrated vectors, binary vectors present some advantages:
No recombination process takes place between the molecules involved.
Instead of a very large, recombinant, disarmed Ti plasmid, small vectors are used,
which increases transfer efficiency from E. coli to Agrobacterium
29. •This vector system is most widely used nowadays.
• Different types of binary vectors have been devised to suit different needs in a plant
transformation process. One example is pGA series vectors.
1.pGA series vectors, which contain:
•an ori derived from RK2 for replication in E. coli and Agrobacterium,
•a tetracycline resistance gene,
•the cis-acting factor required for conjugal transfer,
•the right (RB) and left (LB) T-DNA borders,
• a neomycin phosphotransferase (nptII) gene, which confers resistance to
•kanamycin and G418 in transformed plants, and
•a polylinker site (multicloning site).
•Specific vectors in this series are designed for cloning large fragments (colE1 origin of
replication and phage l cos), analyzing promoters (multiple cloning site immediately
upstream of a promoterless cat gene), and expressing a gene of interest (polylinker
site between a plant promoter and a terminator).