1. ON
A comprehensive study of shuttle vector & binary vector and its
rules of in gene transfer
A-9606/16
DEPARTMENT OF PMB&GE
N.D. University of Agriculture & Technology
Kumarganj, Faizabad

2.  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.
 E. coli supports several type of vector, some natural, some
constructed, which can be grouped as e.g.1) Plasmid 2)
Bacteriophase (Both natural) 3) Cosmid 4) Phasmid 5) Shuttle
vector( Last three constructed by man ) 6)Artificial
chromosome 7) Phagamid
 Plasmid : A plasmid is a double stranded circular DNA
molecule, other then the bacterial chromosome, that is capable
of independent replication and transmission.
 Three Widely studies type are as: F plasmid ( Responsible for
conjugation)R plasmid (Carry gene for resistance to antibiotics)
and Col plasmid ( Code for colicincs).

3. SHUTTLE VECTOR
 A vector (e.g. a plasmid) constructed in
such a way that it can replicate in at least
two different host species (e.g. a
prokaryote and a eukaryote). A DNA
recombined into such a vector can be
tested or manipulated in several cell
types.

4. A shuttle vector designated to replicate in E. coli and
Streptomyces has been constructed follows;
 The modules for DNA replication in Streptomyces and
methylenomycin A resistance are derivated from a
Streptomyces plasmid .
 Replication module for maintenance in E. coli and a gene
for antibiotics resistance are taken from E. coli plasmid .
Shuttle vector have been designated to specifically satisfy
this need, i.e. the initial cloning of DNA insert in E. coli and
subsequent test in the species to which the DNA insert
belong. Most of the eukaryotic vector are, in fact shuttle
vector

6. BINARY VECTOR
 The binary vector system consists of an Agrobacterium strain along
with a disarmed plasmid called vir helper plasmid.
 Both of them are not physically linked.
 The plasmid is said to be "disarmed", since its tumor-inducing
genes located in the T-DNA have been removed.
 Along with T- DNA it can replicate in E. coli and Agrobacterium.
 Two different plasmids employed in binary vector system.
 A wide-host-range small replicon.
 A helper Ti plasmid.

7. A wide-host-range small replicon; 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 :
• Foreign DNA in place of T-DNA,
• The left and right T-DNA borders (or at least the right T-border),
• Markers for selection and maintenance in both E. coli and A.
tumefaciens,
• A selectable marker for plants.
A helper Ti plasmid, harbored in A. tumefaciens, which lacks the
entire T-DNA region but contains an intact vir region.

8. CONSTRUCTION OF BINARY
VECTOR
 Obtain plasmids and other DNA fragments necessary for
constructions of vectors from appropriate sources.
 Combine the bacteria-selectable marker and the plasmid
replication functions for E. coli.
 Insert the plasmid replication functions for A. tumefaciens, if
necessary.
 Insert the plasmid mobilization functions, if necessary.
 Insert the RB, the LB, and the MCS to give the empty vector.
 Construct the expression unit of the selectable marker gene
separately.
 Insert the unit into the empty vector to give the selection vector.
 Construct the expression unit of the reporter gene separately.
 Insert the unit into the selection vector to give the reporter
vector.

10. BASIC STRUCTURE 0F BINARY VECTORE
 T-DNA borders
 Selectable marker genes for plants
 Reporter genes
 Introduction of DNA fragments to T-DNA
 Plasmid replication functions
 Bacterial selection marker
 Plasmid mobilization functions
 Promotars
 3 Signals

12. T-DNA BORDERS
 The RB and the LB are imperfect, direct repeats
of 25 bases to define and delimit T-DNA.
 The RB and the LB are integrated in binary
vectors as DNA fragments cloned from well-
known Ti plasmids, either octopine or nopaline
type.

13. SELECTABLE MARKER GENES FOR PLANTS
 Choice of selectable marker genes is a key factor in plant
transformation.
 Antibiotics or herbicides resistance genes, such as
kanamycin, hygromycin, phosphinothricin, and glyphosate,
are very popular.
 Kanamycin resistance has been most frequently employed
in the transformation of many dicotyledonous plants.
 Hygromycin resistance is the most effective in rice (Oryza
sativa) transformation, whereas phosphinothricin resistance
is the most effective in maize.
 Selectable marker genes are usually driven by constitutive
promoters.

14. REPORTER GENES
Some genes being trasferred produce enzyme
whose activity can be easily detected or used as a
basis of selection for the transformed cells, e.g.
gene for herbicide resistance. However, most gene
need to be tagged with another gene. called
reporter gene
1. An ideal reporter gene
2. Scorable reporter gene
3. Selectable reporter gene

15. INTRODUCTION OF DNA FRAGMENTS TO
T-DNA
 Insertion of genes of interest into appropriate locations
of a binary vector is traditionally carried out by standard
subcloning techniques. Multiple cloning sites, which are
similar or identical to those in pUC, pBluescript, and
other standard vectors, are still very useful in this
regard, but recently constructed vectors are more user
friendly.
 Recognition sites for ‘‘rare cutters,’’ which are
restriction enzymes with long recognition sequences,
are very convenient in this respect because the DNA
fragments that are to be inserted scarcely have such
sites.

16. PLASMID REPLICATION FUNCTIONS
 Binary vectors need replication functions active
in E. coli and A. tumefaciens. Replication functions
active in a wide range of bacteria, such as ones
of plasmid incompatibility group P or W may
be conveniently employed.
 The types of replication functions determine the
copy number and the stability of the plasmids in
bacterial cells.

17. BACTERIAL SELECTION MARKER
 Antibiotic resistance genes in common cloning vectors,
such as genes that can confer resistance to:
 Kanamycin
 Carbenicillin
 Gentamicin
 Spectinomycin
 Chloramphenicol
 Tetracycline
 all employed in plant transformation vectors.
 In the process of plant transformation, A. tumefaciens
should be removed from plant cells by antibiotics after
infection.

18. PLASMID MOBILIZATION FUNCTIONS
 The plasmids with OriT or the bom may be
mobilized from E. coli to A. tumefaciens aided by a
conjugal helper plasmid, such as pRK2013.
 This function is not necessary when vectors are
introduced into A. tumefaciens by electroporation
or freeze-thaw methods, but it is a good idea to
have a wider option because the conjugal
transfer is a very efficient process.

19. PROMOTERS
 Selectable markers need to be expressed in calli, in cells
from those plants that are being regenerated, or
germinating embryos to facilitate plant transformation.
Therefore, promoters for constitutive expression are
preferred. Promoters used mainly for dicotyledonous
plants include the 35S promoter from cauliflower
mosaic virus and promoters derived from Ti plasmids,
such as nopaline synthase (Nos), octopine synthase
(Ocs),mannopine synthase (Mas), gene 1, gene 2, and
gene 7

20. 3' Signal
 DNA fragments of a few hundred bases derived
fromthe 3' ends of the CaMV 35S transcript and
Agrobacterium Nos and otherT-DNA genes are
carried by many of the binary and super-binary
vectors

21. 1.pGA series vectors.
2. pCG series vectors
3. pCIT series
4. pGPTV
5. pBECK2000 series
6. Binary-BAC (BiBAC) vector
7. pGreen series

22. 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.
 A polylinker site (multicloning site).

23. pGPTV (glucuronidase plant transformation
vector) series , which have:
Different plant selectable marker genes close to
the left T-DNA border.
This design overcomes problems inherent with
the preferential right to left border transfer of T-
DNA and improves the chances of having the
gene of interest transferred to the plant cell in
cells expressing the selectable marker gene.

24. Binary-BAC (BiBAC) vector
 Based on a bacterial artificial chromosome
(BAC) vector and is suitable for Agrobacterium-
mediated transformation of high-molecular-
weight DNA.
 Comprises low-copy number origins of
replication for both E . coli and Agrobacterium to
ensure replication of the plasmid as a single-
copy in both bacteria; and
 A helper plasmid carrying additional copies of
vir-genes in order to clone very large T-DNAs
(up to 150 kb) into the plant genome.

25. pGreen series, small plasmids of around 3.2
Kb containing:
A broad host range replication origin (ori pSa)
and a ColE1 origin derived from pUC,
A pSa replicase gene (rep A) that provides
replication functions in trans and is located in a
compatible plasmid (pSoup) in Agrobacterium,
and
Multiple cloning sites based on the
pBlueScript vector, which allow any
arrangement of selectable marker and reporter
genes.

26. TRANSFARMATION PROCUDER
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. ADVANTASE
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. DISVANTASE
 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
unstale in E. coli where both origins are active.

30. REFFERENCE
 An Introduction to Genetic Analysis, Griffiths et al., 7th
ed. ISBN 0-7167-3771-X
 Department of Biological Sciences, Purdue University, West
Lafayette, Indiana 47907–1392
 Lan-Ying Lee and Stanton B. Gelvin(2007) Molecular Cell
Biology, Lodish et al., 6th ed. ISBN 1-4292-0314-5
 Principles of Genetics, Snustad & Simmons, 4th ed. ISBN 0-
471-69939-X
 Sherman, Fred(2007). "9 Yeast
Vectors" http://dbb.urmc.rochester.edu/labs/Sherman_f/yeast
/9.html.
 Slater, Adrian; Scott, Nigel; Fowler, Mark (2008). Plant Biotechnology
the genetic manipulation of plants. Oxford University Press Inc., New
York.