1. Lecture
on
Binary and Shuttle Vectors
by
Prof. Tuhina Verma
Department of Microbiology,
Dr. R.M.L. Avadh University, Ayodhya (U.P.)
2. The binary vector is a shuttle vector, so-called because it is able to
replicate in multiple hosts (e.g. E. coli and Agrobacterium
tumefaciens).
They are artificial cloning vectors that have been created from two
naturally occurring plasmids and has significant use in
biotechnology.
Binary vectors are standard tool in the transformation of higher
plants mediated by Agrobacterium tumefaciens and also in
producing genetically modified plants.
They are mainly developed for purpose of gene transfer in higher
plants.
They are based on pPCV vector (plant cloning vector) series.
Binary Vectors
3. There are several binary vector systems that differ mainly in
the plasmid region that facilitates replication in Agrobacterium.
Commonly used binary vectors include: pBIN19, pPCV002, pPVP,
pGreen.
The pBIN19 plasmid was developed in the 1980s and is one of the first
and most widely used binary vector plasmids.
It is a binary Agrobacterium vector and is used to transform higher
plants. Also known as pBin19 or BIN19 or Bin19 or Bin 19.
The size of pBIN19 binary vector is 11,777 base pairs.
It carries lacZ gene, kanamycin resistance gene, E. coli origin of
replication (ori) and two boundary sequences from T-DNA region of the
Ti plasmid.
4. The two boundary sequences recombine with plant
chromosomal DNA, inserting the segment of DNA between
them into the plant DNA.
Recombinant plant cells are selected by plating on kanamycin
agar and then allowed to regenerate as whole plant.
Recombinant cells are constructed in E. coli, using lacZ
selection system, before transfer to A. tumefaciens and then to
plants.
6. The Binary vectors are constructed by combining the disarmed
plasmid of A. tumefaciens and some E. coli genes.
Hence, two different plasmids are employed in binary vector
system.
Disarmed plasmid means its tumor inducing genes located in
the T-DNA of Agrobacterium is removed.
Along with T-DNA it can replicate both in E. coli and
Agrobacterium strain.
7. Such plasmid contains foreign DNA in place of T-DNA.
Improved pBIN19 vectors have been developed and they are
named as pBINPLUS.
Improvements over pBIN19 include location of the selectable
marker gene at the left T-DNA border, genes for higher copy
number in E. coli, and two rare restriction sites around the
multiple cloning site for easier cloning and analysis of T-DNA
insertions in plant genomes.
12. Final step in construction of a superbinary vector. An intermediate vector with T-DNA
constructed in E. coli is transferred to an A. tumefaciens strain that carries an acceptor vector,
and the cointegrate is created via homologous recombination between shared sequences.
Abbreviations: OriV, origin of vegetative replication of the IncP plasmid; O, origin of replication
of ColE1; Trf, transacting replication function of the IncP plasmid: OriT, origin of transfer of the
IncP plasmid; B, bom site for plasmid transfer of ColE1; C, Cos site of phage lambda.
14. Shuttle Vector
Shuttle vector can propagate in two different host species (pro or eukaryote).
DNA inserted into such vector can be tested/ manipulated in two different
cell types.
The main advantage of these vectors is they can be manipulated in E. coli,
then used in a system which is more difficult or slower to use (e.g. yeast).
Broad host range vectors and used in transfer of genes between two totally
different organisms. It contains one replication origin for each host.
Shuttle vectors are used to rapidly make multiple copies of the gene in E. coli.
Also, used for in vitro experiments and modifications (e.g. mutagenesis, PCR).
One of the most common types of shuttle vectors is the yeast shuttle vector.
Almost all commonly used S. cerevisiae vectors are shuttle vectors.
15. Yeast shuttle vectors have genes that allow replication and selection in
both E. coli & yeast.
The E. coli component of yeast shuttle vector includes origin of
replication and selectable marker gene, e.g. antibiotic resistance, beta
lactamase, , beta galactosidase.
Yeast component of yeast shuttle vector includes an autonomously
replicating sequence (ARS), yeast centromere (CEN), and yeast selectable
marker (e.g. URA3, a gene that encodes an enzyme for uracil synthesis).
Some difficulties were observed in direct cloning in Bacillus sp. So a series
of shuttle vectors were developed that can replicate in both E. coli
and Bacillus subtilis. e.g. pHV14, pHV15, pEV10, pGPB14, pHV1431, etc.
Most of these were constructed as fusions between pBR322 and pC194 or
pUB110.
With such plasmids, E. coli can be used as an efficient intermediate host
for cloning.
19. Yeast artificial chromosomes (YACs) are shuttle-vectors that can be
amplified in bacteria and employed for the cloning and manipulation
of large deoxyribonucleic acid (DNA) inserts (up to 3 Mb pairs) in the
yeast Saccharomyces cerevisiae.
Shuttle plasmid vectors, pHY460 and pHY310, were developed from
streptococcal tetracycline resistance (TcR) plasmid pAMα1 (9.2kb) and
the Escherichia coli vector pACYC177 (3.7kb). The bifunctional
plasmids can replicate and express the TcR gene in both E. coli and B.
subtilis.
Plasmids pHY460 (7.0 kb) and pHY310 (4.8 kb) contain the TcR gene of
pAMα1 and the ampicillin resistance (ApR) gene of pACYC177. Both
plasmids showed high transformation efficiency in both host cells.
pHY460 was maintained stably in B. subtilis and, is a useful shuttle
vector functioning in E. coli and B. subtilis.
20. They are derived from the 2 μm plasmid (yeast) and pBR322
(E. coli).
It is a shuttle vector, which means it can replicate and exists
in both bacterial and yeast system.
The size of the YEp13 is 10.6 kb.
It consists of 2 μm plasmid origin, LEU2 gene, and antibiotic
resistant genes of pBR322 (ampicillin and tetracycline).
It is an episome, i.e. the plasmid can integrate itself into the
yeast chromosome and due to recombination, the LEU2 gene
may get incorporated into the yeast chromosome.
YEp13 (Yeast Episomal plasmid) Shuttle plasmid
21. The LEU2 gene codes for isopropyl malate dehydrogenase
which is necessary for the synthesis of Leucine.
The vector is generally incorporated into an auxotrophic
mutant having a defective LEU2 gene, and thus only those
cells which have this vector would grow.
The plasmid may remain integrated, or a later recombination
event may result in it being excised again.
The transformation efficiency of the vector is 10,000 to
1,00,000 cells per μg.
The copy number is 20–50.
