Recombinant DNA technology

III-B.Sc. Biotechnology
18UBT508 -4 credits

Binary and Shuttle Vector

Prepared by
Dr. V. Bhuvaneshwari,
Associate Professor and Head in Biotechnology UG,
Kongunadu Arts and Science College,
Coimbatore-641029
Shuttle vector
• A shuttle vector is a vector (usually a plasmid) constructed so that it
can propagate in two different host species. Therefore, DNA inserted
into a shuttle vector can be tested or 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).
• Shuttle vectors include plasmids that can propagate in
eukaryotes and prokaryotes (e.g. both
Saccharomyces cerevisiae and Escherichia coli) eg.
pJDB219 or in different species of bacteria (e.g. both E.
coli and Rhodococcus erythropolis). There are also
adenovirus shuttle vectors, which can propagate in E. coli
and mammals.
• Shuttle vectors are frequently used to quickly make
multiple copies of the gene in E. coli (amplification). They
can also be used for in vitro experiments and
modifications (e.g. mutagenesis, PCR)
• Eg: pHV14, pEB10, pHP3 replicate both in Bacillus subtilis
and E.coli
•
Can replicate in E.coli and Pichia
pastoris.
Pichia pastoris has
alcohol oxidase gene, Aox1 which
have a strongly inducible
promoter.
These genes allow Pichia to use
methanol as a carbon and energy
source.
HIS4-selectable marker for
recombinant Pichia
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).
Systems in which T-DNA and
vir genes are located on
separate replicons are called
T-DNA binary systems.
Eg: pBIN19

neomycin phosphotransferase II (NPT II)

•  Binary vector-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:
• 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, (Antibiotic-resistance genes used to select for the
presence of the binary vector in bacteria)
• 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.
• A helper Ti plasmid, harbored in A. tumefaciens, which lacks the
entire T-DNA region but contains an intact vir region.
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.
• There are several binary vector systems that differ mainly in the
plasmid region that facilitates replication in Agrobacterium.
• Commonly used binary vectors include:
• pBIN19, pPVP, pGreen, pGA series vectors, pCG series vectors
Binary vector
without vir genes