Grade XII-PBR 322 and alternate selectable markers

CLONING VECTORS – GENETICALLY ENGINEERED
PLASMID
• Cloning vectors is a small piece of DNA that can be stably maintained in an
organism, and into which a foreign DNA fragment can be inserted for
cloning purposes.
• SIGNIFICANCE OF CLONING VECTORS
• A) USED TO PROPOGATE DNA
• B) USED AS THE VEHICLE FOR TRANSPORTING FOREIGN GENETIC MATERIAL INTO
ANOTHER CELL.
• C) IT FACILITATES AMPLIFICATION A SINGLE COPY DNA MOLECULE INTO MULTIPLE
COPIES.
TYPES OF CLONING VECTORS FOR PROKARYOTES
• CLONING VECTORS
YAC-YEAST
BAC- ARTIFICIAL
PHAGEMIDS BACTERIOPH BACTERIAL BACTERIOPH
COSMIDS PLASMIDS CHROMOSO
AGE ARTIFICIAL AGE
ME.
CHROMOS
OME
COSMIDS AND PHAGEMIDS
• COSMIDS
• TYPE OF HYBRID PLASMID THAT CONTAINS ALPHA LAMBA PHAGE COS SEQUENCE
FROM LAMBA SEQUENCE.
• USED TO BUILD GENOMIC LIBRARY.
• PHAGEMID
• IT IS DNA BASED CLONING VECTOR WHICH HAS BOTH BACTERIOPHAGE AND
PLASMID PROPERTIES.
BACTERIOPHAGE AS A VECTOR
IT CAN ACCEPT VERY LARGE PIECES OF DNA, HAVE HIGH COPY
NUMBER OF THEIR GENOME WITHIN THE BACTERIAL CELLS.
DESIGN OF A VECTOR AT PRESENT DAYS.
• 1) ENGINEERED IN A SUCH WAY THAT IT HELPS EASY LINKING OF
FOREIGN DNA
• 2) IT HELPS IN SELECTION OF RECOMBINANTS AND NON-

RECOMBINANTS.
CLONING VECTORS FOR VARIOUS ORGANISMS.
• CLONING VECTORS
PROKARYOTES PLANT ANIMAL

VECTORS VECTORS
PLASMIDS- PBR322,
PUC18 AGROBACTERIUM
TUMEFACIENS RETEROVIRUS
BACTERIOPHAGES
PBR 322- CHARACTERISTICS OF A
PLASMID
• ORI- ORIGIN OF REPLICATION
➢It initiates replication
➢Responsible to control the copy number of the linked DNA molecule.
• SELECTABLE MARKERS- A VECTOR SHOULD HAVE ATLEAST TWO SELECTABLE
MARKERS.
➢These are genes that holds specific characteristics- eg: antibiotic
resistance
➢It is used to identify transformants and non-transformants
➢It permits the growth of the transformants
PBR 322- CHARACTERISTICS OF A
PLASMID
• Rop-Repressor of primer/Replication of plasmid.
• It is a region which control the replication of a plasmid ( copy the number of the cell)
• CLONING SITES.
These are recognition sites into which foreign DNA is integrated. But these
recognition sites present in selectable marker region is given important as it is
easy to select the transformants and non-transformants.
Always, insert foreign DNA into recognition site

present in selectable markers.
PBR 322
P-PLASMID;B-BOLIVER. R-RODRIGUEZ
322- NUMBER
• IT HAVE 4361 BASE PAIRS
• TWO SELECTABLE MARKERS FOR ANTIBIOTIC RESISTANCE
➢ampR- Ampicillin resistance
➢Tet R- Tetracycline resistance
• RESTRICTION SITES- 8 RESTRICTION SITES
PBR 322- P-PLASMID;B-BOLIVER. R-RODRIGUEZ
322- NUMBER
Two selectable markers-:
Ampicillin and Tetracycline
Restriction sites in PBR 322

Ampicillin- Pst I, Pvu I
Tetracycline- Bam HI, Sal I
Outside the selectable markers- ECORI, ClaI,
HindIII, Pvu II
GENE CLONING PROCESS IN PBR 322
• INSERTIONAL INACTIVATION
• When a foreign DNA is ligated at BAMHI of TETRACYLINE medium, this regions turns
into recombinant DNA(r-DNA) and hence lose resistance against tetracycline.
DO YOU THINK IT WILL HAVE RESISTANCE AGAINST AMPICILLIN?
TRANSFORMANTS AND NON-TRANSFORMANTS
TRANSFORMANTS- BACTERIA /HOST THAT ALLOWED r-DNA to grow
NON-TRANSFORMANTS-
RECOMBINANTS- ONLY ABOUT FOREIGN DNA.

Replica plating
Ampicillin Tetracycline
Non- Non- Transformants will not

Transformants
Transformants Transformants grow due to
will grow
will grow will grow insertional inactivation
HENCE ALWAYS,
ONE SELECTABLE MARKER- ALLOWS THE GROWTH OF TRANSFORMANTS AND HELPS TO IDENTIFY THE
TRANSFORMANTS
ANOTHER SELECTABLE MARKER- ALLOWS THE ALIEN DNA TO GET LIGATED FORMING RECOMBINANT DNA I.E.
INSERTIONAL INACTIVATION.
ALTERNATE SELECTABLE MARKERS OR SCREENABLE
MARKER
• Alternate selectable markers are developed to differentiate recombinants
from non-recombinants on the basis of their ability to produce colour in the
presence of chromogenic substrate(Chromogenic substrates are peptides
that react with proteolytic enzymes under the formation of color. They
are made synthetically and are designed to possess a selectivity
similar to that of the natural substrate for the enzyme)
• Hence when using alternate selectable marker containing vectors, We
perform insertional inactivation only in the regions where enzymes
which acts as a chromogenic substrate is present. Ex: Beta-
galactosidase.
• Beta-galactosidase is the coding sequence in the lacz gene which
produces the chromogenic substrate- X-gal producing blue coloured
colonies.
PUC 18 VECTOR AS AN EXAMPLE
Insertional inactivation in lac Z gene inactivates the beta

galactosidase enzyme.
lac Z--→ Beta galactosidase enzyme-→ X-gal--→ Producing blue

coloured colonies
Replica plating done in ampicillin medium shows blue and white

coloured colonies.
BLUE AND WHITE COLOURED COLONIES
Blue colonies -------→ Non-Transformants which have not taken

the r-DNA and hence the colour is produced.
White colonies--------→ Transformants which have taken the r-

DNA and hence lost the potential to produce the blue colour.

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CLONING VECTORS – GENETICALLY ENGINEERED

• 2) IT HELPS IN SELECTION OF RECOMBINANTS AND NON-

PROKARYOTES PLANT ANIMAL

Always, insert foreign DNA into recognition site

Restriction sites in PBR 322

RECOMBINANTS- ONLY ABOUT FOREIGN DNA.

Non- Non- Transformants will not

Insertional inactivation in lac Z gene inactivates the beta

lac Z--→ Beta galactosidase enzyme-→ X-gal--→ Producing blue

Replica plating done in ampicillin medium shows blue and white

Blue colonies -------→ Non-Transformants which have not taken

White colonies--------→ Transformants which have taken the r-

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