Transformed bacterial cells can contain either recombinant or non-recombinant plasmid DNA after introduction of a ligation mixture. Recombinant plasmid DNA contains a foreign DNA insert with the desired gene. The plasmid replicates within transformed bacteria and is passed on to progeny cells. Transformed bacteria form clones containing the cloned DNA. Selection of transformed cells containing the recombinant plasmid pBR322 is done using its antibiotic resistance genes for tetracycline and ampicillin. Plating on different antibiotic media allows identification of bacteria with the recombinant plasmid, as insertion of foreign DNA into one of the resistance genes makes it non-functional for that antibiotic. This technique enables easy selection of a single, transformed bacterial cell from many.

1. Biotechnology: Selection of transformed cells
Dr. Saji Mariam George
Associate Professor
Assumption College Autonomous
Changanacherry

2. Transformation
• The process of uptake of foreign DNA (DNA insert
– the desired gene from another organism ) by a
cell - Transfer of recombinant plasmid
DNA(recombinant vector or rDNA) to a bacterial
host (e.g. E.coli) .
• Ligation mixture – recombinant and non-
recombinant DNA – introduced to bacterial cells
by Calcium chloride treatment / Electroporation.
• Transformed bacteria will carry either
recombinant plasmid DNA(recombinant vector or
rDNA) or non- recombinant plasmid DNA.

3. • Multiplication of plasmid DNA (vector DNA or
rDNA) occurs within each transformed
bacterium - As the host bacterial cell divides ,
the plasmid vectors are passed on to the
progeny – continue to replicate.
• Numerous cell divisions of a single transformed
bacterium →clone of cells → colony.
• The cloned DNA can be isolated from the clone
of bacterial cells.

4. Recombinant selection by Replica plating
technique using the plasmid vector pBR 322
Selection of cells transformed with the vector
pBR 322 using selectable marker genes
(antibiotic resistance genes) tetʳ (the gene
that gives resistance to the antibiotic
Tetracycline) and ampʳ(the gene that gives
resistance to the antibiotic Ampicillin) .

5. • Restriction enzymes PstI or Pvu I places the
DNA insert (foreign DNA containing the gene
of interest) within the gene ampʳ, that gives
resistance to the antibiotic Ampicillin and
hence it becomes non –functional (That
means, Ampicillin resistance is lost. In other
words, it becomes Ampicillin sensitive).
Hence the transformed bacteria will be
unable to grow in presence of Ampicillin, but
will grow on a medium containing the
antibiotic Tetracycline.

6. • Restriction enzymes Bam HI or Sal I places DNA
insert within the gene tetʳ (the gene for
Tetracycline resistance ). It becomes non –
functional. (That means, Tetracycline resistance
is lost. In other words, it becomes Tetracycline
sensitive). That is, transformed bacteria -
unable to grow in presence of Tetracycline, but
will grow on Ampicillin.
• This feature allows an easy selection of a single
bacterial cell having recombinant pBR 322 (with
the DNA insert) from a large number of other
types of cells.

7. • Transformed E.coli are first plated on agar
medium containing the antibiotic for which
the bacterial cells having the recombinant
plasmid are expected to be resistant– This
eliminates the non- transformed cells.
• The resulting bacterial colonies will possess
either recombinant plasmid vector pBR 322
(with the DNA insert) or unaltered pBR 322
(have no DNA insert).

8. • The bacterial colonies are replica plated on agar plates
containing the other antibiotic.
( the DNA insert is placed within this gene for antibiotic
resistance – it becomes non-functional and lost
antibiotic resistance and become antibiotic sensitive ).
So the bacterial cells with the recombinant pBR322,
with DNA insert, can not grow in this medium).
• So, all the colonies that develop on this plate will
contain the unaltered PBR 322(having no DNA insert –
its gene for antibiotic resistance will be functional).
• The antibiotic sensitive colonies – will have the
recombinant pBR322(with DNA insert) - can be
identified and recovered from the master plate.

9. pBR 322 Plasmid Vector

10. Recombinant selection by Replica plating
technique using the plasmid vector pBR 322

11. THANK YOU