www.le.ac.uk
Transformation and Antibiotic Resistance
www.le.ac.uk/genie
Basic Cloning I
DNA to be inserted
join/ligatePlasmid vector
Recombinant DNA molecule
transform host cell
Host cells are made “competent” to accept plasmids. This can be achieved either:
Chemically (with heat shock)OR
Electrically
Basic Cloning II
Recombinant DNA molecule
ABR
transform host cell
select for cells containing recombinant DNA by growth in presence of antibiotic
Gene cloning – Gene library
A
B
C
XA A A
BBB
C C C
A
A
BB
C
vector
A
B
C
A A A A
B BBB
C C C C
AC
CA
BB
C
AB
C
X XXX X X X
X
X
XX
B
A
Gene cloning – Gene library
Transformation and Selection• Use ligated DNA to
transform bacteria
• Not all ligated DNA maintained in bacteria
• Select for bacterial cells containing vector with insert (screen for recombinants)
Screen for recombinants• Ensure library predominantly recombinants• Simple screen to differentiate recombinants and vector
alone• For instance, blue-white screening using the lacZ gene
• Vector alone able to grow on antibiotic-containing medium
• Screen for recombinants identify lack of insert
• Recombinant grows on antibiotic-containing medium
• Recombinant identified by screen
Blue-White Screening• lacZ encodes β-galactosidase• β-galactosidase converts X-Gal
(colourless) to blue compound• X-Gal
– 5-bromo-4-chloro-3-indolyl β-D-galactopyranoside
• Vector containing lacZ• Insert DNA fragments into
sequence encoding lacZ• Insertional inactivation• β-galactosidase no longer
produced, X-Gal not converted• SCREEN for recombinants
EcoRI EcoRIinsert
insert
lacZ
lacZ
recombinant vector
No LacZ activity
White!
LacZ activity
Blue!
Insertional Inactivation
Insertional Inactivation
TetR
AmpR
pBR322
TetR
TetR
cut with enzyme X
DNA ligase
Ligate
transformation
X
enzyme X
X
TetR, AmpS
enzyme X
XX
KanR
KanR
KanR
,KanR
Recombinant Identification
Insertional inactivation
Phenotype of clone
Physical characteristics of DNA
TetR, AmpS ,KanR
pGLO
Clone that Gene!Rationale of the experiment
Which is which?
Make bacterial clones (transformation)Investigate phenotype of clones (transformants)
Investigate physical characteristics of DNA
vector only recombinant DNA