Download 3c Resources Trans and Ab resistance

Transformation and Antibiotic Resistance
www.le.ac.uk
www.le.ac.uk/genie
Basic Cloning I
DNA to be inserted
Plasmid
vector
join/ligate
transform
host cell
Recombinant DNA molecule
Basic Cloning II
transform
host cell
select for cells
containing
recombinant DNA
by growth in
presence of
antibiotic
ABR
Recombinant
DNA molecule
Host cells are made “competent” to accept plasmids. This can be
achieved either:
Chemically (with heat shock)
OR
Electrically
Gene cloning – Gene library
X
X
X
A
A
C
B
C
C
A
B
X
A
X
A
B
C
A
A
X
X
C
B
B
B
B
A
B
vector
X
B
A
C
X
A
A
C
C
B
X
A
C
C
B
B
B
A
C
C
C
X
X
B
C
A
Gene cloning – Gene library
B
A
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 antibioticcontaining 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
EcoRI
insert
EcoRI
lacZ
– 5-bromo-4-chloro-3-indolyl β-Dgalactopyranoside
• Vector containing lacZ
• Insert DNA fragments into
sequence encoding lacZ
• Insertional inactivation
• β-galactosidase no longer
produced, X-Gal not converted
• SCREEN for recombinants
recombinant
insert
vector
lacZ
No LacZ activity
LacZ activity
White!
Blue!
Insertional Inactivation
Insertional Inactivation
X
X
X
TetR
AmpR
KanR
pBR322
cut with enzyme X
enzyme X
enzyme X
Ligate
TetR
KanR
DNA ligase
KanR
TetR
transformation
TetR, AmpS ,KanR
Recombinant Identification
Insertional inactivation
TetR, AmpS ,KanR
Phenotype of clone
Physical characteristics of DNA
pGLO
vector only
recombinant DNA
Clone that Gene!
Rationale of the experiment
Which is which?
Make bacterial clones (transformation)
Investigate phenotype of clones (transformants)
Investigate physical characteristics of DNA