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 libraryA

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 InactivationTetR

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 inactivationPhenotype of clone

Physical characteristics of DNATetR, 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