pGLO Transformation LABAP LAB 6

http://www.mshri.on.ca/nagy/GFP%20mice.jpg

BIO-RAD lab book

pGLOori

blaGFP

araC

Aequorea victoria: Source of “glowing gene” for this experiment

Jellyfish Gene put into Other CrittersJellyfish Gene put into Other Critters

http://www.technologyreview.com/files/21291/monkey_x600.jpg

http://www.lafuga.de/GFP_pig.jpg

PLASMIDExtrachromosomal DNA

Often carry genes for antibiotic resistance

Can be passed from one bacterium to another

http://www.agen.ufl.edu/~owens/age2062/OnLineBiology/OLBB/www.emc.maricopa.edu/faculty/farabee/BIOBK/14_1.jpg

Bacterial Transformation

Plasmids

Chromosomal DNA

Bacterial Cell

The uptake of DNA

pGLO plasmid

bla (beta-lactamase)- On all time- Makes protein that breaks down ampicillin- Provides ampicillin resistance

GFP-Green Fluorescent Protein- Glows green in fluorescent light

ARABINOSE OPERON(INDUCIBLE)Turns on when arabinose sugar is presentAllows bacteria to digest this sugar

pGLOori

blaGFP

araCOri-PlasmidReplicationgenes

ARABINOSE OPERON REGULATION

araC

RNA Polymerase

Effector (Arabinose)

B

B

B

A

A

A

D

D

D

araC

araC

ara Operon

INDUCIBLE OPERONPRESENCE OF ARABINOSE TURNS ON GENES TOMAKE ENZYMES TO DIGEST ARABINOSE

pGLO Regulation

araC

RNA Polymerase

Effector (Arabinose)

B

B

B

A

A

A

D

D

D

araC

araC

ara Operon

GFP Gene

GFP Gene

GFP Gene

GFP GENE HAS BEEN ADDED TO ara OPERON

WHEN ARABINOSE IS PRESENT, OPERON IS TURNED ON andGFP GENEIS EXPRESSED TOO

Cells “glow” on mediawith arabinose

Reasons for Each Transformation Step

CaCl2 treatment

Positive charge of Ca2+ ions neutralizes: • negative charge of DNA

phosphates • negative charge of

membrane phospholipids

Ca++

Ca++

OCH2

O

P O

O

O Base

CH2

O

P

O

O

O

Base

OH

Sugar

Sugar

OCa++

Incubation on ice slows fluid cell membranes

Heat-shock increases permeability of cell membrane

Nutrient broth incubation allows beta lactamase expression

Reasons for Each Transformation Step

Selection for plasmid uptake

• Antibiotic becomes a selecting agent– only bacteria with the plasmid will grow on

antibiotic (ampicillin) plate

LB/amp plateLB plate

all bacteria growonly transformed

bacteria grow

a

aa a

aa

aa

aa

aaa a

a

cloning

a a

Transformation Results

All cells grow since there is no antibiotic on the plate

LB PLATELuria Broth

+

- PGLO = NO Plasmid

→

Transformation Results

NO GROWTHCells without plasmid don’t have antibiotic resistance. Can’t grow on media with antibiotic added.

LB/AMP PLATELuria Broth with antibiotic

+

- PGLO = NO plasmid

→

Transformation Results

LAWNCells with plasmid have antibiotic resistance gene so can grow on media with antibiotic

LB/AMP PLATELuria Broth with antibiotic

+

+ PGLO = Plasmid added

→

Transformation Results

Cells with pGLO plasmid GROW & GLOW-can grow on media with antibiotic

GLOW on media with arabinose (turns on GFP gene)

LB/AMP/ARA PLATELuria Broth+ antibiotic|+ arabinose

+

+ PGLO = Plasmid added

→