1. pGLO Transformation LAB AP BIO LAB 6
ori
bla
GFP
araC
BIO-RAD lab book

2. PLASMID Extrachromosomal DNA
Often carry genes for antibiotic resistance
Can be passed from one bacterium to another

3. pGLO plasmid Contains genes for:
Image from: BIO-RAD lab book
pGLO plasmid Contains genes for:
- GFP = Green fluorescent protein
taken from jellyfish (Aequorea victoria) glows green in UV- light
- ori = Origin of replication allows plasmid to replicate itself
- araC = codes for arabinase
= enzyme to break down arabinose sugar
Turns on when arabinose sugar is present (INDUCIBLE)
- bla On all time
makes enzyme beta-lactamase that breaks down ampicillin
provides antibiotic resistance (=ampR)

4. pGLO plasmid genetically engineered plasmid
Image from: BIO-RAD lab book
pGLO plasmid
genetically engineered plasmid
used in biotechnology as a vector creating genetically modified organisms.
contains several “reporter“ genes green fluorescent protein (GFP) – glows under UV light ampicillin resistance gene (ampR) – allows growth on media containing ampicillin
produces an observable phenotype to help identify cells that contain the plasmid (AND ANY OTHER GENES THAT SCIENTISTS ATTACH TO THE PLASMID!)

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

6. put into Other Critters
Jellyfish Gene
put into Other Critters

7. Bacterial Transformation
Uptake of DNA from environment Changes phenotype
Image modified from BIORAD pGLO lab manual

8. pGLO LAB SUPPLIES FOAM tube holder/float 4 - flip top microtubes
Blue- Transforming solution (CaCl2)
Yellow- LB nutrient broth
Pink- label +
Purple- label -
1- colored eraser (to ID your tubes in water bath)
1-pkg yellow innoculating loops
2- Sterile pipettes
4 poured agar plates
1 - LB
2 - LB/amp
1- LB/amp/ara
PERMANENT MARKER
Cup with crushed ice

9. PLATE ABBREVIAIONS LB (LURIA & BERTANI) BROTH
contains nutrients for bacterial growth
AMPICILLIN (amp)
Antibiotic that kills bacteria
ARABINOSE (ara) = sugar used for food if glucose is unavailable

10. LABEL TUBES purple = + pGLO pink = - pGLO
Images from BIORAD pGLO lab manual

11. solution to pGLO + and – tubes
Transformation solution (CaCl2)
Use sterile pipette to
add 250µL transformation
solution to pGLO + and – tubes
Images from BIORAD pGLO lab manual

12. Get your TUBES on ICE!
Images from BIORAD pGLO lab manual

13. INNOCULATE TUBES WITH E. coli BACTERIA
Images from BIORAD pGLO lab manual
INNOCULATE TUBES WITH E. coli BACTERIA
Pick ONE colony Twirl loop in +pGLO tube
GET NEW LOOP Pick ONE colony Twirl loop in –pGLO tube
USE SPECIAL GARBAGE BAG FOR DISPOSAL OF USED SUPPLIES FOR THIS LAB

14. EXAMINE pGLO plasmid DNA
Use UV light to examine pGLO plasmid vial DOES IT GLOW?
UV light can be harmful to your eyes!
GFP = Green Fluorescent Protein
isolated from jellyfish
USED AS A GENETIC TOOL

15. PLASMID DNA TRANSFER THIS STEP IS CRUCIAL!
Look closely to make sure you have a film of solution across the ring.
(Similar to soapy film when you blow bubbles)
ADD PLASMID TO + TUBE
DO NOT ADD PLASMID TO - TUBE
Images from BIORAD pGLO lab manual

16. Put rack on ICE for 10 MIN!
Images from BIORAD pGLO lab manual

17. WHILE TUBES COOL CHECK LABELS ON PLATES
LB (Luria and Bertani) – broth & agar
provides nutrients for bacterial growth
LB/amp
Nutrients + ampicillin (antibiotic)
LB/amp/ara
Nutrients + ampicillin + arabinose sugar
+ plasmid will be added NO plasmid added
Images from BIORAD pGLO lab manual

18. MAKING CELLS COMPETENT “COMPETENT” cells have the ability to pick up plasmids
TRANSFORMATION SOLUTION (CaCl2)
Positive charge of Ca++ ions neutralizes:
~ negative charge of DNA phosphates
~ negative charge of membrane phospholipids
HEAT SHOCK
Increases permeability of cell membranes so plasmid can enter
Image modified from BIORAD pGLO lab manual

19. SHOCKING INCREASES UPTAKE OF FOREIGN DNA (PLASMID)
OSMOTIC SHOCK = Transformation solution CaCl2
HEAT SHOCK increases the permeability of the cell membrane to DNA
RAPID TEMPERATURE CHANGE is the key
2 MINUTES
50 SECONDS
Images from BIORAD pGLO lab manual

20. Place foam rack with + and – tubes on desktop
Images from BIORAD pGLO lab manual
Place foam rack with + and – tubes on desktop
Use new sterile pipette to add 250 µL LB broth to + tube
Use new sterile pipette to add 250 µL LB broth to – tube
Incubate at ROOM TEMPERATURE for 10 min

21. TAP TUBE WITH FINGER TO MIX!
Use NEW STERILE pipette for each vial
to add 100 uL bacterial suspension
to CORRECT DISH
(CHECK LABELS!)
Use a NEW STERILE LOOP FOR EACH PLATE
to spread suspension evenly on surface of plate
DON’T DIG INTO AGAR!
QUICKLY REPLACE LIDS
Images from BIORAD pGLO lab manual

22. HOW TO INNOCULATE A PLATE
Images from BIORAD pGLO lab manual

23. FLIP PLATES UPSIDE DOWN STACK AND TAPE LABEL WITH YOUR GROUP NAME PLACE IN INCUBATOR
Images from BIORAD pGLO lab manual

24. MAKE A PREDICTION
WHAT WILL HAPPEN?

26. Selection for plasmid uptake
Slide from Kim Foglia
Selection for plasmid uptake
Antibiotic becomes a selecting agent
only bacteria with the plasmid will grow on antibiotic (ampicillin) plate
only transformed bacteria grow
all bacteria grow a a a a a a a a a a a a a a a a a
LB plate
LB/amp plate
cloning

27. +pGLO LB/amp +pGLO LB/amp/ara -pGLO LB/amp -pGLO LB

28. Transformation Results
LB PLATE Luria Broth +
- PGLO = NO Plasmid →
All cells grow since there is no antibiotic on the plate

29. Transformation Results
LB/AMP PLATE Luria Broth with antibiotic +
- PGLO = NO plasmid →
NO GROWTH Cells without plasmid don’t have antibiotic resistance. Can’t grow on media with antibiotic added.

30. Transformation Results
LB/AMP PLATE Luria Broth with antibiotic +
+ PGLO = Plasmid added →
LAWN Cells with plasmid have antibiotic resistance gene so can grow on media with antibiotic

31. Transformation Results
Cells with pGLO plasmid GROW & GLOW -can grow on media with antibiotic
ONLY GLOW on media with arabinose (turns on GFP gene)
LB/AMP/ARA PLATE Luria Broth + antibiotic| + arabinose +
+ PGLO = Plasmid added →

32. Inducible operon: lactose
Slide from Kim Foglia
Inducible operon: lactose
Digestive pathway model
GLUCOSE is food of choice
Don’t need lactose digesting enzymes
Gene is turned off
RNA
polymerase
repressor
TATA
gene1
gene2
gene3
gene4
DNA
promoter
operator
repressor
ACTIVE repressor protein
Slide from Kim Foglia

33. Inducible operon: lactose
Slide from Kim Foglia
Inducible operon: lactose
lac
Digestive pathway model
When lactose is present, binds to lac repressor protein & triggers repressor to release DNA
induces transcription
RNA
polymerase
repressor
TATA
lac
gene1
gene2
gene3
gene4
DNA
promoter
operator 1 2 3 4
mRNA
enzyme1
enzyme2
enzyme3
enzyme4
repressor
repressor protein
lactose
lac
conformational change in repressor protein makes it
INACTIVE!
repressor
lactose – repressor protein
complex
lac

34. Lactose operon What happens when lactose is present?
Slide from Kim Foglia
Lactose operon
What happens when lactose is present?
Need to make lactose-digesting enzymes
Lactose is allosteric regulator of repressor protein
Slide from Kim Foglia

35. promoter operator Slide from Kim Foglia http://explorebiology.com RNA
polymerase
TATA
gene1
gene2
gene3
gene4
DNA
promoter
operator

36. ARABINOSE OPERON REGULATION
Slide from Kim Foglia
ARABINOSE OPERON REGULATION
= INDUCIBLE OPERON
PRESENCE OF ARABINOSE TURNS ON GENES
WHICH MAKE ENZYMESTO DIGEST ARABINOSE (along with pGLO gene)
Adding ARABINOSE to media makes bacteria GLOW