Pglo experiment
• What is ampicillin?
• A cell wall inhibiting antibiotic
• What happens to normal bacteria that are grown on
agar plates with ampicillin in the agar?
• The bacterial cells won’t grow on the agar.
Pglo Experiment (continued)
• What is beta-lactamase?
• An enzyme that breaks down ampicillin.
• What happens to bacterial cells that produce the
beta-lactamase enzyme (because the have the betalactamase gene) when you grow them on ampicillin
agar?
• They grow!
Pglo Experiment (continued)
•
•
•
•
What is GFP?
A green fluorescent protein (it glows green under UV light)
Where is the GFP gene located in our experiment?
On the pGlo plasmid
Pglo Experiment (continued)
• How do we manipulate Ecoli to take up the
pglo plasmid (become transformed)?
• Heat shock
• Osmotic shock (transformation solution)
Pglo Experiment (continued)
• What is ara?
• A sugar that must be present in order for the
GFP gene to be expressed and form a GFP
protein.
Pglo Experiment (continued)
We have 4 plates in this experiment
a. - DNA LB
b. -DNA LB/amp
c. + DNA LB/amp
d. + DNA LB/amp/ara
What is the purpose of the –DNA LB plate?
Which plates should be compared to determine if the bacteria have acquired
the amp resistance gene?
Which plates should be compared to demonstrate that arabinose is required
for the expression of the GFP gene?
Which plate is the control for the + DNA LB/amp/ara plate?
Pglo Experiment (continued)
• What do these results tell you about bacterial
transformation with the Pglo plasmid
Employee Resource Manual
• Plasmids
• Restriction Endonucleases
• Agarose Gel Electrophoresis
Plasmids
• Small extrachromosomal pieces of DNA found
in some bacterial species
• May carry additional genes (such as antibiotic
resistance)
• Can be genetically modified and used as
vectors for genetic engineering
PUC 18-Plasmid
What is Lac Z?
• It is the gene that codes for the enzyme Bgalactosidase
• What does B-galactosidase do?
• It hydrolyzes sugars including lactose and X-gal
What is X-gal?
• An artificial sugar that some bacteria can hydrolyze
• What determines whether a bacterium can hydrolyze
X-gal?
• The bacterium must have the enzyme Bgalactosidase
• How can we tell if a bacterium has hydrolyzed X-gal?
• The bacterial colonies will turn blue. If they
don’t hydrolyze X-gal, they will remain white.
Restriction Endonucleases
• Produced by some bacteria as a defense
against virus infection
• Cleave DNA at specific bases sequences
(different recognition site for each different
enzyme)
• Can be used to join DNA from 2 different
sources (plasmid DNA and genomic DNA)
ECOR1
Agarose Gel Electrophoresis
• Separates DNA based upon size differences
• DNA is pulled through a gel by an electric
current
• (-) charged DNA is pulled to the positive pole
of the apparatus.
• Smaller pieces of DNA migrate through the gel
faster than larger pieces of DNA
Agarose Gel Electrophoresis
Procell
Procell in Action
What is your first job assignment?
• Clone the H gene (use a bacteria to make
copies of the gene for us)
What kind of bacteria do we use to
clone the H gene?
• E.coli (lacZ(-), amp sensitve)
Where is the H gene located?
• Lambda virus
How do you get the cloned gene into the
bacteria so the bacteria can copy it?
• Transform E.coli (lacZ(-), amp sensitve) with
PUC 18-lambda plasmid (heat shock and
osmotic shock)
Lambda virus genes have
been inserted into the plasmids here
How do you get lambda genes into
PUC 18 plasmid?
• Incubate PUC-18 and lambda with EcoRI,
ligate products
How many different plasmids do you get when you mix
PUC 18 and lambda, both of which have been ECOR1
and then ligated?
7
Would all 7 of the plasmids be
recombinant (have lambda DNA)? No!
How do you tell if bacteria have been
transformed successfully with PUC-18 plasmid?
• They will grow on amp agar.
How can you distinguish whether plasmids that
transformed bacteria were recombinant (lambda and
PUC-18) or nonrecombinant (pUC-18 only)?
Plate the transformed cells on Xgal-amp agar
E.Coli transformed
With nonrecombinant
Plasmids (PUC-18)
E.Coli transformed
With recombinant
Plasmids (PUC-18/lambda)