Transcript Bacterial Transformation - Tamalpais Union High School

Bacterial
Transformation
What is transformation?

Changing the genes and phenotype of a
bacteria by uptake of foreign/new DNA

Let’s review bacterial DNA first…
Bacterial genome

Bacteria are
prokaryotes—no
nucleus.


The area where
DNA is located is
called the nucleoid
DNA is organized
in one double
stranded circular
molecule
What is carried on the Plasmid?



The plasmid contains genes
necessary for survival and
can be passed from one
bacteria to another
Color Marker gene- Betagalactosidase-produces
enzyme that breaks down
lactose
Antibiotic Resistance: Some
bacteria have genes coding
for enzymes that destroy
certain antibiotics!
pBLU plasmid
B-galactosidase gene
Ampicillin
resistance gene
The transformation lab…

Our plasmid—pBlu
plasmid
RNA


Into E. coli
(scary?…no!)
Our plasmid contains
genes for:


Enzyme that
breaks down XGal to make IndoBlu
amp= ampicillin (an
antibiotic) resistance
Beta-galactosidase-an
enzyme that converts XGal  Indo Blu
Protein that
allows for
antibiotic
resistance
RNA
How do we get the plasmid
inside of the bacteria?
1.
Obtain E. Coli
bacteria cells +
Add to CaCl2
(helps plasmid
attach to
bacteria)
2.
Add plasmid to
same microtube
1. E. Coli
2. pBlu
plasmid
How do we get the plasmid
inside the bacteria?

Wait…and then
3. Heat shock! This
temporarily opens
pores to allow the
plasmid to enter the
bacteria…timing is
critical!!!
Growing the bacteria

After they have received
the plasmid…

Placed on a growth
media and allowed to
grow.
How will we know if the bacteria
actually got the plasmid??

Any ideas?

We can grow the bacteria on a plate:



That contains ampicillin and X-Gal
Regular bacterial medium
What do you predict will happen in each?
What will we
observe???
Predict
pBlu (+/-plasmid)
LB
pBlu (+ plasmid)
LB/AMP
pBlu (- plasmid)
LB/AMP
pBlu (- plasmid)
LB/AMP
/X-gal
pBlu (- plasmid)
LB/AMP
/X-gal