Transcript Bacterial Transformation - Eastern Regional 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

Antibiotic Resistance:
Some bacteria have
genes coding for
enzymes that destroy
certain antibiotics!
The transformation lab…

Our plasmid—pBlu
plasmid
RNA


Into E. coli
(scary?…no!)
Our plasmid contains
genes for:


AMP= ampicillin (an
antibiotic) resistance
Beta-galactosidase-an
enzyme that converts XGal  Indo Blu
Protein that
allows for
antibiotic
resistance
RNA
Enzyme that breaks down
X-Gal to make Indo Blu
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?
Predict
pBlu
Amp
X-Gal
pBlu
Regular
Control
Amp
X-Gal
Control
Regular
What will we
observe???