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

Into E. coli (scary?…no!)

Our plasmid contains genes for: AMP= ampicillin (an

antibiotic) resistance Beta-galactosidase-an

enzyme that converts X-Gal Indo Blu

Protein that allows for antibiotic resistance

Enzyme that breaks down X-Gal to make Indo Blu

RNA

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. Coli2. 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

pBlu

Control

Control

Amp

X-Gal

Regular

Amp

X-Gal

Regular

What will we observe???