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- Beta- galactosidase-produces enzyme that breaks down lactose Antibiotic Resistance: Some bacteria have genes coding for enzymes that destroy certain antibiotics!

pBLU plasmid Ampicillin resistance gene B-galactosidase gene

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

How do we get the plasmid inside of the bacteria? 1. Obtain E. Coli bacteria cells + Add to CaCl 2 (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 (+/-plasmid) pBlu (+ plasmid) pBlu (- plasmid) What will we observe??? LB LB/AMP LB/AMP /X-gal