160 likes | 231 Views
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
E N D
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 B-galactosidase gene Ampicillin resistance 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 RNA Enzyme that breaks down X-Gal to make Indo- Blu RNA Protein that allows for antibiotic resistance
How do we get the plasmid inside of the bacteria? 2. pBlu plasmid • Obtain E. Coli bacteria cells + Add to CaCl2 (helps plasmid attach to bacteria) • Add plasmid to same microtube 1. E. Coli
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?
pBlu (+/-plasmid) pBlu (+ plasmid) pBlu (- plasmid) pBlu (- plasmid) pBlu (- plasmid) Predict What will we observe??? LB LB/AMP LB/AMP LB/AMP/X-gal LB/AMP/X-gal