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Bacterial Transformation

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

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Bacterial Transformation

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  1. Bacterial Transformation

  2. What is transformation? • Changing the genes and phenotype of a bacteria by uptake of foreign/new DNA • Let’s review bacterial DNA first…

  3. 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

  4. 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!

  5. 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 Protein that allows for antibiotic resistance RNA Enzyme that breaks down X-Gal to make Indo Blu

  6. 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

  7. 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!!!

  8. Growing the bacteria • After they have received the plasmid… • Placed on a growth media and allowed to grow.

  9. 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?

  10. pBlu pBlu Control Control Predict What will we observe??? Amp X-Gal Regular Amp X-Gal Regular

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