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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???