Transforming Bacteria

Step 1. Use electroporation to insert plasmid containing a gene for kanamycin resistance into E. coli.

Step 2. Allow E. coli to grow on a media containing kanamycin.

Will bacteria without a plasmid grow on plates containing kanamycin?

Will bacteria with the plasmid grow on plates containing kanamycin?

Step 3. Determine if the bacteria have a plasmid containing R. sphaeroides DNA.

The EcoRI site lies in the middle of the gene for the alpha peptide. Plasmids containing R. sphaeroides DNA will not be able to produce alpha peptide. The strain of bacteria are also unable to make the alpha peptide. Recall that the alpha peptide is an important component of beta galactosidase without this peptide functional beta galactosidase is not produced.

Will bacteria with plasmids containing R. sphaeroides DNA produce beta galactosidase?

Will bacteria with plasmids lacking R. shpaeroides DNA produce beta galactosidase?

Step 4. Isolation of the plasmids from bacteria that have R. sphaeroides DNA. Our ability to do this depends on the nature of the DNA molecule you studied earlier.


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