Making Recombinant Plasmids

1. Making Recombinant Plasmids

2. Pseudomonas fluorescens

3. Now make recombinant from a bacteria Pseudomonas fluorescens’ entire gDNA is broken Then broken pieces are mixed with lots of commercial plasmids and restriction enzymes Segments of the gDNA attach to the plasmids.

4. Imagine the entire P.f. gDNA put into thousands of plasmids

5. Then the plasmids are allowed to enter commercially available E. coli.

6. But a problem arises . . . Some E coli don’t take on plasmids. Draw that (show 5 E coli) So we have thousands of E coli WITH plasmids and thousands WITHOUT them.

7. Solution! The plasmids we used have a protein that resists the antibiotic kanamycin. All the E coli those WITH and those WITHOUT are put in a broth that has _____________ in it. The E coli ______________ die while the E coli ____________ survive. ANSWERS: kanamycin; without plasmids; with plasmids

9. All the pieces of Pseudomonas fluorescens’ genomic DNA: have been _________ into plasmids. Inserted And then the plasmids went in ________ E. coli bacteria—18,432 E. coli to be precise! And each of the 18,432 E. coli were ________ in a broth until there were ____________. Cultured or grown; millions And each of those 18,432 E. coli has a distinct subset of the ___________ from Pseudomonas flourescens. Genomic DNA or gDNA. We will call it a P.f. sequence The actual ______ ______ of that P.f. are unknown. Base pairs

10. Today, we’ll take some of those 18,432 E coli and let them multiply. Then we’ll have millions of copies of a little piece of P.f. gDNA Later we’ll sequence that!