Investigation 8: Bacterial Transformation

Transformation starts with bacteria sitting in cold calcium chloride, which neutralizes charge repulsion between the negatively charged DNA and the negatively charged membrane. A brief heat shock, usually 42 degrees C for about 45 to 90 seconds followed by an immediate return to cold, creates a thermal gradient that pushes the membrane to form temporary pores. Plasmid DNA nearby gets pulled in through those pores before the membrane reseals. Only a small fraction of cells in the culture actually take up a plasmid, so once the bacteria are plated, only those transformed cells carry the ampicillin resistance gene and the GFP gene that sit on the plasmid. That is why just some colonies survive and glow green under UV light, not the whole plate.

Four plates isolate what each variable is doing. Plate 1, LB only, no plasmid, no antibiotic, confirms the bacteria are alive and healthy. Plate 2, LB plus ampicillin, no plasmid, confirms the antibiotic actually kills untransformed cells. Plate 3, LB plus ampicillin, with plasmid, is the real transformation test, since only cells that took up the plasmid's resistance gene survive here. Plate 4, LB only, with plasmid, shows all plasmid-containing cells can grow without selection pressure, giving a baseline count. Comparing colony counts across these plates is what lets you trust the result on the selection plate.

This investigation is the course's clearest hands-on example of a calculation science practice: counting colonies, dividing by DNA mass, and reasoning about what a result actually proves. It connects directly to gel electrophoresis investigations, where you again separate what DNA is present from what a band pattern is supposed to mean.