Final answer:
The transformation protocol involves making bacterial cell membranes permeable to allow the uptake of plasmid DNA, either by using heat shock with CaCl₂ treatment, or by electroporation which uses an electric pulse to create pores in the cell membrane. In yeast cells, lithium acetate and PEG are used to permeabilize the cell wall for DNA entry. Transformation is generally a low-yield procedure.
Step-by-step explanation:
The transformation protocol is a biotechnological method used to introduce plasmid DNA into bacterial cells. This process is crucial for recombinant DNA technology, where bacteria must synthesize plasmid DNA. To perform the transformation, the bacterial cell wall, an apolar structure that impedes the entry of polar molecules like DNA, must be rendered permeable. One common method for transformation is heat shock, where bacterial cells are treated with CaCl₂ before being subjected to a sudden increase in temperature. This treatment allows chloride ions to penetrate the cell, leading to a 'swelling' effect. A subsequent heat shock at 42°C causes the formation of pores in the cell membrane, facilitating DNA uptake.
In yeast cells, like those of Saccharomyces cerevisiae, the cell wall can be made permeable by treatment with lithium chloride or lithium acetate, and the osmotic pressure can be modulated further with polyethylene glycol (PEG), enhancing the uptake of DNA. The alternative method, especially for eukaryotic cells, is electroporation. This involves an electric pulse that temporarily disrupts the phospholipid bilayer, creating pores through which DNA can enter. The electric pulse also causes a charge difference across the cell membrane, which helps draw the negatively charged DNA molecules into the cell interior. It is important to note that transformation typically has a low yield, with only a fraction of cells successfully taking up the plasmid DNA despite utilizing either heat shock or electroporation techniques.