Getting Cloned Genes into Bacteria by Transformation

GETTING CLONED GENES INTO BACTERIA BY TRANSFORMATION

Once the gene of interest is cloned into a vector, the construct can be put back into a bacterial cell through a process called transformation (Fig. 3.18). Here the “naked” DNA that was constructed in the laboratory is mixed with competent E. coli cells. To make the cells competent, that is, able to take up naked DNA, the cell wall must be temporarily opened up. In the laboratory, E. coli cells are treated with high concentrations of calcium ions on ice, and then shocked at a higher temperature for a few minutes. Most of the cells die during the treatment, but some survive and take up the DNA. Another method to make E. coli cells competent is to expose them to a high-voltage shock. Electroporation opens the cell wall, allowing the DNA to enter. This method is much faster and more versatile. Electroporation is used for other types of bacteria as well as yeast.

When a mixture of different clones is transformed into bacteria as in a gene library (see later discussion), cells that take up more than one construct usually lose one of them. For example, if genes A and B are both cloned into the same kind of vector and both cloned genes get into the same bacterial cell, the bacteria will lose one plasmid and

keep the other. This is due to plasmid incompatibility, which prevents one bacterial cell from harboring two of the same type of plasmid. Incompatibility stems from conflicts between two plasmids with identical or related origins of replication. Only one is allowed to replicate in any given cell. If a researcher wants a cell to have two cloned genes, then two different types of plasmids could be used, or both genes could be put onto the same plasmid.