3.8 Selecting Transformed Cells
Remember that only a small percentage of the bacterial cells may have taken up the plasmid DNA and not all of the plasmids will have the gene we wished to insert. Some transformed cells may contain original plasmid that closed upon itself without incorporating any of the new DNA--and not all of the DNA that might be inserted in the plasmid may be intact and functional. We will see later that when we are making a gene library, the one gene that interests us may be only one of thousands present in the DNA sample that we have cloned into the plasmids. We clearly need very sensitive and accurate methods for screening for just the transformed cells that we want.
In even the simplest cases, the transformation mixture will contain large numbers of untransformed cells, usually some cells that are transformed with only the original plasmid, and a few cells that are transformed with the complete vector containing the gene of interest. There are several procedures that may be used to create very efficient selection techniques:
Include in the plasmid a gene that is required for growth of the transformed host cell--usually a gene for resistance to an antibiotic. If the plasmid brings in a gene for antibiotic resistance, then normal host cells will die if the antibiotic is added to the culture and only transformed cells will survive.
Include in the plasmid a gene that will yield colored colonies when a suitable substrate is added. Only those colonies that are transformed by plasmids that have a new gene for an enzyme that will convert a substrate to a colored produce will be colored. Normal cell colonies will appear white and colorless.
A radioactive nucleic acid probe (such as 32P-mRNA) may be used to detect those colonies that have the new DNA gene. For this procedure, we must isolate the mRNA of the gene or chemically synthesize in radioactive form a small section of the DNA sequence which we know to be a part of the gene. Of course, we do not always have this information.
Antibodies (often labeled with radioactivity) that react with the protein product of the gene may be used to determine which colonies contain the new gene. For this procedure to work, the transformed cell must make mRNA copies of the new gene and then translate these messages into new protein products. For example, E. coli containing the complete gene for proinsulin will make large quantities of human insulin. These cells could be detected using anti-insulin antibody. The general techniques for these procedures will be described under "replica plating" and other refinements are described in the Nicholl text.