Selectable Markers and Competent Host Cells

Once a suitable vector has been engineered, two further practical problems remain: how to tell, among all the host cells exposed to the vector, which ones actually picked up a recombinant (insert-carrying) plasmid rather than an empty or self-ligated one; and how to get the host cell to take up the plasmid DNA in the first place, since a bacterial cell membrane does not normally allow large DNA molecules to pass through freely.

The first problem is solved using the selectable marker system built into vectors like pBR322, through the trick of insertional inactivation introduced in the previous section. If the foreign gene has been inserted within the tetR gene of pBR322, any host cell carrying a true recombinant plasmid will have lost tetracycline resistance while keeping ampicillin resistance, whereas a host cell carrying only the unmodified vector (no insert) will retain resistance to both antibiotics. By first growing host colonies on an ampicillin-containing medium (which kills any cell that received no plasmid at all) and then replica-plating those surviving colonies onto a second medium containing both ampicillin and tetracycline, a researcher can identify recombinant colonies as exactly those that grow on the ampicillin plate but fail to grow on the ampicillin-plus-tetracycline plate. This comparison-based selection is simple, reliable, and does not require sequencing or any specialised equipment. …