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Additional Methods for Screening and Selection of Recombinants

Antibiotic resistance

This is one of the simplest selection methods. The plasmid of our interest should contain a specific gene for antibiotic resistance. For example, plasmid pBR322 contains the resistance for ampicillin and tetracycline. If the cells are successfully transformed (if they have taken up the plasmid), they will be resistant to both of these antibiotics, so other unwanted cells which do not contain our gene of interest can be easily eliminated simply by placing the cells on agar medium which contains either one or both of these antibiotics. This method works well also on the mammalian cells.

Insertional inactivation

Insertional inactivation helps us detect the target DNA because it disrupts the coding sequence once it has recombined. For example, if the target DNA gets inserted into the sequence coding for ampicillin resistance (because specific restriction enzyme cuts exactly at that position), it renders the cell which has successfully transformed sensitive to the ampicillin. What we can do next is grow cells in the culturing medium and wait for the colonies to form. We can then make a replica plate, expose it to the ampicillin and mark which colonies are sensitive, so we can work with them on the original plate.

Plaque morphology

There are some λ vectors (bacteriophage type of vectors) like λgt10 which encode the cI gene. The cI gene codes for the cI repressor proteins which allow phage lambda to transform the bacteria and start the lysogenic cell cycle. This basically means that the phage will live quietly in the cell, integrated into its genome (as a prophage) instead of multiplying itself, destroying the cell and exiting to attack the surrounding cells (lytic cycle). Plaques derived from the cI+ vectors will be turbid because some cells in the lysogenic cycle have survived. However, if the cI gene is inactivated during the process (due to the insertion of our fragment), the plaques formed will be clear and easily distinguishable from the turbid non-recombinants.