Question

Alpha-complementation is fundamental for the blue-white colony screening during cloning experiments. There are several modifications to both the plasmid and E-coli necessary to make this methodology work.

b) Although alpha-complementation and/or blue-white screening are efficient models for colony screening in cloning experiments, they are not"false-positive" proof systems. One of the limitations arises from the use of ampicillin as a selectible marker in liquid cultures: a portion (sometimes significant) of the cells without a plasmid can survive, giving poor yielding plasmid preps, protein expression results, etc. Discuss ONE of the several ways this can happen during your experiment.

Answer 1

The plasmid which contain the ampicilin selectable marker gene can hydrolysed and inactivates the antibiotic ampicilin. The plasmid contains the bla gene which can express beta lactamase. Now, if some instances, such as long saturation period for the bacterial culture, beta lactamase which is secreted by bacteria can built up extracellularly thus inactivating the amplicilin in the liquid medium. As a result the selection pressure caused by ampicilin get decreased resulting in cells without the plasmid.These cells are responsible for poor yeild during plasmid preparation or protein expression.