Question

Intro of rdna into a host cells and selection methods

Answer 1

Answer:

Under laboratory conditions, the isolated plasmids are introduced into bacterial host cells via a process called transformation (Figure 10.4). One of two principal means is generally applied to achieve an experimentally feasible efficiency of transformation:

Prior to the transformation procedure, bacterial cells can be made „competent” via treatment with solutions containing bivalent cations. Plasmids are then introduced into host cells via a transformation procedure involving co-incubation and a subsequent heat shock step.

Plasmids can also be introduced into the host cell via electroporation. During this procedure, cells co-incubated with plasmids are exposed to short pulses of electric shock.

Introduction of plasmids into the host cell and their amplification therein

Figure 10.4. Introduction of plasmids into the host cell and their amplification therein

The extent of competence, which can be defined for a given set of experimental conditions, is specified as the number of plasmid-containing colonies grown after transformation (cfu, colony forming unit) extrapolated to units of mass of plasmids used for transformation (typically specified in cfu/µg plasmid).

Even with the maintenance of the above conditions facilitating the introduction of plasmids into the host cells, only a very small fraction of bacterial cells will stably take up a plasmid. The identification of transformed—i.e. plasmid-containing—cells is made possible by the utilisation of selection marker genes (Figure 10.5). The most commonly used selection markers confer resistance to certain antibiotics. The most commonly used antibiotics and the enzymes inactivating these antibiotics are listed below.