质粒稳定性测试

Immediately before indcution, it is advisable to test the culture to determine the fraction of cells that still carry the target plasmid. This involves plating of cells on four different plates.

Remarks

In the presence of IPTG, cells carrying a protein production plasmid do not grow because have dedicated all their resources to the production of the recombinant protein instead of cell maintainance.

In the presence of the pLysS vector, IPTG also prevents colony formation except with certain vector (such as pET-3 and some vectors carrying the T7 lac promoter). In the presence of pLysE, IPTG usually does not prevent colony formation unless the target protein is toxic.

In a typical culture useful for producing target protein, almost all cells will form colonies both on the LB plate and on the LB plate + antibiotic less than 2% of the cells will form a colony on the LB plate + IPTG; and less than 0.01% will form a colony on the LB plate + antibiotic + IPTG.

With unstable target plasmids, the fraction of cells that have lost the plasmid will be reflected by an increase in colonies on the LB plate + IPTG and a decrease on ther LB plate + antibiotic.

Protocol