Relationship Between Opacity of Transformed E. coli Colonies and Over-Expression of the Recombinant Transcript

Abstract

Following transformation with recombinant plasmid clones, E. coli BL21(DE3) often produced extremely opaque colonies on a standard semisolid agar plate, as compared with the translucent colonies produced by normal, untransformed bacteria. A standard BL21(DE3) culture consisted of two kinds of cells: one kind produced translucent colonies and the other produced opaque colonies upon transformation by recombinant plasmids. The translucent-generating phenotype often switched to the opaque-generating phenotype, which was irreversible. Opacity in the BL21(DE3) background was correlated to a higher preinduced level of T7 RNA polymerase, presumably through a stable and inheritable genetic change. In all E. coli strains tested, a robust transcription of the recombinant gene from the plasmid clone was found to be an essential prerequisite for very high opacity; translation of the RNA was not required. The degree of opacity was also determined by the nature of the insert in a given strain background. Colony opacity generally, but not invariably, correlated with a smaller colony size on semisolid agar and a reduced growth rate in liquid culture.