Relationship between opacity of transformed E. coli colonies and over-expression of the recombinant transcript.
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 every 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.
Barik
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Department of Biochemistry and Molecular Biology, University of South Alabama, College of Medicine, Mobile 36688-0002, USA. barik@sungcg.usouthal.edu
D017123:Bacteriophage T7 / Q000201:enzymology; D001483:Base Sequence; D003001:Cloning, Molecular; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D010641:Phenotype; D011994:Recombinant Proteins / Q000096:biosynthesis; D011995:Recombination, Genetic / Q000235:genetics*; D014170:Transformation, Genetic
D011994:Recombinant Proteins; D012321:DNA-Directed RNA Polymerases