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Author(s) -
Brakmann Susanne,
Grzeszik Sandra
Publication year - 2001
Publication title -
chembiochem
Language(s) - English
Resource type - Reports
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/1439-7633(20010302)2:3<153::aid-cbic153>3.0.co;2-b
Subject(s) - t7 rna polymerase , mutant , transcription (linguistics) , rna polymerase , directed evolution , biology , green fluorescent protein , fluorescence , rna , genetics , microbiology and biotechnology , gene , escherichia coli , bacteriophage , physics , linguistics , philosophy , quantum mechanics
The cover picture shows a schematic representation of the positive genetic selection of an error‐prone T7 RNA polymerase (T7 RNAP) mutant (shown in green). A system of two compatible plasmids (blue) carrying a T7 RNAP mutant library (bottom left) or an inactive mutant of the gene coding for tetracycline resistance under the exclusive control of a T7 promoter (top) rewarded inaccurate transcription with the survival of bacteria (symbolized by the smileys in the flask). Following a single round of selection, the survivors expressed a T7 RNAP variant with a nucleotide substitution error rate at least 20‐fold greater than that of the wild‐type enzyme. The heterogeneity of RNA products synthesized by this enzyme was demonstrated in vitro, and—most impressively—in vivo by employing the transcription of Green Fluorescent Protein (GFP): In contrast to the uniform distribution of fluorescence that was observed after transcription by the wild‐type T7 RNAP (left inset diagram), transcription by the mutant enzyme induced large deviations from the standard fluorescence behavior (right inset diagram), which were attributed to contributions of GFP mutants with altered excitation wavelengths. More about this successful application of the principle of directed evolution can be found in the paper by S. Brakmann and S. Grzeszik on p. 212 ff.