Fig. 2

Functionality of TEV-tagged protein variants in vivo. Upper and middle panels (a-d): functionality of plasmid encoded TEV-tagged protein variants in vivo. Variants were expressed from their natural promoter on low copy plasmids. a TpiA, b GpsA, c Adk, d Adk variants isolated from an insertion library around PSII. Insertion positions and corresponding permissive stretches (PSs) are given for each variant. Functionality was evaluated as the ability of a certain variant to support growth of the corresponding knockout strain on different carbon sources at 37 °C. Experiments were done in biological duplicates ± SD. Lower panel (e and f): functionality of chromosomally encoded TEV-tagged proteins variants in vivo. e Indicated strains carrying a TEV-tag on the chromosome were grown in LB medium or M9 glucose with casamino acids at 32 °C, and growth rates were compared to the appropriate parent strain (Ec or Ec*) which was used for chromosomal integration; in case of TpiAL70 the strain has an additional STOP codon in amn (Ec*) resulting in a translational knockout. f Growth rates on M9 succinate of strains carrying a TEV-tag in the α- (AtpA) and β- (AtpD) subunits of ATP synthase. A functional ATP synthase is essential for growth on the non-fermentable carbon source succinate. Strains having AtpA or AtpD replaced by a kanamycin cassette fail to grow on succinate. Experiments were done in triplicate ± SD