Figure 2. A positive feedback loop decouples expression capacity from host-specific transcription.

(a) The T7 RNAP and the GFP cassettes of UBER are organized in divergent orientation on opposite DNA strands. The priming pr is a 456 bp DNA sequence of eukaryotic origin with 40% GC content that has low basal promoter activity in E. coli. Its basal activity can be increased by inserting a host-specific J23100 promoter upstream of the T7 pr. T7 RNAP PFL strength can be tuned by tuning the translation rate of the T7 RNAP RBS. (b) The top graph shows the average steady-state GFP fluorescences for UBER variants with and without the positive feedback loop, using either the (high) J23100 priming promoter or the (low) 456 nucleotides nonspecific promoter and either a high or low T7 RNAP translation rate. The bottom graph shows the corresponding specific growth rates of these UBER variants. Variants that lack (−) the positive feedback loop do not have a T7 pr driving T7 RNAP expression, causing T7 RNAP to be solely expressed by basal transcription from the priming pr. Variants with (+) the positive feedback loop use a T7 pr to self-amplify T7 RNAP expression. The predicted T7 RNAP translation rates are either low (97, 328, 97 and 91 a.u.) or high (7,656 and 8,378 a.u.) in left to right order. Data points and bars are the means and s.d. of three measurements. (c) Solid lines: time-course model solutions showing GFP levels corresponding to the characterized UBER variants both (red) with and (blue) without the positive feedback loop. Dotted lines: corresponding time-course model solutions if T7RNAP toxicity is not considered in the model. The model solution for the high/high UBER variant shows very high T7 RNAP expression levels; this UBER variant could not be successfully constructed. Inset: dark green, the steady-state GFP levels with and without the positive feedback loop are shown. Inset: light green, corresponding solutions if T7 RNAP toxicity is not considered in the model.