Fig. 1: Development of the Proof-of-concept Csr-regulated BUFFER Gate (cBUFFER). | Nature Communications

Fig. 1: Development of the Proof-of-concept Csr-regulated BUFFER Gate (cBUFFER).

From: Rewiring native post-transcriptional global regulators to achieve designer, multi-layered genetic circuits

Fig. 1

A Native post-transcriptional interactions of the Csr Network (left), and the hypothesized engineered system (right). In the engineered BUFFER Gate, natively expressed CsrA repressed a synthetic target through an engineered 5’ UTR sequence, and target translation is activated through induction of the sponge sRNA, CsrB. B Genetic circuit diagram of cBUFFER – the target of interest gfpmut3 is repressed by CsrA through the engineered glgC 5’ UTR sequence and CsrB transcription is activated through IPTG induction. These genetic components were expressed from a ColE1 ori-based plasmid (Copy Number 10–15). C Time course of initial cBUFFER fluorescence for induced ( + IPTG) and uninduced (-IPTG). The initial cBUFFER utilized the wild type CsrB sequence. D Titration response curve of initial cBUFFER fluorescence two hours post-induction. E. Fluorescence of cBUFFER systems using different versions of the CsrB sRNA. Mutants A1 and A3 contain deletions of the RNase E degradation site initially identified in Vakulskas et al. 2016 NAR. F Time course of optimized cBUFFER fluorescence for induced ( + IPTG) and uninduced (-IPTG) samples. The optimized version uses the CsrB A3 mutant. Samples were grown in biological triplicate, and data presented are the mean values +/- the standard deviation, represented as the error bars.

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