Supplementary Figure 8: Biomolecular feedback controller.

(a) Modular structure of the feedback system. The feedback system was placed on a medium copy number plasmid with the ampicillin resistance marker. The feedback is composed of a dCas9 cassette with constitutive expression from a synthetic promoter and of a downstream burden-responsive cassette. In the burden-responsive cassette the burden-responsive htpG1 promoter controls the production of a sgRNA, in this case targeting the pBAD core promoter. The system was conceived with a modular structure where the htpG1 promoter and the gRNA can be easily replaced using rare restriction enzymes flanking each module. (b) Burden imposed by the molecular feedback controller on DH10B cells. Top panels show the growth rates (left panel) and GFP production rates (right panel) 1 hour post induction for uninduced DH10B cells (with integrated capacity monitor) when no construct is present (DH10B), and uninduced cells containing both pSB1C3-H2 and the feedback system plasmids (PdCas9-X), or uninduced cells containing just pSB1C3-H2 (H2). The burden imposed by the feedback system when promoters of different strength are present upstream of dCas9 is shown (PdCas9-1 to 6). Bottom panels show growth rates, as well as GFP and mCherry production rates when the same constructs are induced with arabinose. H2 is a previously-designed and tested construct, similar to pSB1C3-H3, but with a stronger RBS and so imposing even higher levels of burden. For a full description of H2, please see Ceroni et al, Nature Methods 2015. Dots show single measurements and bar show the mean value. Error bars show the standard deviation of three biologically independent repeats.