Fig. 5: Feedforward ribosome controller compensates for burden caused by activation of the RFP gene in a common laboratory strain and across different nominal growth rates.

a CL system’s genetic construct (P_IFFL_RelA_2) co-expresses RFP and SpoTH via the AHL inducible Plux promoter. The SpoTH RBS is fixed to RBS 2 (Supplementary note 5). RelA+ is expressed using the SAL inducible Psal promoter. b Circuit diagram depicting the effect of activating RFP (AHL input) on ribosomes and growth rate for the open loop (OL) or closed loop (CL) systems. In the OL system, SpoTH is not present, so there is only the upper path from AHL to growth rate. In the CL system, AHL also activates SpoTH production and hence upregulates ribosome concentration and growth rate. Dashed edges represent sequestration of free ribosomes by a protein’s expression. RelA+ activation via SAL sets the basal level of ppGpp and thus the nominal growth rate⁶⁴. c–e Growth rate normalized by the growth rate at 0 nM AHL (nominal growth rate shown in the inset) versus the RFP production rate for the OL in CF944 (c), CF945, (d), and CF946 (e) and CL systems in TOP10. For the CL system, RelA+ expression is set to match the growth rate of the OL strain. The inset shows the nominal growth rate with no AHL induction. Data for all RBS’s of the CL system tested, AHL induction concentrations used, and GFP production data are shown in Supplementary Fig. 6 and Supplementary Fig. 7. Data are shown as the mean ± one standard deviation (N = 3, three biological replicates). Individual experimental values are presented as gray dots. All experiments were performed with glycerol as the sole carbon source. The complete experimental protocol is provided in the Materials and Methods section. Plasmid description, plasmid map, and essential DNA sequences are provided in Supplementary Information section Plasmid maps and DNA sequences.