Supplementary Figure 14: Burden-driven feedback control in a 24-h batch time course.

(a) Constructs FB, FB2 and FB5 from Figure 3c were selected for a 24 hour batch growth time course after addition of 1% arabinose to the cultures containing the feedback system and pSB1C3-H2 construct (H2). The culture density (left panel), total VioB-mCherry production per cell (middle panel) and total VioB-mCherry yield in growth flasks are compared for time points from 16 to 24 hours. Plots show how increased growth due to alleviation of burden can compensate the decreased VioB-mCherry production per cell, leading to higher total protein yields over time. Interestingly, looking at Figure S15 at 20 hours it is possible to compare the mCherry per cell values measured using the plate reader with the corresponding values obtained through flow cytometry measurement. Both plate reader and flow cytometry assays confirm H2 (no feedback control) and FB2 as the constructs producing the most VioB-mCherry per cell, while FB5 produces a lower amount of VioB-mCherry per cell, and FB (the closed-loop feedback controller) produces the least amount per cell. (b) Earlier time points of a 24 hour time-course experiment are shown. Data correspond to a time-course from 0 to 12 hours for culture density (left) and total VioB-mCherry yield (right) in growth flasks. To avoid artefacts due to measurements and normalisation with very low ODs, this experiment was started at a higher initial OD.