In Vivo Feedback Control of an Antithetic Molecular-Titration Motif in Escherichia coli Using Microfluidics

Abstract

We study both in silico and in vivo the real-time feedback control of a molecular titration motif that has been earmarked as a fundamental component of antithetic and multicellular feedback control schemes in E. coli. We show that an external feedback control strategy can successfully regulate the average fluorescence output of a bacterial cell population to a desired constant level in real-time. We also provide in silico evidence that the same strategy can be used to track a time-varying reference signal where the set-point is switched to a different value halfway through the experiment. We use the experimental data to refine and parameterize an in silico model of the motif that can be used as an error computation module in future embedded or multicellular control experiments.

Cheetah: A Computational Toolkit for Cybergenetic Control

“…Once trained, Cheetah segmentation masks were generated and used to calculate the number of cells and average GFP fluorescence per cell (Figure B,C). These results were compared to similar analyses using segmentation masks generated by ChipSeg that we , and others , have previously implemented in a similar experimental setup (Supplementary Movie 1; Methods).…”

“…Results from this experiment and related replica showed that the platform was able to accurately control average mCherry fluorescence in the cells for the duration of the experiment (Figures 3C, 3D; Supplementary Figure 1; Supplementary Movie 3). To evaluate the performance of the control experiment we measured the Integral Square Error (ISE) 41 for both controlled and uncontrolled chambers (i.e. those that received the same input in open loop).…”

“…Results from this experiment and related replica showed that the platform was able to accurately control average mCherry fluorescence in the cells for the duration of the experiment (Figure 3C,D; Supplementary Figure 1; Supplementary Movie 3). To evaluate the performance of the control experiment, we measured the Integral Square Error (ISE) 41 for both controlled and uncontrolled chambers (i.e., those that received the same input in open loop). Across the three closed-loop experiments, we found that the ISE values were lower for living cells in the controlled chambers when compared to living cells in the uncontrolled chambers that received the same input signals (Supplementary Table 1), confirming the effectiveness of our simple control strategy.…”

Model Predictive Control of Cancer Cellular Dynamics: A New Strategy for Therapy Design

“…Three main approaches have been proven to be effective for the control of different processes (such as gene expression and cell proliferation), namely: i) open-or closed-loop controllers embedded into cells by means of synthetic gene networks [2][3][4][5][6]; ii) external controllers, where the controlled processes are within cells, while the controller (either at single cell or cell-population level) and the actuation functions are implemented externally via microfluidics-optogenetics/microscopy-flow cytometry platforms and adequate algorithms for online cell output quantification and control [7][8][9][10][11][12][13][14][15][16]; iii) multicellular control, where both the control and actuation functions are embedded into cellular consortia [17][18][19][20][21]. Plenty of examples of embedded controllers have been engineered across different cellular chassis; instead, applications of external and multicellular controllers in mammalian cells are scarce and either just theoretical or limited to proof of concepts.…”

ChipSeg: An Automatic Tool to Segment Bacterial and Mammalian Cells Cultured in Microfluidic Devices

“…The same core functions are implemented for both cell types, making the code flexible for other chassis and applications. The algorithm is easy to use and shows robust segmentation results in external feedback control experiments with microfluidics/microscopy platforms; − ChipSeg can be easily adapted for open-loop experiments and other cell types/experimental settings.…”