Implementation of cell-free biological networks at steady state

Abstract

Living cells maintain a steady state of biochemical reaction rates by exchanging energy and matter with the environment. These exchanges usually do not occur in in vitro systems, which consequently go to chemical equilibrium. This in turn has severely constrained the complexity of biological networks that can be implemented in vitro. We developed nanoliter-scale microfluidic reactors that exchange reagents at dilution rates matching those of dividing bacteria. In these reactors we achieved transcription and translation at steady state for 30 h and implemented diverse regulatory mechanisms on the transcriptional, translational, and posttranslational levels, including RNA polymerases, transcriptional repression, translational activation, and proteolysis. We constructed and implemented an in vitro genetic oscillator and mapped its phase diagram showing that steady-state conditions were necessary to produce oscillations. This reactor-based approach will allow testing of whether fundamental limits exist to in vitro network complexity.

Keywords: cell-free protein synthesis; computational biology; minimal artificial cell; synthetic biology.

Fig. 1.

ITT under steady-state conditions. (A) Function of a microfluidic nanoreactor for continuous ITT. At each dilution step, the supply channel is flushed with fresh reagent. A peristaltic pump meters a specific volume into the reaction ring. After both ITT mix and DNA have been added, another peristaltic pump mixes the reaction. (B) Experimental setup and analysis. (C) Model of EGFP synthesis in the reactor. Relative transcriptional (TX) and translational (TL) activities decrease at constant rates. In the continuous reaction (blue arrows), all modeled species are diluted at a constant rate, and DNA as well as relative TX and TL activities are replaced at the same rate. (D) Model predictions for a batch and a continuous reaction. Predictions were for 18.3 nM DNA and dilutions of 32% every 15 min. (E) Model of the repressilator (25) under three reaction conditions (SI Appendix). We show the concentration of one of the repressor proteins (R).

Fig. 2.

Steady-state ITT. (A) Dilution conditions for the experiments in this figure. Experimental RNA and protein concentrations (solid lines, Left axes) and model prediction (dashed lines, Right axes) for (B) long-term ITT at different dilution rates, (C) a transient switch to batch conditions (shaded area), and (D) oscillating DNA template concentrations (shaded area, water added; white area, DNA added). DNA template concentration, 10 nM (B and C); maximum 8.2 nM (D).

Fig. 3.

Regulation at the transcriptional, translational, and posttranslational levels. Solid lines, experimental data; dashed lines, controls. DNA template of the regulator was transiently present (gray shaded area). Reporter (EGFP) DNA template was present at constant concentration. For a detailed summary of concentrations and controls, see SI Appendix, Table S2. (A) Transcriptional activation by T3RNAP and σ⁷⁰. E. coli RNAP core enzyme was present in the reaction mix. Controls: wrong activator. (B) Transcriptional repression by TetR. Promoters transcribed by three different RNA polymerases were tested in the presence of their respective polymerase. Controls: promoter without repressor binding site. (C) Activation of translation by RNA molecules. Controls: wrong activator. (D) Protein degradation by ClpXP protease. Controls: no degradation tag (ssrA), gray lines; only one protease subunit expressed, broken lines.

Fig. 4.

Steady-state ITT conditions allow implementation of a genetic oscillator. (A) The oscillator network consists of three DNA templates: T3RNAP, supD amber suppressor tRNA, and TetR repressor. The TetR operator in the T3tet promoter and the amber stop codon in the tetR gene are indicated as red and blue boxes, respectively. Reporters for the two promoters in the network: yellow fluorescent protein (citrine) and cyan fluorescent protein (cerulean). (B) Phase diagram of the oscillator at different supD DNA template concentrations and different dilution rates. Oscillations (diamond symbols) occurred over a narrow range of dilution rates, which also determined the period of oscillations (fill color). Two other general behaviors were observed: one fluorescence peak and then low fluorescence (cross) or the system immediately reached a stable steady state (circles). (C) Cerulean (blue) and citrine (yellow) example traces.