A Multilayer Microfluidic Platform for the Conduction of Prolonged Cell-Free Gene Expression

Abstract

The limitations of cell-based synthetic biology are becoming increasingly apparent as researchers aim to develop larger and more complex synthetic genetic regulatory circuits. The analysis of synthetic genetic regulatory networks in vivo is time consuming and suffers from a lack of environmental control, with exogenous synthetic components interacting with host processes resulting in undesired behavior. To overcome these issues, cell-free characterization of novel circuitry is becoming more prevalent. In vitro transcription and translation (IVTT) mixtures allow the regulation of the experimental environment and can be optimized for each unique system. The protocols presented here detail the fabrication of a multilayer microfluidic device that can be utilized to sustain IVTT reactions for prolonged durations. In contrast to batch reactions, where resources are depleted over time and (by-) products accumulate, the use of microfluidic devices allows the replenishment of resources as well as the removal of reaction products. In this manner, the cellular environment is emulated by maintaining an out-of-equilibrium environment in which the dynamic behavior of gene circuits can be investigated over extended periods of time. To fully exploit the multilayer microfluidic device, hardware and software have been integrated to automate the IVTT reactions. By combining IVTT reactions with the microfluidic platform presented here, it becomes possible to comprehensively analyze complex network behaviors, furthering our understanding of the mechanisms that regulate cellular processes.

CFPU: A Cell-Free Processing Unit for High-Throughput, Automated In Vitro Circuit Characterization in Steady-State Conditions

“…Though the experimental setup behind the CFPU device is somewhat complex, it enables the study and characterization of dynamic gene circuits in a high-throughput and fully automated manner. Additional resources have been published that provide greater detail for fabricating two-layer PDMS devices and building hardware for controlling the on-chip pneumatic valves [28,29]. Apart from device fabrication and the experimental setup, another potential hurdle is access to a microarray robot.…”

Bottom-Up Construction of Complex Biomolecular Systems With Cell-Free Synthetic Biology

“…In 2013, Niederholtmeyer et al reported a two-layer PDMS device with eight independent nano-reactors that exchanged reagents at dilution rates similar to those of growing bacteria. Using this device, steady-state TX-TL reactions could be maintained for up to 30 h, enabling the first in vitro implementation of genetic oscillator circuits (Niederholtmeyer et al, 2013;van der Linden et al, 2019) (Figure 3B). Using the same device, Yelleswarapu et al (2018) recently demonstrated the construction of synthetic oscillating networks using sigma-factor-based regulation of native RNAP in E. coli lysate.…”

Cell-Free Characterization of Coherent Feed-Forward Loop-Based Synthetic Genetic Circuits

“…Characterization of the temporal behavior of CFFLs requires the introduction of input pulses of varying durations, which requires a method to dynamically add and eliminate DNA species in TXTL reactions. Here, we utilized semi-continuous microfluidic flow reactors 23,24,44 in combination with TXTL reactions to implement and characterize the synthetic CFFL 1 and its reference motif, taking advantage of the controlled inflow and outflow capabilities of the reactors to implement variable length inputs (Figure 4a). After 3 or 4 hours of pre-equilibration without input DNA species, during which all constitutive and background expression equilibrated, persistent step inputs were applied to both the CFFL and reference circuits (Figure 4b).…”

“…The microfluidic semi-continuous flow reactors were produced using standard soft lithography methods. 23,44 Semi-continuous flow IVTT reactions were performed according to the protocol previously described by van der Linden et al 44 , with adapted Labview control software to enable configuration of timevarying input signals. The 1.54x IVTT reaction mixture was stored on a water-cooled peltier element during the experiment to maintain reactivity of the solution, whereas other reactants were stored in tubing in the incubation chamber (29 °C).…”

“…30 We use the modularity of our design to implement several CFFL variants with orthogonal posttranscriptional toehold switch-based regulation 27 and, inspired by naturally occurring feed-forward architectures, 43 combine the CFFL variants into a topologically more complex feed-forward circuit. We characterize both the background suppression and temporal filtering functions of the synthetic CFFL circuit using a microfluidic semi-continuous flow reactor to sustain prolonged TXTL reactions, [23][24][25]44 complemented with in silico experiments. Our analysis reveals that the synthetic CFFL can effectively reduce background expression of components, increasing the fold-change of circuits for a wide range of circuit parameters.…”