Name: a multilayer microfluidic platform for the conduction of prolonged cell-free gene expression
Uploaded: 2019-10-06T12:30:00
Duration: 11 min 23 s
Description: Watch this Scientific Journal Video about A Multilayer Microfluidic Platform for the Conduction of Prolonged Cell-Free Gene Expression at JoVE.com

This work was supported by the European Research Council, ERC (project n. 677313 BioCircuit) an NWO-VIDI grant from the Netherlands Organization for Scientific Research (NWO, 723.016.003), funding from the Ministry of Education, Culture and Science (Gravity programs, 024.001.035 &#38; 024.003.013), the Human Frontier Science Program Grant RGP0032/2015, the European Research Council under the European Union&#8217;s Horizon 2020 research and innovation program Grant 723106, and a Swiss National Science Foundation Grant 200021_182019.

The authors declare that they have no competing financial interests.

A PDMS-based multilayer microfluidic device has been presented, and its capability to sustain IVTT reactions for prolonged periods of time has been demonstrated. Although well-suited for this specific example, this technology can conceivably be used for numerous other applications. The additional control over fluid flow - paired with the ability to continuously replenish reaction reagents whilst removing (by)products - is ideal for continuous synthesis reactions, the investigation of various dynamic behaviors, and the simultaneous conduction of multiple variations of a single reaction.
Despite the relatively straightforward fabrication process of PDMS based devices, the use thereof requires an extensive hardware setup. Comprising valve arrays, pressure regulators, pressure pumps, incubators, and cooling units, the transition from fabrication to use is not elementary, and requires a significant initial investment. In addition, the ability to consistently set-up and perform successful experiments with these devices requires a significant time-investment; a point which this manuscript aims to address. However, once in place, the entire setup can be modified for a range of purposes. Furthermore, the hardware setup comprises numerous modular elements, each of which can be expanded to allow more complex microfluidic device designs to be employed. Additionally, the modular design enables the replacement of hardware components by similarly functioning alternatives, such that users are not limited to the specific setup described here48,49.
Variability between individual devices, and in the external conditions (such as pressure fluctuations) can result in inaccuracies when performing experiments using these devices. To address this issue, a calibration of the system should be performed prior to each experiment, providing a unique Refresh Ratio for each of the reactors. Whilst the calibration addresses the device-to-device and experiment-to-experiment variations, it is a time consuming process and not flawless. Fluids with differing viscosities will not flow with the same rate when exposed to identical pressure, and as such performing the calibration with multiple reagents may not yield identical Refresh Ratios. This effect is attenuated by utilising three control channels to peristaltically pump the reagents into the microfluidic device, as opposed to regulating the flow by varying the supplied pressure only. As a last resort in cases where the disparity in viscosity is very large, a unique Refresh Ratio can be implemented for each individual reagent by performing multiple calibration experiments.
The use of a peristaltic pump to inject reagents into the microfluidic device attenuates the effects of using solutions with varying viscosities, however it also creates a secondary problem. Using discrete steps to pump fluids into the microfluidic device, means that the resolution of injections into a single reactor, is limited by the volume injected when performing a single pump cycle. Within our research this value - determined during the calibration - is approximately equal to 1%, indicating that a single pump cycle displaces approximately 1% of the reactor volume (about 0.1 nL). As such, displacing 30% of the reactor volume requires the execution of 30 pump cycles, with 23 pump cycles of the IVTT reaction solution being added, and only 7 pump cycles of DNA or ultrapure water being added. Although sufficient for our research, alternative experimental protocols may encounter problems when attempting to add larger numbers of unique reagents, use a lower Refresh Fraction, or add smaller volumes of a single reagent to a reactor. In such cases, the microfluidic device design can be adapted to provide reactors with a larger volume. An example of such is reported in Niederholtmeyer et al.36.
Crucially, the device outlined within this manuscript allows reactions to be sustained for prolonged durations resulting in steady-state transcription and translation rates. By periodically injecting new reagents into the reactors - and removing reaction (by)products - the reactions are sustained and complex dynamic behaviors can be monitored. In this way, a platform has been created that - to some extent - mimics the cellular environment. Furthermore, this platform enables the exploration of the system dynamics, by adapting the period between injections and the specific composition of the injections. As a result, these multilayer microfluidic devices are a powerful tool for the characterisation and optimisation of novel synthetic networks which display complex dynamic behavior.

Cells are able to sense and respond to their environment using complex dynamic regulatory networks1,2. The field of synthetic biology utilizes our knowledge of the naturally occurring components comprising these networks to engineer biological systems that can expand the functionality of cells3,4. Conversely, it is also possible to further our understanding of the natural networks governing life by designing simplified, synthetic analogues of existing circuits or by forward-engineering biological systems which exhibit naturally occurring behaviors. The de novo engineering of such biological systems is performed in a bottom-up fashion where novel genetic circuits or signalling pathways are engineered in a rational manner, using well-defined parts5,6. Combining the rational design of networks with the design of biologically relevant systems enables the in-depth characterization and study of biological regulatory systems with various levels of abstraction7.
The pioneering works of Elowitz and Leibler8 and Gardner et al.9 were the first to demonstrate the successful introduction of synthetic genetic networks into cellular hosts. In the following decade, numerous researchers have continued to build on these initial successes despite the emergence of several limitations regarding the introduction of synthetic circuits into cells7,10,11,12. Ideally, the introduction of synthetic circuits into cellular hosts should occur in a modular fashion. Unfortunately, the complexity of the cellular environment makes this particularly challenging, with the function of many parts and networks being highly context dependent12,13,14. As a result, networks often experience undesired interactions with native host componentry which can affect the function of the synthetic circuit. Similarly, components of the exogenous network can inhibit host processes, compete for shared resources within the host, and influence growth kinetics15,16,17. Consequently, in order to rationally design and predict the behavior of synthetic networks in an in vivo environment, a comprehensive model of all host and circuit-specific dynamics is required18.
A viable alternative to the use of cellular hosts for the characterization of synthetic networks is the application of in vitro transcription and translation (IVTT) technologies. Acting as a testbed for synthetic networks, reactions are performed in solutions comprising all the components required to enable gene expression19,20,21. In this manner, a biologically relevant, albeit artificial, environment is created within which synthetic networks can be tested22,23,24,25,26,27,28. A major advantage of using IVTT solutions is the ability to perform reactions under user-specified conditions, with researchers able to tune the precise composition of each reaction2. Furthermore, the cell-free approach enables high-throughput testing of synthetic networks, since it removes the need to perform time-consuming cellular cloning steps. As a result, the duration of successive design - build - test cycles is significantly reduced29,30,31,32. The design cycle can be further accelerated by utilizing cell-free cloning techniques such as the Gibson assembly to rapidly engineer novel networks, and by constructing networks from linear DNA templates which - unlike the plasmids required for in vivo testing - can be amplified via polymerase chain reactions (PCR)33,34.
Batch reactions are the simplest method by which IVTT reactions can be performed, requiring a single reaction vessel wherein all of the reaction components are combined35. Such reactions are sufficient for protein expression and basic circuit testing yet prove insufficient when attempting to study the long-term dynamic behavior of a network. Over the course of a batch reaction, reagents are either depleted or undergo degradation resulting in a continuous decrease of the transcription and translation rates. Furthermore, as reactions progress by-products accumulate that can interfere with - or completely inhibit - the correct functioning of the network. Ultimately, the use of batch reactors limits the dynamic behavior which can be observed, with negative regulation being particularly challenging to implement5,36.
The versatility of IVTT systems enables multiple alternative methods by which prolonged IVTT reactions can be performed, ranging from continuous flow to droplet based methods as well as simpler dialysis approaches2,30,37,38,39,40. The application of microfluidic devices offers users increased control over their reactions whilst increasing the throughput and minimizing costs35,41,42, with each specific approach having its own advantages. The use of continuous flow can be easily optimized for increasing expression yields however, the inability to effectively remove specific reaction products makes the study of dynamic behavior non-trivial39. Whilst employing droplet based microfluidic systems allows high-throughput screening of novel networks, the difficulty of supplying fresh reagents to the reaction results in the droplets resembling small volume batch reactions43. Dialysis based reactors allow the introduction of fresh reagents as well as the removal of some reaction products however, RNA molecules and larger proteins accumulate within the reactor, being too large to diffuse through the membrane pores. Furthermore, large volumes of reagents are required to sustain these reactions for prolonged periods30,44. In 2013, Maerkl et al. presented a multi-layered microfluidic device designed specifically for conducting prolonged IVTT reactions36,45. The use of multi-layered microfluidic devices permits direct control over fluid flow, allowing for the redirection of flow as well as the isolation of fluid in specific regions of the device46,47. These isolated regions can function as independent nanoliter-scale reaction chambers wherein IVTT reactions can be performed. Over the course of a single IVTT reaction, periodic injections of fresh reagents into the reactor are used to replenish IVTT components and DNA templates. Simultaneously, an equal volume of the old reaction solution is displaced, removing reaction products. In this manner, an out-of-equilibrium environment is maintained where the basal transcription and translation rates remain in steady-state, prolonging the lifetime of IVTT reactions and allowing rich dynamic behaviors to occur. By applying this approach, researchers are able to investigate the kinetic rates of the individual processes occurring within a specific circuit, aiding in the forward-engineering of novel genetic networks. For instance, Niederholtmeyer et al. implemented this approach to characterize various elements of a genetic ring oscillator, determining the kinetic rates thereof36. In further studies, Yelleswarapu et al. showed that the kinetic rates of sigma factor 28 (&#963;28) determined under batch conditions were insufficient to describe the behavior of a &#963;28-based oscillator, and that the addition of flow-based data improved model predictions of the network behavior22.
The goal of this manuscript is to present a complete protocol for the fabrication of multilayer microfluidic devices capable of performing long-term IVTT reactions. In addition, this manuscript will describe all of the hardware and software required to perform prolonged IVTT reactions. The actuation of the microfluidic device - necessary to control the flow of fluids therein - is achieved using a series of pneumatic valves which connect directly to the microfluidic devices via lengths of tubing. In turn, the pneumatic valves are controlled via a custom-built virtual control interface. Fluid flow within the microfluidic devices is achieved using continuous pressure which is provided by a commercially available pressure regulation system. IVTT reactions are typically performed between 29 &#176;C and 37 &#176;C and a microscope incubator is used to regulate the temperature during reactions. However, the functionality of the IVTT mixture gradually degrades when stored above 4 &#176;C. As such, this manuscript will expand on the off-chip cooling system used to cool the IVTT mixture prior to injection into the microfluidic device. In conclusion, this manuscript provides a comprehensive overview of the procedures required to successfully perform prolonged IVTT reactions using a microfluidic flow reactor such that other researchers will be able to replicate this technology with relative ease.

<table>
	<tbody>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>VWR</td>
			<td>20063.365</td>
			<td></td>
		</tr>
		<tr>
			<td>AZ 40 XT</td>
			<td>Merck KGaA (Darmstadt, Germany)</td>
			<td>-</td>
			<td>Positive Photoresist</td>
		</tr>
		<tr>
			<td>AZ 726 MIF Developer</td>
			<td>Merck KGaA (Darmstadt, Germany)</td>
			<td>-</td>
			<td>Developer Positive Photoresist</td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>Merck KGaA (Darmstadt, Germany)</td>
			<td>109634</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope slides</td>
			<td>VWR</td>
			<td>ECN 631-1550</td>
			<td></td>
		</tr>
		<tr>
			<td>mr Dev 600</td>
			<td>Microresist Technology GmbH (Berlin, Germany)</td>
			<td>-</td>
			<td>Developer Negative Photoresist</td>
		</tr>
		<tr>
			<td>Silicon Free Heat Sink Grease</td>
			<td>Circuit Works</td>
			<td>CW7270</td>
			<td>Thermal Compound</td>
		</tr>
		<tr>
			<td>Silicon wafers</td>
			<td>Silicon Materials</td>
			<td>-</td>
			<td>&#60;1-0-0&#62; orientation, 100 mm diameter, 525 &#181;m thickness</td>
		</tr>
		<tr>
			<td>SU-8 3050</td>
			<td>Microchem Corp. (Newton, MA)</td>
			<td>-</td>
			<td>Negative Photoresist</td>
		</tr>
		<tr>
			<td>Sylgard 184 Elastomer Kit (PDMS)</td>
			<td>The Dow Chemical Company</td>
			<td>01317318</td>
			<td></td>
		</tr>
		<tr>
			<td>trichloro(1H,1H,2H,2H-perfluorooctyl)silane</td>
			<td>Sigma-Aldrich</td>
			<td>448931-10G</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>4 channel digital input/output module</td>
			<td>WAGO Kontakttechnik GmbH</td>
			<td>750-504</td>
			<td>8x</td>
		</tr>
		<tr>
			<td>Camera lens</td>
			<td>The Imaging Source</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Compression fitting</td>
			<td>Koolance, Inc.</td>
			<td>FIT-V06X10</td>
			<td>Fitting for tubing with 6mm ID and 10mm OD. 4x</td>
		</tr>
		<tr>
			<td>Controller end module</td>
			<td>WAGO Kontakttechnik GmbH</td>
			<td>750-600</td>
			<td></td>
		</tr>
		<tr>
			<td>Device connecting tubing</td>
			<td>Saint-Gobain Performance Plastics</td>
			<td>AAD04103</td>
			<td>0.02&#34; ID, 0.06&#34; OD, Tygon Tubing (ND-100-80)</td>
		</tr>
		<tr>
			<td>Device connector pins</td>
			<td>Unimed SA (Lausanne, Switserland)</td>
			<td>200.010-A</td>
			<td>AISI 304 tubing, 0.35mm ID, 0.65mm OD, 8mm L</td>
		</tr>
		<tr>
			<td>Ethernet Controller</td>
			<td>WAGO Kontakttechnik GmbH</td>
			<td>750-881</td>
			<td></td>
		</tr>
		<tr>
			<td>Female bus connector</td>
			<td>Encitech</td>
			<td>DTCK15-DBS-K</td>
			<td>15 pole female bus connector</td>
		</tr>
		<tr>
			<td>Fluid reservoirs</td>
			<td>Fluigent</td>
			<td>Fluiwell-4C</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluigent pressure system</td>
			<td>Fluigent</td>
			<td>MFCS-EZ</td>
			<td>0 - 345 mbar</td>
		</tr>
		<tr>
			<td>Hg short arc lamp</td>
			<td>Advanced Radiation Corporation</td>
			<td>-</td>
			<td>350W</td>
		</tr>
		<tr>
			<td>Hot plate</td>
			<td>Torrey Pines Scientific</td>
			<td>HP61</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted microscope</td>
			<td>Nikon Instruments</td>
			<td>Eclipse Ti-E</td>
			<td></td>
		</tr>
		<tr>
			<td>LabVIEW Software</td>
			<td>de Greef Lab, Eindhoven University of Technology</td>
			<td colspan="2">https://github.com/tfadgreef/Microfluidic-Device-Control-Software</td>
		</tr>
		<tr>
			<td>Liquid coolant</td>
			<td>Koolance, Inc.</td>
			<td>LIQ-705CL-B</td>
			<td></td>
		</tr>
		<tr>
			<td>Luer stubs</td>
			<td>Instech Laboratories, Inc.</td>
			<td>LS23</td>
			<td></td>
		</tr>
		<tr>
			<td>Male Luer to barb connectors</td>
			<td>Cole Parmer</td>
			<td>45505-32</td>
			<td>3/32&#34; ID</td>
		</tr>
		<tr>
			<td>Matlab Software</td>
			<td>de Greef Lab, Eindhoven University of Technology</td>
			<td colspan="2">https://github.com/tfadgreef/Microfluidic-Device-Control-Software</td>
		</tr>
		<tr>
			<td>Microcamera</td>
			<td>The Imaging Source</td>
			<td>DMK 42AUC03</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope camera</td>
			<td>Hamamatsu Photonics</td>
			<td>OrcaFlash4.0 V2 (C11440-22CU)</td>
			<td></td>
		</tr>
		<tr>
			<td>Orbital shaker</td>
			<td>Cole Parmer</td>
			<td>EW-513000-05</td>
			<td></td>
		</tr>
		<tr>
			<td>Oven</td>
			<td>Thermo Scientific</td>
			<td>Heraeus T6P 50045757</td>
			<td></td>
		</tr>
		<tr>
			<td>Oxygen plasma asher</td>
			<td>Quorum Technologies</td>
			<td>K1050X</td>
			<td></td>
		</tr>
		<tr>
			<td>PDMS puncher</td>
			<td>SYNEO</td>
			<td>Accu-Pucnh MP10</td>
			<td></td>
		</tr>
		<tr>
			<td>PEEK tubing</td>
			<td>Trajan</td>
			<td>1301005001-5F</td>
			<td>0.005&#34; ID, 1/32&#34; OD, Red</td>
		</tr>
		<tr>
			<td>Peltier element</td>
			<td>European Thermodynamics</td>
			<td>APH-127-10-25-S</td>
			<td></td>
		</tr>
		<tr>
			<td>Peltier temperature controller</td>
			<td>Warner Instruments</td>
			<td>CL-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Photomask</td>
			<td>CAD/Art Services, Inc.</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Photomask Design</td>
			<td>Maerkl Lab, EPFL</td>
			<td>https://zenodo.org/record/886937#.XBzpA8-2nOQ</td>
			<td></td>
		</tr>
		<tr>
			<td>Pneumatic valve array</td>
			<td>FESTO</td>
			<td>-</td>
			<td>1x 22 valve array and 1x 8 valve array, Normally closed valves.</td>
		</tr>
		<tr>
			<td>Power adapter</td>
			<td>Koolance, Inc.</td>
			<td>ADT-EX004S</td>
			<td>110/220V AC Power Adapter</td>
		</tr>
		<tr>
			<td>PTFE tubing</td>
			<td>Cole Parmer</td>
			<td>06417-21</td>
			<td>#24 AWG Thin Wall PTFE</td>
		</tr>
		<tr>
			<td>Punching pin</td>
			<td>SYNEO</td>
			<td>CR0320245N21R4</td>
			<td>OD: 0.032&#34; (0.8128 mm), ID: 0.024&#34; (0.6090 mm)</td>
		</tr>
		<tr>
			<td>PVC Tubing</td>
			<td>Koolance, Inc.</td>
			<td>HOS-06CL</td>
			<td>6 mm ID, 10 mm OD</td>
		</tr>
		<tr>
			<td>Single edge blades</td>
			<td>GEM Scientific</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Soft tubing</td>
			<td>Fluigent</td>
			<td>-</td>
			<td>Supplied with fluid reservoirs. (1 mm ID, 3mm OD)</td>
		</tr>
		<tr>
			<td>Spin coater</td>
			<td>Laurell Technologies Corporation</td>
			<td>WS-650MZ-23NPPB</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereo microscope</td>
			<td>Olympus Corporation</td>
			<td>SZ61</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermistor cable</td>
			<td>Warner Instruments</td>
			<td>TA-29</td>
			<td>Cable with bead thermistor</td>
		</tr>
		<tr>
			<td>UV exposure system</td>
			<td>ABM, USA</td>
			<td>-</td>
			<td>Near UV Exposure System, 350W</td>
		</tr>
		<tr>
			<td>Vacuum pump</td>
			<td>Vacuumbrand GmbH</td>
			<td>MD1C</td>
			<td></td>
		</tr>
		<tr>
			<td>Water cooled cold plate block</td>
			<td>Koolance, Inc.</td>
			<td>PLT-UN40F</td>
			<td></td>
		</tr>
		<tr>
			<td>Water cooler</td>
			<td>Koolance, Inc.</td>
			<td>EX2-755</td>
			<td></td>
		</tr>
		<tr>
			<td>Weighing scales</td>
			<td>Sartorius</td>
			<td>M-prove</td>
			<td></td>
		</tr>
	</tbody>
</table>

a multilayer microfluidic platform for the conduction of prolonged cell-free gene expression

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.

Watch this Scientific Journal Video about A Multilayer Microfluidic Platform for the Conduction of Prolonged Cell-Free Gene Expression at JoVE.com

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

The fabrication process of a PDMS-based, multilayer, microfluidic device that allows in vitro transcription and translation (IVTT) reactions to be performed over prolonged periods is described. Furthermore, a comprehensive overview of the hardware and software required to automate and maintain these reactions for prolonged durations is provided.

The limitations of cell-based synthetic biology are becoming increasingly apparent as researchers aim to develop larger and more complex synthetic genetic ...

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