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

Prototyping synthetic gene networks is greatly improved by the use of cell-free gene expression systems. Our protocol describes the fabrication process for multilayer microfluidic flow reactor and shows that such a device can be used for prolonged cell-free expression of GFP. Cell-free protein expressions in combination with microfluidic flow reactors provide the rapid prototyping platform for the design of synthetic biological devices.

This entire setup is highly versatile and can be adapted for the conduction of any biochemical or chemical reactions requiring high levels of control. Ensuring the stability of the cell-free reaction solution is critical and difficult to achieve. Be sure to focus on providing sufficient cooling and utilize the tubing specified in this protocol.

Fabricating and preparing the experimental platform requires the connection of numerous individual components, a process which can be difficult to follow without visual aids. To begin, follow along in the accompanying text protocol to fabricate the microfluidic device and to set up the pneumatic control and flow pressure regulation which will be used for controlling the valves critical to this device. To set up the off-chip cooling, begin by coiling a length of PTFE tubing onto the cold face of a Peltier element and secure the coil with tape.

Ensure that one end of the PTFE tubing is connected to reservoirs of the flow layer pressure control system and that the other end protrudes no more than one centimeter from the Peltier surface. Next insert a five to 10 centimeter length of PEEK tubing into the protruding end of the PTFE tubing. Apply a sufficient amount of thermal compound onto the hot face of the Peltier element and place it onto the cold plate of the water block.

Ensure that the tubing, Peltier element, and cooling block are in direct contact with one another at all times. Next connect the Peltier element to the temperature controller using a serial bus connector so that the voltage supplied to the Peltier can be regulated. Then, securely place a thermistor on the Peltier surface and connect its output to the temperature controller.

After turning on the water cooler, adapt the voltage supplied to the Peltier until the temperature is stable at four degrees Celsius. For each control channel of the microfluidic device cut a length of standard tubing that is one meter long. At one end, insert the pin of a 23 gauge, one half inch Luer stub and at the other end insert a stainless steel connecting pin.

Connect the Luer stub to a male Luer 3/32 inch barbed nylon connector and insert the barb of the connector into a length of polyurethane tubing. Then insert this polyurethane tubing directly into one of the solenoid valves. Next attach a 23 gauge half inch Luer stub to a syringe and insert this into a three to four centimeter long piece of standard tubing.

Place the open end of this tubing into a reservoir of ultra pure water and fill the syringe with ultra pure water. Number each control channel of the microfluidic device as shown here. For channels four through 29, find the corresponding tubing and insert the metal pin into the open end of the tubing attached to the syringe.

Then inject water into the control channel tubing until half the length has been filled. Next, disconnect the tubing from the syringe and insert the stainless steel connector pin into the corresponding hole of the microfluidic device. Repeat this step for all of the control channels.

Now, use the control interface to open all of the solenoid valves. This will pressurize the fluid within the control channel tubing forcing it into the microfluidic device and closing all of the membrane-based valves within the device. For each of the uncooled reagents cut a meter length of standard tubing to connect the reservoir outlet to the microfluidic device inlets.

Take one end of the tubing and insert this into the reservoir ensuring that the tubing reaches the base of the reservoir. The reservoir tubing outlet should be tightened so that an airtight seal is achieved. Then, insert a stainless connection pin into the open end of the tubing.

Next, attach a 23 gauge half inch Luer stub to the end of a one milliliter syringe. Add a short length of standard tubing to the Luer stub. Place the end of the tubing into the desired reagent solution and fill the syringe with the reagent.

Then insert the stainless steel connector pin into the polyurethane tubing connected to the syringe and fill the tubing with the reagent. When using small reaction volumes, the reagent will not enter the reservoir and the tubing itself will act as the reservoir. When finished, disconnect the syringe and insert the connector pin into one of the flow layer inlet holes of the microfluidic device.

Then apply pressure to each of the reservoirs using the pressure regulator software to force the reagents into the microfluidic device. Ensure that the water cooler and Peltier elements have been turned on with the surface temperature of the Peltier set to four degrees Celsius. Mount the cooling setup as close to the microfluidic device as possible to minimize the uncooled volume between the Peltier and the device inlet.

Then connect the one the one milliliter syringe to a 23 gauge half inch Luer stub with a short length of standard tubing attached to the end. Draw in the to be cooled reagent to fill the syringe. Next connect the PEEK tubing to the syringe via the connective tubing and apply constant pressure to the syringe forcing the reagent through the PEEK tubing and into the PTFE tubing.

Finally disconnect the PEEK tubing from the syringe and insert it directly into of the flow channel inlets of the microfluidic device. When pressure is applied, the cooled reagent will be forced into the microfluidic device. Ensure the microfluidic device is secure on the microscope stage with all control and flow layer tubing attached and close any openings on the incubator.

Next set the ambient temperature of the incubator to 29 degrees Celsius. Then insure that the cooling system has been turned on and is set to four degrees Celsius prior to initiating the experiment. Check that the pressures applied to the flow channel pressure regulator is set to 800 millibar and using the software, set the output pressure of each individual flow channel to between 20 and 100 millibar.

Check that pressures applied to the control channel solenoid valves is one bar for channels one through eight and three bar for channels nine through 29. Next, close the outlet of the device by pressurizing channel 29 and simultaneously depressurize control channels one through three and 15 through 28. Then selectively depressurize the control channels of the multiplexer to allow a single selected reagent to flow into the device.

Use the microscope to monitor the removal of air and subsequently ensure that all reagents flow correctly without introducing air bubbles. Using the provided software package, set up the data fields related to the calibration process as described in the accompanying text protocol. Subsequently determine the fluid volume displaced from each reactor during a single inflow step by executing the calibration protocol.

Follow along with the steps presented by the control software to complete the analysis of the calibration experiment and to determine the refresh ratio of each ring reactor in the microfluidic device. Finally, set the required values for the desired experiment within the virtual control interface. Initiate the experimental protocol by pressing the perform experiment button in the control interface.

During a calibration experiment the reactors are filled with a fluorophore whose intensity is recorded after each dilution. The decrease in the fluorescence intensity per dilution reveals the volume of the reactor ring displaced for the set number of inflow steps. This volume is termed the refresh ratio.

The average refresh ratio and standard deviation is shown for each dilution step in red. Seven of the eight reactors show very similar behavior, however one reactor shows fluctuations after the seventh dilution cycle. This highlights the need for unique refresh ratios for each of the reactors.

The prolonged in vitro transcript and translation reaction shown here had 30%of the reactor volume displaced every 14.6 minutes. Two reactors of the microfluidic device where used as blanks. All the other reactors comprised 75%in vitro transcription and translation reaction solution and 25%of either ultra pure water or 2.5 nanomolar linear DNA templates coding for the expression of the deGFP protein.

In all four reactors where DNA was added, there was clear deGFP expression. One reactor displays a lower fluorescence signal. This could be caused by disparity in flow resulting in less DNA entering the reactor or due to variations in the reactor dimensions.

After 14 hours, a sudden increase is seen in the signal of the reactors containing DNA. This is caused by an air bubble entering the flow layer of the microfluidic device. Upon resumption of flow, the experiment returns to its previous fluorescence intensity.

Cell-free reaction solutions are subject to degradation over time unless they are sufficiently cooled, make the cooling process described in this protocol of critical importance. Due to the versatility of the platform, a variety of biochemical and chemical reactions can be conducted in a precise and controlled manner. The application of microfluidic flow reactors has accelerated successive design build test cycles for prototyping double genetic circuits.