Sealable Femtoliter Chamber Arrays for Cell-free Biology

The overall goal of this procedure is to confine cell-free protein synthesis reactions in cellular relevant volumes. This is accomplished by first fabricating A-P-D-M-S device using layered soft lithography techniques. Then a cell-free protein expression reaction is mixed, introduced into the device, and an ensemble of isolated reactions are formed within an array of micro fabricated chambers.

Next, the device's imaged using time-lapse fluorescent microscopy. The final step is measuring the fluorescent intensity of each well over time. Ultimately, these fluorescence traces are used to show protein expression noise in confined cell-free reactions.

The main advantage of this technique over existing methods, such as studying noise in live cells is that synthetic systems may be easily probed for conditions such as confinement, which can't be easily controlled in living systems. To begin the microfabrication process, first make a 20 to one weight ratio of PDMS mixture by measuring 10 grams of PDMS base and 0.5 grams of curing agent in a small plastic dish. In another dish, weigh a five to one PDMS mixture for an amount enough to fill a control valve.

Master mold up to one centimeter thick. Mix both parts evenly with a spatula and place the dishes inside a vacuum Desiccate for 20 to 30 minutes to degas all bubbles from the mixture while degassing. Place the control valve master mold into a glass container.

Pour the Degas five to one PDMS mixture onto the control valve mold. Transfer the glass container into the vacuum desiccate and degas the PDMS for another 20 to 30 minutes. Next, place the membrane master onto a spin coder, Chuck and carefully pour an aliquot of the soft 20 to one PDMS onto the wafer.

Spin the wafer for 45 seconds to form a thin coat of PDMS with an approximate thickness of 30 to 60 microns after degassing. The control valve master partially cure both the spin coated PDMS wafer and the control valve PDMS in an oven at 80 degrees Celsius for six minutes and 15 minutes respectively. Inspect the extent of curing by gently pressing the PDMS block with tweezers or a spatula.

If under cured, extend the incubation time for a few more minutes. At 80 degrees Celsius, a partially cured PDMS block should hold its form When pressed with a combination of a scalpel and a pair of tweezers, carefully cut and separate the PDMS blocks from the control valve master, then punch the inlet holes through the PDMS blocks using a 0.75 millimeter core punch. Clear all intra channel debris by inserting a blunt ended 23 gauge needle inside each hole, and remove exterior debris on the PDMS surface with cellophane tape.

To prepare the surfaces for PDMS to PDMS bonding, place the spin coated PDMS wafer onto the microscope stage. Move the stage and locate the PDMS bonding alignment marks on the wafer to ease the complexity of the subsequent manual alignment process. These visual alignment aid features should be pre-designed to be adjacent to the chips reaction chambers.

Once the alignment marks on the spin coated wafer have been located, use crossover tweezers to carefully place the control valve PDMS block over the spin coated wafer with slow and steady movements. Locate the corresponding alignment marks on the top control valve PDMS block. When the top PDMS block is optically aligned with the corresponding alignment features on the spin coated wafer, gently press the two PDMS surfaces into contact with one another.

Inspect the aligned structure under the microscope to ensure both the reaction chambers and the inlet channels are visible inside the rectangular control valve region. Repeat the alignment and interfacial contact procedure for other device components. After all components have been aligned and upon passing visual inspection, place the PDMS components into an oven and cure at 80 degrees Celsius for two hours.

This incubation step will form a permanent P-D-M-S-P-D-M-S bonding interface afterwards. Take the bonded PDMS structure out of the oven and cool to room temperature on the bench. Trim the PDMS structure and pull the bonded PDMS chip away from the silicone master using a 0.75 millimeter core punch.

Punch the microfluidic inlet and outlet holes. Remove all intra port debris with a blunt 23 gauge needle for the final stages of the chip making process. Place the PDMS structure and a clean number zero glass cover slip inside the plasma chamber with the PDMS membrane and the glass bonding interfaces both facing upwards plasma.

Treat both items at 10.5 watts for 20 seconds with air as the gas source. Immediately remove the glass cover slip and the PDMS block from the plasma cleaner. After treatment, carefully flip the PDMS block over and bring the PDMS membrane surface into contact with the plasma treated side of the cover slip.

Finally, cure the completed device at 80 degrees Celsius for two hours to create a permanent glass PDMS bonding interface. Prior to the experiment, hydrate the PDMS device by either boiling it in deionized water for hour or soaking the device overnight in sterile room temperature water. In addition, equilibrate the temperature of the inverted microscopes environmental chamber at 30 degrees Celsius.

Next mount the hydrated PDMS chip onto the microscope stage holder with cellophane tape to prevent excessive chip dehydration during the incubation steps of the experiment. Wrap the edges of the device with wet tissue paper to actuate the PDMS control valves and to control the sample loading process inside the main reaction. Channel two closed loop voltage pressure transducers should be used to modulate the nitrogen gas pressure for each process.

Furthermore, a combination of 24 gauge tubings and male to male lure lock adapters should be used to simplify the fluidic connections between the transducers and the chip to construct the control valve fluidics connections. First to dedicate one pressure transducer for this task, starting from the transducer end, the first portion of the fluidic path consists of a nons symmetric 24 gauge lure tubing connected to a male to male lure adapter, which is then connected to a sharp 23 gauge needle, which is inserted into a four milliliter glass vial water reservoir fitted with a septum cap. Next, the second portion of the control valve path consists of a sharp 23 gauge needle, followed by a male to male lure adapter, which is connected to a symmetric 24 gauge tubing and trailed with a set of male to male lure adapters attached to a blunt 23 gauge needle.

To complete the control valve assembly, the sharp 23 gauge needle is inserted into the water reservoir, whereas the blunt needle terminus will be inserted into the control valve inlet of the PDMS device. Switching to the reaction chamber inlet setup, the main sample loop connector set consists of nons symmetric 24 gauge tubing attached to a male to male lure adapter, followed by a blunt 23 gauge needle inserted into 24 gauge tubing and terminated with a 23 gauge blunt needle directly inserted into the tube without a lure lock, the proximal female lure end will be attached to either the syringe or a second pressure transducer during the sample loading phase of the protocol. Whereas the distal blunt needle terminus will be connected to the reaction chamber inlet of the PDMS device using a cell-free protein expression system or CFPS for short, follow the manufacturer's protocol and prepare all necessary reagents on ice or in an ice block for this microfluidic system.

A typical CFPS reaction volume for constitutive GFP expression in e coli whole cell extracts is around 25 microliters in each assay. The DNA plasmid should be added last to prevent premature expression of fluorescent proteins. Sample loading onto the microfluidic chip consists of five main steps.

To start, attach a one milliliter syringe to the female lure terminus of the sample loop connector. Set Next, insert the blunt needle terminus of the connector set into a tube containing the CFPS reaction and draw the fully assembled reaction mixture into the tubing when the entire reaction mixture has been loaded into the sample loop. Withdraw the blunt needle terminus from the micro centrifuge tube and insert the needle into the reaction chamber inlet of the chip on the fourth step.

Disconnect the syringe from the sample loop and attach the liberated female lure terminus of the sample loop to a male lure adapter set that is connected to a second pressure transducer. Finally activate the second pressure transducer to initiate sample loading. When the microfluidic channel has been filled with the CFPS reactants, remove the blunt needle from the PDMS inlet port.

Now insert the blunt needle connected to the first pressure transducer and water reservoir into the control valve channel inlet. After sample loading, open the environmental chamber and fix the PDMS device onto the microscope stage. Locate the reaction chambers with a 100 x oil immersion objective.

Under bright field illumination, actuate the control valve pressure transducer to 20 PSI. Visual evidence of valve actuation can be seen by observing changes in contrast in the PDMS layer. Adjust the focus such that the focal plane is at the bottom PDMS class interface of the device.

Finally, set the data acquisition frequency to one image per minute and begin fluorescent microscopy. Continue to collect data until the protein synthesis in all wells has reached a steady state fluorescence. At the moment of the first image acquisition, check the lab timer and record the time elapsed since the introduction of DNA into the reaction After performing fluorescent microscopy, begin the data analysis process by opening all photographs with an image processing software such as image J.Select the interior of the reaction chambers as the region of interest or ROI For all images, the total fluorescence from the selection will be the raw fluorescence intensity trace.

Using the ROI manager choose regions of interest around the interior region of each reaction chamber in the toolbox, set the auto ROI properties to areas corresponding to the interior of each reaction chamber. Then check, add-on, click and select all chambers on the screen. Finally, highlight all ROIs.

In the ROI manager. Use the multi mesure function to determine the mean fluorescent intensity of each ROI throughout the entire image stack. With standard PDMS fabrication techniques, the GFP synthesis within each femto liter well is completely isolated from its immediate environment and can be individually interrogated by imaging and photobleaching techniques, all by user-defined actuation of the PDMS control valve.

With a vast array of wells each approaching the intracellular volume of a single bacterium, the stochastic nature of gene expression on a single cell level can be assayed by quantitation of intra well fluorescence as a function of time. This can be compared to the same cell-free protein expression assay performed at a larger reaction volume of 15 microliters where the stochastic nature of the expression system is hidden behind the ensemble averaging of numerous fluorescence signals comparing the protein expression profile between an ensemble of live bacteria versus individual single well cell-free reactions. Although the fluctuation magnitudes are similar, auto correlation decays more quickly in the cell-free expression profiles.

The auto correlation time in the cells is controlled by growth and dilution of the GFP growth doesn't happen in the cell-free systems. And instead, auto correlation time is controlled by decay of the protein synthesis rate. After watching this video, you should have a good understanding how to use synthetic micro fabricated platforms as models to better understand how crowding and confinement can impact biological processes.