Supplementary Material
Optimization of a Miniaturized Fluid Array Device for Cell-Free Protein Synthesis
Kirsten Jackson1, Shouguang Jin2, and Z. Hugh Fan1,3,4*
1
J. Crayton Pruitt Family Department of Biomedical Engineering,
University of Florida, P.O. Box 116131, Gainesville, FL 32611, USA
2
Department of Molecular Genetics and Microbiology,
University of Florida, P.O. Box 100266, Gainesville, FL 32610, USA
3
Interdisciplinary Microsystems Group, Department of Mechanical and Aerospace Engineering,
University of Florida, P.O. Box 116250, Gainesville, FL, 32611, USA
4
Department of Chemistry,
University of Florida, P.O. Box 117200, Gainesville, FL 32611, USA
*Author to whom correspondence should be addressed at hfan@ufl.edu.
Supplementary Material S1: Vertically-Oriented µFAD Fabrication
In short, the 12 reaction chamber pieces and 13 feeding chamber pieces were milled from a 6.35
mm thick sheet of black polycarbonate (McMaster-Carr, Atlanta, GA, USA). Note that the
µFAD used for luciferase expression was fabricated with white polycarbonate to reflect light and
enhance luminescent signal output. Dialysis membranes with a molecular weight cutoff of 6-8
kilo Daltons (kDa) (Spectrum Labs, Rancho Dominguez, CA, USA) were cut using a cutting
plotter (Craft ROBO Pro, Graphtec, Irvine, CA, USA) and adhered to the polycarbonate pieces
using polydimethylsiloxane (PDMS) (Sylgard 184, Dow Corning, Midland, MI, USA) through a
microstamping technique (Chueh et al., 2007). The polycarbonate pieces and dialysis membranes
were assembled such that the reaction chamber pieces were sandwiched by dialysis membranes
in a clamping frame. The assembled device was then placed in a 70°C oven for four hours to cure
the PDMS and then left at room temperature overnight. Finally, the device was removed from
the clamping frame and placed in a device holder made from milled black polycarbonate.
Supplementary Material S2: Wheat Germ Lysate Reaction and Feeding Solution
Compositions
The reaction solution was prepared by combining 15 µl of wheat germ lysate, 15 µl reaction mix,
4 µl of amino acids, and 1 µl of methionine with 15 µl of the DNA vector (2 µg) in nuclease-free
water. The feeding solution was prepared by mixing 900 µl of feeding mix with 80 µl of amino
acids and 20 µl of methionine.
Supplementary Material S3: PURE System Reaction and Feeding Solution Reagent
Concentrations
The reaction solution consists of the published reagents concentrations of 2.7 μM IF1, 0.40 μM
IF2, 1.5 μM IF3, 0.26 μM EF-G, 0.92 μM EF-Tu, 0.66 μM EF-Ts, 0.25 μM RF1, 0.24 μM RF2,
0.17 μM RF3, 0.50 μM RRF, 1900 U/ml AlaRS, 2500 U/ml ArgRS, 20 mg/ml AsnRs, 2500
U/ml AspRs, 630 U/ml CysRs, 1300 U/ml GlnRs, 1900 U/ml GluRs, 5000 U/ml GlyRs, 630
U/ml HisRs, 2500 U/ml IleRS, 3800 U/ml LeuRS, 3800 U/ml LysRS, 6300 U/ml MetRS, 1300
U/ml of PheRS, 1300 U/ml ProRS, 1900 U/ml SerRS, 1300 U/ml ThrRS, 630 U/ml TrpRS, 630
U/ml TyrRS, 3100 U/ml ValRS, 4500 U/ml MTF, 1.2 μM ribosomes, 4.0 μg/ml creatine kinase,
3.0 μg/ml myokinase, 1.1 μg/ml nucleoside-diphosphate kinase, 2.0 units/ml pyrophosphatase,
10 μg/ml T7 RNA polymerase, 2 mM ATP, 2 mM GTP, 1 mM CTP, 1 mM UTP, 20 mM
creatine phosphate, 50 mM Hepes-KOH (pH 7.6), 100 mM potassium glutamate, 13 mM
magnesium acetate, 2 mM spermidine, 1 mM dithiothreitol (DTT), 0.3 mM 20 amino acids, 10
µg/ml 10-formyl-5,6,7,8-tetrahydrofolic acid, 56 A260/ml tRNA mix, and 10 ng/µl DNA vector
(Shimizu et al., 2005; Shimizu and Ueda, 2010). The feeding solution has the previously
optimized reagent concentrations of 4 mM ATP, 4 mM GTP, 1 mM CTP, 1 mM UTP, 20 mM
creatine phosphate, 50 mM Hepes-KOH (pH 7.6), 100 mM potassium glutamate, 17 mM
magnesium acetate, 2 mM spermidine, 1 mM DTT, 0.5 mM 20 amino acids, and 10 µg/ml 10formyl-5,6,7,8-tetrahydrofolic acid (Jackson et al., 2014; Shimizu et al., 2005; Shimizu and
Ueda, 2010).
Supplementary Material S4: pIVEX 2.4d-tPA Vector Design and Construction
The primer sequences for PCR are 5’AGGGAGCGGCCGCATGTCTTACCAAGTGATCTGCAGAGATG-3’ and 5’TCGTTCGGATCCTCACGGTCGCATGTTGTCACGAATCC-3’ (Integrated DNA
Technologies, Coralville, IA, USA). The PCR product was cloned into the pIVEX 2.4d vector (5
Prime) using the Not1 and BamH1 restriction enzyme sites. The tPA vector was verified through
restriction enzyme digestion and agarose gel electrophoresis, and the tPA gene sequence was
confirmed by Sanger Sequencing. The gene for tPA consists of 1581 base pairs and codes for the
complete 527 amino acid tPA protein.
Supplementary Material S5: Materials and Experimental Conditions for Studies on
Experimental Parameters
For studies on the effects of experimental parameters on protein expression, control experiments
involved the µFAD being placed in room temperature (20°C) for four hours. With feeding
solution stirring, micro magnetic stirring bars with a length of 2 mm and a width of 2 mm (Flea
Micro Spinbar Magnetic Stirring Bars, Bel-Art Scienceware, Wayne, NJ, USA) were placed in
the feeding solution chambers, and the device was placed on a stirring plate with a stirring speed
of 150 RPM. To achieve an ambient temperature of 37°C for studying temperature effects, the
device was placed in an incubator for the entire incubation time.
Supplementary Material S6: Protein Detection in the Microplate Reader
GUS was detected by adding 30 µl of 100 µM 4-methylumbelliferyl-ß-D-glucuronide (MUGIcU,
Marker Gene Technologies, Eugene, OR), followed by 15 min of incubation and fluorescence
measurement using excitation and emission filters of 355 and 460 nm, respectively. Expressed
luciferase was detected by injecting 30 µl of luciferase assay reagent (LAR, Promega) using the
microplate reader, shaking the device for 2 sec, and measuring luminescence for 10 sec. GFP
was detected fluorescently using excitation and emission filters of 485 and 535 nm, respectively.
The presence of LacZ was determined by adding 30 µl of 200 µM fluorescein mono-ß-Dgalactopyranoside (FMGal) (Marker Gene Technologies), incubating for 15 minutes, and
measuring the resulting fluorescence signal with excitation and emission filters of 485 and 535
nm, respectively. tPA was detected using a fluorescent functional activity kit by adding 30 µl of
tPA substrate (SensoLyte AMC tPA Activity Assay Kit, AnaSpec, Fremont, CA, USA),
incubating for 10 min, and measuring the fluorescence signal with excitation and emission filters
of 355 and 460 nm, respectively. Additionally, expressed tPA was confirmed by His-tag
purification (His-Spin Protein Miniprep, Zymo Research, Irvine, CA, USA) according to the
manufacturer’s instructions and sodium dodecyl sulfate polyacrylamide gel electrophoresis
(SDS-PAGE).
Supplementary Figure 1. Change in detected GFP concentration within the reaction and
feeding (inset) solutions over time. At each time point, the fluorescence signal of the reaction
and feeding solutions were monitored by removing 10 µl of each solution. The solutions were
replenished with an equal volume of 112.5 µg/ml of GFP for the reaction solution and nucleasefree water for the feeding solution to maintain chamber volumes. The detected GFP
concentrations at each time point were adjusted according to the serial dilutions.
Supplementary Figure 2. Schematic of the microstamping technique utilized towards the
assembly of the µFAD. In the first (1) step, PDMS is prepared according to the manufacturer’s
instructions and spun onto a flat, sacrificial layer in a spin coater. Next (2), a thin layer of PDMS
is transferred to the feeding chamber device piece by stamping the surface into the PDMS. The
feeding chamber piece is then removed, and one of the surfaces of the reaction chamber device
piece is brought into contact with the PDMS layer (3). Finally (4), the feeding and reaction
chamber pieces are assembled by sandwiching a dialysis membrane between the pieces with the
PDMS-covered surfaces interfacing with the dialysis membrane.
Supplementary Figure 3. Calibration curve and trendline for purified recombinant GUS. A
simple linear regression was then performed to determine the equation of the trendline and
coefficient of determination (R2 value) for the resulting data.
Supplementary Figure 4. Calibration curve and trendline for purified recombinant tPA. A
simple linear regression was then performed to determine the equation of the trendline and
coefficient of determination (R2 value) for the resulting data.
Supplementary Figure 5. SDS-PAGE results for cell-free synthesized tPA. The three lanes
include a molecular weight ladder (L) with weight units of kDA, the negative control (-)
consisting of the CFPS solution devoid of tPA following His-tag purification, and the positive
control (+) of tPA from the CFPS system following His-tag purification. For this gel, 300 µl of
reaction solution was purified to 100 µl. The band for the expressed tPA is identified by the red
arrow.