1. Paper-Based Synthetic Gene Networks
Keith Pardee, Alexander A. Green, Tom Ferrante, D. Ewen Cameron, Ajay DaleyKeyser, Peng Yin, James J. Collins 
Cell 
Volume 159, Issue 4, Pages (November 2014)
DOI: /j.cell
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2. Cell  , DOI: ( /j.cell )
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3. Figure 1 Paper-Based Synthetic Gene Networks
(A) The enzymes of transcription and translation are combined with engineered gene circuits and then embedded and freeze dried onto paper to create stable and portable synthetic gene networks outside of the cell. These networks are genetically encoded tools with trigger, regulatory transducer, and output elements.
(B) GFP expression in solution phase from fresh and freeze-dried PT7 cell-free reactions.
(C) Freeze-dried pellets of the PT7 cell-free expression system are stable for over a year at room temperature, yielding GFP expression in solution phase when rehydrated.
(D) A schematic of the constitutive GFP expression constructs used on paper.
(E) Images of paper discs following a 2 hr incubation and maximum fold-change measurements of constitutive paper-based GFP expression from freeze-dried S30, S30 T7, and PT7 cell-free systems during the first 90 min of incubation.
(F) Schematic of tetO regulation for GFP and mCherry.
(G) Images of paper discs following a 2 hr incubation and maximum fold-change measurements of GFP and mCherry from the tetO-regulated promoter during the first 2 hr of incubation ± aTc inducer (11 μM) from freeze-dried S30 reactions. BF indicates bright-field images, and F indicates fluorescence images.
Error bars represent SD.
Cell  , DOI: ( /j.cell )
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4. Figure 2 Freeze-Dried RNA-Actuated Gene Circuits on Paper
(A) Schematic of the RNA-based toehold switch regulating translation of GFP.
(B) Images of paper-based GFP expression from eight toehold switches (A–H) in the PT7 cell-free system, ± complementary trigger RNAs following a 2 hr incubation.
(C) Maximum fold-change measurement of GFP expression from paper-based toehold switches A-H during the first 90 min of incubation.
(D) RNA-actuated expression of GFP, Venus, mCherry, and Cerulean fluorescent proteins from toehold switch H on paper and quartz microfiber discs following a 2 hr incubation.
(E) Bright field and fluorescence images of an orthogonality screen between toehold switches and trigger RNAs using paper-based reactions arrayed in a 384-well plate. Images collected after the overnight data collection.
(F) Quantification of fluorescence over time from paper discs containing switch G (bottom row of the array). All data were generated from freeze-dried, cell-free reactions embedded into paper with their respective gene circuits. Trigger RNA concentration used for toehold switch activation was 5 μM. BF indicates bright-field images, and F indicates fluorescence images.
Error bars represent SD.
Cell  , DOI: ( /j.cell )
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5. Figure 3 Colorimetric Output from Paper-Based Synthetic Gene Networks
(A) A schematic of the modified, LacZ-expressing toehold switches used to generate colorimetric outputs.
(B) Images of the paper-based, colorimetric output from toehold switches ± complementary RNA triggers following a 2 hr incubation.
(C) Maximum fold-change measurements from LacZ toehold switches during the first 2 hr of incubation. Fold induction based on the rate of color change from LacZ toehold switches.
(D) The paper-based development of color from LacZ toehold switch D over 60 min.
(E) Color intensities from (D) were converted to a ratio of blue over yellow (red + green channels) channels and graphed over time.
(F) Schematic describing the process of arraying synthetic gene networks on paper using printed arrays.
(G) A 25-reaction printed array (14 × 14 mm) of toehold switch E, containing positive reactions (purple) and negative control reactions (yellow) distributed in a checkerboard pattern following a 2 hr incubation.
(H) Schematic of the low-cost, electronic optical reader developed to read colorimetric output from paper-based synthetic gene networks. Paper reactions held in a chip between an LED light source (570 nm) and electronic sensor.
(I) Time-course data from the electronic optical reader of toehold switch G in the presence of 0, 30, 300 and 3,000 nM trigger RNA. Data were collected every 10 s with SD indicated by line thickness, and all data were generated from freeze-dried, cell-free reactions embedded into paper with their respective gene circuits. Trigger RNA concentrations used for toehold switch activation were 5 μM or as specified.
Error bars represent SD.
Cell  , DOI: ( /j.cell )
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6. Figure 4 Application of Paper-Based Synthetic Gene Networks
(A) Schematic of the paper-based mRNA sensors based on toehold switches.
(B) Images and fold-change measurements of a paper-based mCherry mRNA sensor in the presence and absence of full-length target mRNA, following a 2 hr incubation. GFP is produced in response to detection of mCherry mRNA.
(C) Images and fold-change measurements of a paper-based GFP mRNA sensor in the presence and absence of full-length target mRNA. mCherry is produced in response to detection of GFP mRNA.
(D) Maximum fold change during the first 90 min of the LacZ-mediated color output rate from sensors for mRNAs encoding resistance to spectinomycin, chloramphenicol, ampicillin, and kanamycin antibiotics using a plate reader.
(E) Maximum fold change during the first 90 min of the color output rate from the ampicillin resistance sensor using the in-house electronic optical reader over a titration of mRNA concentrations.
(F) Images of paper discs following a 2 hr incubation and fold-change measurement of constitutive paper-based GFP expression from a freeze-dried HeLa cell extract.
(G) Schematic of the FRET-based mechanism used in the glucose nanosensor.
(H) Using an average of the data collected every 10 min between 390 min and 490 min, the 528 nm fluorescence is reduced in response to glucose binding to the FRET-based glucose nanosensor expressed on paper. All data were generated from freeze-dried, cell-free reactions embedded into paper with their respective gene circuits.
Error bars represent SD.
Cell  , DOI: ( /j.cell )
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7. Figure 5
Rapid Prototyping of Paper-Based RNA Sensors for Sequences from Sudan and Zaire Strains of the Ebola Virus
(A) Schematic of the generation of Ebola RNA sensors. Sensors with the same letter were targeted to identical windows in the Ebola nucleoprotein mRNAs of their respective strains.
(B) Twenty-four toehold switch-based RNA sensors were constructed and tested in a 12 hr period. Based on the RNA segment windows (A–L), maximum fold change during the first 90 min at 37°C is reported for both the Sudan and Zaire strains of the virus. Fold change rate is determined from the slope of absorbance at 570 nm over time (− control, + 3000 nM RNA trigger).
(C) Composite image of the 240 paper-based reactions used to test the 24 sensors after data collection overnight. Control and untriggered toehold sensors remain yellow, and activated toehold sensors have turned purple.
(D) Sequence specificity tested for four Sudan and four Zaire sensors from the original set of 24. Each of the four sensors targeting Sudan sequences were treated with 3,000 nM of off-target RNA sequence from the complementing Zaire RNA sequence and vice versa.
(E) Fold change of the color output rate of sensors SD and ZH over a titration of RNA concentrations. All data were generated from freeze-dried, cell-free reactions embedded into paper. DNA containing the sensor and RNA triggers was added during rehydration.
Error bars represent SD.
Cell  , DOI: ( /j.cell )
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8. Figure 6 Paper-Based Converging Transcriptional Cascade
(A) Schematic of the genetically encoded components that convert transcription from E. coli RNAP into transcription from T3 RNAP and/or T7 RNAP. Expression of these new RNAPs drives the transcription of previously dormant GFP constructs containing T3 or T7 promoters, and alternatively, they can drive expression of a toehold switch and trigger pair under the regulation of T7 and T3, respectively.
(B) Images and fold-change measurements of paper-based T3 GFP expression with and without the T3 cascade module.
(C) Images and fold-change measurements of paper-based T7 GFP expression with and without the T7 cascade module.
(D) Images and fold-change measurements of paper-based T3/T7-dependent GFP expression with and without the T3 and T7 cascade modules. All data were generated from freeze-dried, cell-free reactions embedded into paper with their respective GFP expression constructs; cascade modules were added as DNA components at rehydration. Data were collected after an overnight incubation.
Error bars represent SD.
Cell  , DOI: ( /j.cell )
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9. Figure S1 Related to Figure 1
(A) GFP expression from freeze-dried pellets of S30 cell extract.
(B) GFP expression from freeze-dried pellets of S30 T7 cell extracts.
(C) Time course expression of tetO-regulated GFP on freeze-dried paper discs.
(D) Time course expression of tetO-regulated mCherry on freeze-dried paper discs.
(E) aTc induction of tetO-GFP in the absence and presence of TetR supplementation. Fold change was calculated by comparing each induced reaction to the matching uninduced reaction.
(F) aTc induction of tetO-mCherry in the absence and presence of TetR supplementation. Error bars represent SD. Fold change was calculated by comparing each induced reaction to the matching uninduced reaction. RFU, relative fluorescence units.
(G) Overview of experimental workflow that describes the order of operations for Figures 1, 2, 3, 4, 5, and 6.
Cell  , DOI: ( /j.cell )
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10. Figure S2 Related to Figure 2
(A) Paper-based regulation of GFP expression from toehold switches A – H in freeze-dried paper-based reactions. The red dots indicate the time point from which maximum fold change calculations reported in Figure 2C were taken. RNA trigger concentration 5 uM.
(B) Titration of RNA trigger for toehold switch D in solution phase reactions. Values presented are the average of data at the 360 min time point.
(C) Comparison of toehold switch induction in E. coli and on freeze-dried, paper-based reactions. Dynamic data collected from paper and E. coli-based reactions of toehold switches C, D and E.
(D) Paper-based regulation of GFP, Venus, mCherry and Cerulean fluorescent proteins by toehold switch H over a time course. The red dots indicate the time point from which fold change calculations reported in Figure S2F were taken.
(E) Images of quartz microfiber discs each embedded with three toehold switches (switch H_GFP, switch A_Cerulean and switch C_mCherry). The four discs carrying these switches were rehydrated and incubated with either water or trigger RNAs for H, A or C. The individual activation of these switches was imaged using the green, blue and red fluorescence channels.
(F) Maximum fold change measurement of Venus, mCherry and Cerulean expression from toehold switches H during the first two hours of incubation.
(G) Heat map of GFP expression during the first 60 min of the orthogonality screen for GFP expressing toehold switches on paper.
(H) Heat map of GFP expression during the first 60 min of the orthogonality screen for GFP expressing toehold switches on quartz microfiber. Values presented are the average of either triplicate or quadruplicate data. Error bars represent SD. RFU, relative fluorescence units.
Cell  , DOI: ( /j.cell )
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11. Figure S3 Related to Figure 3
(A) Regulation of LacZ toehold switch colorimetric response from freeze-dried, paper-based reactions over time. Colorimetric response was quantified as a measure of absorbance at 570 nm. The red dots indicate the time point from which the fold change LacZ rate calculations reported in Figure 3C were taken. The rate was calculated as Sn = (Tn+1-Tn)/10, where T is the normalized data at a time point (Tn) and the time point 10 min later (Tn+1), and Sn is the slope reported for Tn.
(B) Composite image of orthogonality screen of LacZ colorimetric toehold switch reactions on paper discs arrayed in a 384-well plate.
(C) Schematic of tetO regulation for chitinase.
(D) Maximum fold change of the colorimetric tetO_chitinase output at 420 min.
(E) Bright field and 410 nm absorbance images of the tetO_chitinase system embedded into paper after overnight incubation, ± aTc induction. Chitinase expression leads to the cleavage of a colorless precursor to generate a yellow product, which was quantified by monitoring absorbance at 410 nm.
(F) Time course evolution of the paper-based colorimetric reaction in Figure S3D as measured by 410 nm absorbance.
(G) Components used in the fabrication of the in-house optical electronic reader. 570 nm LED light source and luminosity sensors are coordinated to the Arduino Micro through two multiplexers.
(H) Line drawings used to cut the reader housing from black acrylic using a laser cutter. Values presented are the average of either triplicate or quadruplicate data. Error bars represent SD.
Cell  , DOI: ( /j.cell )
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12. Figure S4 Related to Figure 4
(A) Titration of full-length target mRNA for paper-based antibiotic resistance gene mRNA sensors with LacZ output. Summary of rate-based fold change for LacZ mRNA sensors for kanamycin, spectinomycin, chloramphenicol and ampicillin resistance gene (Plate reader, Abs 570 nm). Error bars represent SD.
(B) Time course data for the ampicillin resistance sensor from the in-house electronic optical reader. Values presented are the average of either triplicate or quadruplicate data.
(C) Fluorescence spectrum of FRET-based glucose nanosensor in the presence and absence of 10 mM glucose. Using freeze-dried, cell-free expression in HeLa cell extracts, the 528 nm fluorescence peak of the nanosensor in solution is suppressed in the presence of glucose as previously reported (Takanaga and Frommer, 2010). RFU, relative fluorescence units.
Cell  , DOI: ( /j.cell )
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13. Figure S5
Pretreating Matrix Materials with Blocking Agents Improves the Efficiency of GFP Expression, Related to Experimental Procedures
(A) Toehold switch H_GFP reactions on 2 mm paper discs that have been blocked with 5% BSA, 5% Triton X-100, 5% PEG 8k, 5% Tween-20 or washed with water.
(B–D) (B) Toehold switch B_GFP on paper discs treated as above, (C) Toehold switch H_GFP on 2 mm quartz microfiber discs treated as above and (D) Toehold switch B_GFP on quartz microfiber discs treated as above.
Cell  , DOI: ( /j.cell )
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