Paper-based synthetic gene networks

Abstract

Synthetic gene networks have wide-ranging uses in reprogramming and rewiring organisms. To date, there has not been a way to harness the vast potential of these networks beyond the constraints of a laboratory or in vivo environment. Here, we present an in vitro paper-based platform that provides an alternate, versatile venue for synthetic biologists to operate and a much-needed medium for the safe deployment of engineered gene circuits beyond the lab. Commercially available cell-free systems are freeze dried onto paper, enabling the inexpensive, sterile, and abiotic distribution of synthetic-biology-based technologies for the clinic, global health, industry, research, and education. For field use, we create circuits with colorimetric outputs for detection by eye and fabricate a low-cost, electronic optical interface. We demonstrate this technology with small-molecule and RNA actuation of genetic switches, rapid prototyping of complex gene circuits, and programmable in vitro diagnostics, including glucose sensors and strain-specific Ebola virus sensors.

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 two hour 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 minutes of incubation. (F) Schematic of tetO regulation for GFP and mCherry. (G) Images of paper discs following a two hour incubation, and maximum fold-change measurements of GFP and mCherry from the tetO-regulated promoter during the first two hours of incubation, +/ − aTc inducer (11 μM), from freeze-dried S30 reactions. BF indicates bright field images and F indicates fluorescence images.

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 two hour incubation. (C) Maximum fold-change measurement of GFP expression from paper-based toehold switches A–H during the first 90 minutes 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 two hour 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.

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 two hour incubation. (C) Maximum fold-change measurements from LacZ toehold switches during the first two hours 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 minutes. (E) Color intensities from panel 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 two hour 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 3000 nM trigger RNA. Data were collected every 10 seconds, with standard deviation 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.

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 two hour 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 minutes 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 minutes 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 two hour 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 minutes between 390 min – 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.

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 hour period. Based on the RNA segment windows (A–L), maximum fold change during the first 90 minutes 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 3000 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 were added during rehydration.

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 drive 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.