Dataset DOI: 10.5061/dryad.xksn02vws

Description of the data and file structure

File: Cell_free_paper_Final_Processed_Data_reformatted.xlsx 

Our datasets, comprised of many recombinased-based genetic circuits designed to operate in a cell-free environment, support the 6 major data figures in our manuscript.

Figure 1: Schematic diagrams of cell-free recombinase circuits

Figure 2:   Characterization of cell-free recombinase excision circuits

Figure 3:  Characterization of activation TF-implemented recombinase genetic circuits in a cell-free condition

Figure 4:  2-input-2-output circuits for sensing two chemicals in the cell-free condition.  The two chemicals are Zn ion and tetracycline (tet).  Zn controls the expression of PhiC recombinase, whereas Tet controls the expression of Cre.

Figure 5: 2-input-4-output genetic decoder performance in cell free environment

Figure 6: Paper-based recombinase circuits and memory recording platform

Figure 7: Paper-based multi-input multi-output circuits

All of the data used to generate the figures can be found in Cell free paper final processed data reformatted.xlsx.  Each tab in the Excel file corresponds to a sub-figure, labeled with the tab name.

(For vast majority of the data) Every plot except for Figure 6, 7A, and Supplementary Figure 4C was generated using a plate reader, either measuring fluorescence or nanoluc (nluc) luminescence produced by the various genetic circuits under cell-free conditions. The Unit for fluorescence is an arbitrary unit (AU).  The unit for luminescence is also AU.  The final unit plotted is an "normalized translational output", which is detailed below. For plate reader measurements, each data cell in the Excel sheet represents a fluorescence reading from a well on a 96-well plate. Each column is labeled at the top for the first 2 cells, except for Fig 2B, 3D, 6B, Supp 1B, and Supp 8B, where only the 1 cell contains the label.  All tabulated data contains appropriate row and column identifiers used in the manuscript figures. For a detailed description and diagram of the genetic circuits, please refer to the manuscript.

Concentrations of plasmid DNA, linear DNA, purified proteins, and chemical inducers used in the cell-free reaction are listed in the Tables S5–S31.

Days in Figure 6B refer to days after rehydration of the paper-based circuit.

For Supp Fig 2, the rows and columns are labeled to correspond to the matrix table shown in Supp Fig 2.

Figures 6 and 7A were generated from manually counting colonies on agar plates.  It is expressed as a percentage of colonies considered positive.

Supplementary Figure 4C contains the calculation for generating the Area under curve (AUC) plots, using the data in Supplementary Figure 4A.

Plate reader measurement

NEB PURExpress In Vitro Protein Synthesis Kit (New England Biolabs, E6800S) supplemented with RNase Inhibitor (Sigma-Aldrich, 3335402001) was used to generate data for cell-free experiments based on the manufacturer’s protocol. The concentrations of plasmids and linear DNAs are listed in the Supplement. The plasmids, DNAs, aTFs, and small molecules are first premixed before combining with the cell-free reaction mix. Then, 5μL of cell-free circuits were incubated in each well of a 384-well clear bottom black plate (Corning, 3542) at 37°C for 5hr. An endpoint measurement, or dynamic measurement, was performed on a plate reader (SpectraMax M5, Molecular Devices) with an excitation wavelength of 580nm and emission wavelength of 611nm for mCherry, an excitation wavelength of 485nm and emission wavelength of 525nm for GFP, and an excitation wavelength of 381nm and emission wavelength of 445nm for BFP. Nano-Glo Luciferase Assay (Promega, N1110) was used for NLuc luminescence detection. Nano-Glo Luciferase Assay Substrate was diluted 1:50 by Nano-Flo Luciferase Assay Buffer. The luminescence level was measured immediately after a volume of the diluted substrate equal to 6X the volume of the cell-free reaction was added to each well.

Plate reader quantification and MEF standardization

A Texas Red Dye (Invitrogen, D1828) was used to convert arbitrary mCherry fluorescence intensity into nanomolar equivalent Texas Red Dye (labeled as normalized translational expression). A serial dilution was prepared with PBS from a 500 μM stock solution. The samples were prepared with triplicates, and fluorescence values were measured at an excitation wavelength of 580nm and emission wavelength of 611nm on a plate reader (SpectraMax M5, Molecular Devices). Fluorescence for a concentration in which a single replicate saturated at the plate reader was excluded from the analysis. The rest of the measurements were averaged and formed a linear regression line, which is used to convert the measured arbitrary fluorescence value to the concentration of the Texas Red Dye. Standard curves were created with the same process for each plate reader for data collection to normalize the results. The same process was used to convert GFP fluorescence intensity into nanomolar equivalent Fluorescein (Sigma-Aldrich, F6377) and NanoLuc luminescence intensity into nanomolar equivalent NanoLuc purified protein (Promega, E499A). All fluorescence and luminescence outputs are normalized to the nanomolar equivalent of corresponding dye or proteins and then present on the figure as “Normalized Translational Output (NTR).”

Paper-based recombinase circuits

Paper discs with lyophilized CRIBOS system were placed into the wells of a 384-well clear-bottom black plate (Corning, 3542). On Day 0, 2 μL of pre-warmed autoclaved DI water was added directly to the paper disc in each well to activate the cell-free reaction. The 384-well plate was then kept at room temperature with the plate lid covered. At different time points post reaction activation, DNA stored on the paper discs was eluted using 5 μL of pre-warmed miniprep elution buffer (Epoch Life Sciences, 2160250). The eluted DNA was subsequently diluted 1:2 with an equal volume of elution buffer. To retrieve biological information, 2.5 μL of the diluted DNA was used to transform 50 μL of bacteria.