A Multiplexed Cell-Free Assay to Screen for Antimicrobial Peptides in Double Emulsion Droplets

Data underlying the figures in the publication “A Multiplexed Cell-Free Assay to Screen for Antimicrobial Peptides in Double Emulsion Droplets”, published in Angew. Chem. Int. Ed., 2022, e202114632.

https://onlinelibrary.wiley.com/doi/10.1002/anie.202114632

Table of contents:

1. Figure 1b: Bright-field image of the double emulsions droplets produced on the microfluidic chip (scale bar 40 μm).

2. Figure 1c: Source video of the image in Figure 1c. Overlaid fluorescence and bright-field image of a double emulsion in a hydrodynamic trap, containing LUVs loaded with a self-quenching concentration of SRB in the cell-free extract, showing background fluorescence (scale bar 20 μm).

3. Figure 2a: Excel file containing the experimental data for Figure 2a. Cell-free protein production. Cell-free production of sfGFP in double emulsion (DE) droplets. The expression and folding of sfGFP was confirmed by the increase of fluorescence at 516 nm (ex. 488 nm). The dashed ribbon represents standard deviation (n=150).

4. Figure 2c: Excel files containing the experimental data for Figure 2c. Mean fluorescence intensities of b) after incubation at room temperature for 16 hours. no DNA: DEs without any alpha-hemolys in plasmid DNA(n=107), α-HL:DEs with the alpha-hemolys in plasmid DNA(n=258), SDS: double emulsions without any alpha-hemolys in plasmid DNA, exposed to a solution of 0.5% SDS in buffer throughout the incubation (n=204).

5. Figure 2d: Excel file containing the experimental data for Figure 2d. Fluorophore leakage kinetics from mammalian-like LUVs with SRB and from bacteria-like LUVs with 6-FAM, induced by the cell-free expression of pneumolysin in a 384 well-plate, starting at time 0. Fractional fluorescence (fF) is calculated by setting the zero level to the vesicle fluorescence in the absence of DNA, and the maximum level of fluorescence, scaled to a value of 1, to the value obtained by lysing the vesicles with 0.5% SDS. Solid lines represent the average of three independent reactions visible below.

6. Figures 2e and 2f: FACS data for Figures 2e and 2f.

7. Figure 3a: Excel file containing the experimental data for Figure 3a.   Fluorophore leakage kinetics from mammalian-like LUVs with SRB and bacteria-like LUVs with 6-FAM, induced by the cell-free expression of meucin-25 in a 384 well-plate. Each well contained 8 nM of plasmid (Supporting Information Table 1). Solid lines represent the average of three technical replicates displayed as well (the lines are overlapping, thus not visible).

8. Figure 3c: Excel file containing the experimental data for Figure 3c.   Bacterial viability assay with increasing meucin-25 concentrations, measured by flow cytometry. Propidium iodide (PI) cannot pass intact bacterial membranes and only intercalates the DNA of permeabilized dead bacteria (“PI positive”). Constitutively expressed sfGFP proteins normally efficiently retained in intact bacterial cells (“GFPpositive”) but lost in suitably permeabilized cells. Error bars indicate standard deviation (n=10000).

9. Figure SI_2: Excel files containing the experimental data for Supplementary Figure 2.

10. Figure SI_3: Excel file containing the experimental data for Supplementary Figure 3.

11. Figure SI_4a: Excel files containing the experimental data for Supplementary Figure 4a.

12. Figure SI_4b: Excel files containing the experimental data for Supplementary Figure 4b.

13. Figure SI_5: Excel files containing the experimental data for Supplementary Figure 5.

14. Figure SI_6: Excel files containing the experimental data for Supplementary Figure 6.