DOI: 10.5061/dryad.573n5tbmt
title: Programmable assembly of mechanically robust and functional polymer–spore biocomposites in organic solvent

description: Microbial biocomposites offer genetically programmable and regenerative
 functionality, but their mechanical tunability remains limited by the mild
 conditions required for biological activity and viability. Here, we report
 the programmable self-assembly of Bacillus subtilis spores with
 benzalcyanoacetate (BCA)-functionalized polymers to form robust composites
 exhibiting tunable viscoelastic and tensile properties. Surface-exposed
 cysteines on the spore coat react with BCA motifs, forming dynamic
 thia-Michael networks with Young’s moduli of &gt;100 MPa. Systematic
 variation of BCA reactivity and comonomer-dependent polymer dynamics
 enabled control over stiffness, stress-relaxation behavior, microscale
 morphology, and covalent biocontainment. Incorporation of engineered
 spores confers catalytic function that can be regenerated following
 solvent-triggered disassembly. This work establishes a modular platform
 for constructing biocomposites that are both mechanically and genetically
 programmable, bridging the synthetic and biological domains through
 molecularly defined interfaces.
description: # Programmable assembly of mechanically robust and functional
 polymer–spore biocomposites in organic solvent Dataset DOI:
 [10.5061/dryad.573n5tbmt](10.5061/dryad.573n5tbmt) ## Description of the
 data and file structure The dataset folder contains tabular data and TIFF
 files that are organized into folders that correspond to each figure set
 found in the journal article and supporting information. For example, the
 folder named **Figure 1** contains data that was used to create Figure 1.
 Below are the background and table of contents pertaining to each dataset
 folder. More detailed descriptions of experimental methods can be found in
 the main text and supporting information. ### Files and variables ###
 File: Dryad.zip #### **Folder: Figure 1** **Description:** Data in Figure
 1 were collected to investigate whether the key chemical motif
 benzalcyanoacetate (BCA) can covalently bind to cysteine residues on spore
 surface proteins.  Fluorescent nitrobenzoxadiazole–BCA (NBD–BCA) probes
 were synthesized and incubated with Bacillus subtilis spores in dimethyl
 sulfoxide (DMSO, 120 µM) at 25 °C and 800 rpm for 20 h to examine whether
 thia-Michael (tM) linkages can be formed on the spore surface proteins. A
 control probe without BCA, NBD–OH, was also incubated with spores as a
 control. Aryl substituents (R = CF~3~, H, OMe) on the BCA were varied to
 test the hypothesis that the extent of spore labeling can be adjusted
 based on the electronic profile of the BCA. TIFF files correspond to the
 fluorescence microscopy images of spores after incubation with the probes
 and washing with DMSO and were set to the same brightness contrast setting
 to visualize the relative differences in spore surface fluorescence that
 should represent relative spore labeling differences between different
 probes. Normalized fluorescence was calculated by subtracting  (bulk
 fluorescence of spore samples - supernatant fluorescence) / (OD600 of
 spore samples - OD600 of supernatant), of triplicate spore samples are
 tabulated in .txt files for spore labeling and are unitless, relative
 measurements. Methyl esters of BCA small molecules were also synthesized
 and incubated in DMSO with B. subtilis spores to test whether similar
 covalent bonding patterns can be observed in NMR and Rman spectroscopy.
 ^1^H NMR spectroscopy was used to confirm diagnostic peaks belonging to
 the expected BCA–spore tM adducts could be detected. Numerical values of
 ^1^H NMR signal intensities against chemical shifts were tabulated in .txt
 files for DMSO-d~6~ samples of BCA–CF~3~, BCA–CF~3~ with equimolar amounts
 of 1-octanethiol, and 200 mM of BCA–CF~3~ with 1 mg/mL of B. subtilis
 spores.  Raman spectroscopy was used to detect a characteristic shift in
 the CN peak associated with tM adduct formation. Numerical values of Raman
 signal intensities against Raman shifts were tabulated in .txt files for
 DMSO-d~6~ samples of 1 mg/ mL B. subtilis spores with 200 mM each of
 BCA–CF~3~, BCA–H, and BCA–OMe (n = 7 for BCA–CF~3~, n = 5 for BCA–H and
 BCA–OMe). *Table of contents:* 1mg/mL&200mM_NMR Detection of Spore
 Surface tM Bond (CF3).txt 120uM_5wash_NBD-CF3_gfp.TIFF
 120uM_5wash_NBD-OMe_gfp.TIFF 120uM_5wash_NBD-OH_gfp.TIFF
 120uM_5wash_NBD-H_gfp.TIFF Average_BCA-CF3-200mM-PY79-1mgmL-DMSO-spore.txt
 Average_BCA-OMe-200mM-PY79-1mgmL-DMSO-spore.txt
 Average_BCA-H-200mM-PY79-1mgmL-DMSO-spore.txt
 NMR_BCA-CF3+1-octanethiol.txt NMR of BCA-CF3.txt
 SporeLabeling_Triplciate_NFL_120uM.txt #### Folder: Figure 2
 **Background**: Data in Figure 2 were collected to demonstrate the
 assembly of B. subtilis spores with synthetic polymers bearing the BCA
 motif. A fluorescent BCA–bearing polymer (P1–NBD) was synthesized and
 incubated with B. subtilis spores in DMSO (1.2 µM) at 25 °C and 800 rpm
 for 20h to examine whether polymer–spores linkages can facilitate
 micron-scale assemblies that are observable under the fluorescence
 microscope. The TIFF file corresponds to the fluorescence microscopy image
 of the polymer–assemblies after the incubation and washing with DMSO.  To
 create macroscopic assemblies that could be characterized under shear
 rheology experiments, lyophilized B. subtilis spores were mixed with 30%
 w/v DMSO solutions of BCA–bearing polymers (P3 and P4) and were
 characterized after 2 days of benchtop incubation. Rheological
 characterizations were performed at 25 °C on a Discovery series HR-2
 hybrid rheometer from TA instruments with a 8 mm diameter parallel plate
 geometry. Frequency sweep experiments were performed under 1% strain
 amplitude and helped determine the relative shear stiffness of the
 materials depending on their BCA and comonomer side chain structure.
 Numerical value of storage (G') and loss (G") moduli over a
 range of angular frequencies (rad/s) were tabuled in .txt files with names
 beginning with "2d_DMSO leak_...". Characteristic relaxation
 times of materials made with EGMEMA-based polymers were calculated by
 taking the inverse of the crossover frequency. Hammett correlation
 analysis was performed by plotting log(𝜏R,R/𝜏R,H) against Hammett
 constants 𝜎+ of the chosen aryl substituents. Numerical values of
 log(𝜏R,R/𝜏R,H) and the corresponding 𝜎+ were tabulated in a .txt file
 beginning with "Hammett...". *Table of contents:* 2d_DMSO
 Leak_MMA_CF3 vs H vs OMe.txt 2d_DMSO Leak_CF3_MMA vs EGMEMA vs HEMA.txt
 2d_DMSO Leak_EGMEMA_CF3 vs H vs OMe.txt Hammett +_LOG_Relaxation
 time_2d_DMSO Leak_EGMEMA_CF3 vs H vs OMe.txt WT_M90-NBD-CF3_gfp.TIFF ####
 Folder: Figure 3 **Background**: Dry biocomposite materials were made to
 demonstrate molecular-level control over the polymer–spore interface to
 program material properties, as characterized through tensile testing,
 spore leakage, and optical and scanning electron microscopy (SEM)
 experiments. To fabricate the biocomposite, lyophilized B. subtilis spores
 were mixed with 30% w/v DMSO solutions of various BCA–bearing polymers
 (P4, P7, P8, P9, P10, and P11) before being transferred to a
 polytetrafluoroethylene (PTFE) dog bone mold and were incubated overnight.
 The entire PTFE mold was then submerged in ethanol for two hours for
 solvent exchange, and the material was taken out of the mold and placed on
 a PTFE sheet for 24 hours to dry for tensile testing. Materials were
 characterized by tensile testing, and stiffness (Young's moduli) was
 used as a method to determine differences in their mechanical properties.
 Young's moduli of biocomposite materials were determined by the slope
 of the linear region of the stress-strain curve. Numerical values of
 Young's moduli were tabulated in .txt files containing the label
 "Young's Modulus". In addition, toughness was determined by
 the area under the stress-strain curve before fragmentation. Numerical
 values of toughness was tabulated in a .txt file with the label
 "Toughness". To assess the covalent biocontainment ability of
 the biocomposite materials, various material fragments were incubated in
 PBS (pH = 7.16) and shaken at 250 rpm and 37 °C for two days. Numerical
 values of solution turbidity (OD600), indicative of the extent of spore
 leakage, were measured at 16 and 40 hours and were tabulated in a .txt
 file with the label "Spore Leakage". Optical microscopy images
 were taken of the organogel precursors to assess the relative extents of
 spore integration with the polymer phase. TIFF files of the microscopy
 images were saved with the label "Optical_...". To assess the
 retention of polymer–spore interactiosn after the solvent switching and
 drying procedure, SEM images were taken of material fragments. TIFF files
 of SEM images were saved with the label "SEM_..." *Table of
 contents:* Optical_EG90CF3+SPORES.tiff Optical_EG90OME+SPORES.tiff
 Optical_EG90H+SPORES.tiff SEM_EG90_H_Spores.tif SEM_EG90_OMe_Spores.tif
 SEM_EG90_CF3_Spores.tif Triplicate_Young's Modulus_EG90-R_0414.txt
 Spore Leakage_Kinetics.txt Triplicate_Toughness_CF3_dispersity.txt
 Triplicate_Young's Modulus_CF3_spore mass.txt Triplicate_Young's
 Modulus_CF3_%BCA.txt Triplicate_Young's Modulus_CF3_dispersity.txt
 #### Folder: Figure 4 **Background**: The reversible nature of the
 BCA–thiol binding suggested a possible material disassembly pathway that
 is entropically favored in select organic solvents. Material fragments
 were thus submerged in DMSO or acetone, and the extent and efficacy of
 disassembly were characterized by optical microscopy of the mixture
 and ^1^H NMR spectroscopy of the recovered polymer. In addition, growth
 curves of the recovered spores were measured by OD600 to determine whether
 spore viability was impacted by solvent exposure. ^1^H NMR spectra of the
 recovered polymers were compared to the pristine polymer NMR spectra to
 visualize whether they could be recovered from the disassembly process.
 Numerical values of ^1^H NMR signal intensities over a range of chemical
 shifts were tabulated in .txt files with the label "NMR_...".
 The extent of disassembly was visualized by microscopy images of spores
 recovered from the disassembly attempts and were saved as TIFF files
 beginning with "Disassembly_...". To enable mass tracking,
 materials were first lyophilized and large scale disassemblies were
 attempted. Microscopy images of spores recovered were saved as TIFF files
 beginning with "Lyophilized_...". Spore viability was assessed
 using solution turbidity (OD600) tracking over time after material
 disassembly using spores recoevered from acetone-driven partial
 disassemblies. Numerical values of OD600 over 12 hours were tabulated in a
 .txt file beginning with "Recovered WT_...". *Table of
 contents:* Recovered WT_Grrowth Curve_100 uL.txt
 Disassembly_ACETONE_0.5h_trans.tiff Disassembly_DMSO_0.5h_trans.tiff
 Lyophilized material large scale disassembly_CF3_DMSO_day14.TIFF
 Lyophilized material large scale disassembly_H_DMSO_day7.TIFF Lyophilized
 material large scale disassembly_OMe_DMSO_day7.TIFF NMR_Acetone
 Disassembly_EG90CF3.txt NMR_DMSO Disassembly_EG90CF3.txt
 NMR_Pristine_EG90CF3.txt #### Folder: Figure 5 **Background**: Engineered
 APEX2-displaying spores were incorporated into the biocomposite materials
 to investigate whether bio-derived functionality could be installed into
 the hybrid system. Because organic solvents are known to damage cells and
 enzymes, the stability and activity retention of the engineered spores
 were tested by incubating them in various organic solvents and running
 activity assays. Once an ideal condition was identified, materials were
 made to demonstrate the catalytic activity of the biocomposite system. In
 addition, engineered spores were recovered and renewed to show that any
 loss in activity could be recovered by an autonomous growth process.
 Spores were incubated in various organic solvents overnight, removed from
 solvents, and resuspended in PBS at 1 mg/mL concentration with 0.1 mM
 Amplex Red and 0.2 mM hydrogen peroxide and shaken at 205 cpm and 37 °C
 for 15 hours to assess the retention of the spore's catalytic
 activity. The relative Amplex Red conversion, indicative of the activity
 level of the enzyme-displaying spore, were assesed by normalizing against
 a spore without organic solvent exposure (bulk fluorescence of the
 sample/bulk fluorescence of the spore without organic solvent
 exposure)(100%). Numerical values of the normalized conversion were
 tabulated in a .txt file starting with "Organic Solvent
 Incubation_". In addition to the enzyme activity retention, the
 effect of organic solvents on the morphology of APEX2 spores were observed
 under optical microscopy. Microscopy images were saved as TIFF files
 beginning with "TIEDAPEX2_INCUBATION_...". The activity assay
 and spore morphology experiments helped determine that acetone is the
 least harmful of the solvents. In addition, acetone could dissolve the
 polymer and help facilitate polymer–spore bond formation for effective
 material fabrication. Catalytic biocomposite materials were thus
 fabricated in acetone and were tested for their ability to convert Amplex
 Red to resorufin. Numerical values of fluorescence spectroscopy
 measurements, indicative of catalytic material activity, were tabulated in
 .txt files beginning with "Acetone Material_..." for the first
 reaction cycle and "Cycle 2_..." for the second reaction cycle.
 Repeated activity showed the reusability of the material, although the
 second cycle was much slower in catalysis. Catalysis was also attempted in
 the presence of organic solvent in the reaction mixture to show its
 potential benefit in increasing substrate scope for more hydrophobic
 compounds. 12.5% acetone in PBS mixtures were used to show a lower yet
 significant activity level of the material. Because the reaction kinetics
 slowed down and the natural decomposition of Amplex Red became
 significant, bulk fluorescence values of the control mixture without the
 material were subtracted from the bulk fluorescence of the reaction
 mixture with the material. Numerical values of this corrected,
 "deltaFluorescence Intensity" were tabulated in a .txt file
 beginning with "12.5%...". Because diminished activity was
 observed in both the repeated biocatalysis and the 12.5% organic solvent
 experiments, materials were partially disassembled in acetone to recover
 and renew spores to demonstrate the recovery of catalytic activity.
 Pristine spores and renewed spores were incubated in PBS at 1 mg/mL with
 0.1 mM Amplex Red and 0.2 mM hydrogen peroxide at 205 cpm and 37 °C for 16
 hours to compare the renewed spore activity against the expected full
 catalytic activity. Numerical values of fluorescence intensity over time
 were tabulated in a .txt file beginning with "Renewed*..."
 *Table of contents:* TIEDAPEX2_INCUBATION_PBS_trans.TIFF 12.5%
 Acetone_Corrected.txt Acetone Material_TIED APEX2_PBS_Material vs
 Control.txt Cycle 2_Triplicate_Days_Acetone Material_TIED
 APEX2_PBS_Material vs Control.txt Organic Solvent
 Incubation_TIEDAPEX2_Normalized Conversion.txt Renewed APEX2 vs
 Pristine.txt TIEDAPEX2_INCUBATION_ACETONE_trans.TIFF
 TIEDAPEX2_INCUBATION_DMSO_trans.TIFF TIEDAPEX2_INCUBATION_ETOH_trans.TIFF
 #### Folder: SI **Background**: The supporting information (SI) includes a
 comprehensive list of data that are both included and not included in the
 main article. BCA Reactivity: ^1^H NMR analysis of the tM reaction to
 determine the effects of dilution, exogeneous BCA, aryl substituent,
 solvent, and exogeneous thiol on the equilibrium. Calculated values used
 for Hammett plots and k~a~ vs. k~d~ plots. Biocatalysis: determination of
 catalytic efficacy of spores and biocomposite materials in various solvent
 conditions. Morphology of engineered spores after exposure to solvents.
 Disassembly: Optical micrographs showing polymer–spore suspensions in
 various conditions. GPC: GPC traces of polymers used in this project.
 Material microscopy: optical and SEM images of polymer and spore mixtures
 (materials). Rheology: G' and G" values of the materials over a
 range of angular frequency at 1% shear strain and calculated values of
 relaxation times. Solvent Removal: mass tracking of materials over time to
 obverse solvent leakage and evaporation. Spore Labeling: detection of tM
 bond formation on spores using NMR and fluorescence measurements,
 including relevant standard curves and control samples. Spore Viability:
 Growth curve of the B. subtilis from material surface germination. Stress
 Strain Curve: stress strain curves from tensile testing. *Table of
 contents:* BCA Reactivity_%Dissociation by Dilution (concentration)
 Effect-R.txt BCA Reactivity_BCA exchange OMe vs CF3.txt BCA
 Reactivity_BCA-thiol-R_Acetone.txt BCA Reactivity_BCA-thiol-R_DMSO.txt BCA
 Reactivity_Exogeneous Thiol_Reversibility_2 equivalents.txt BCA
 Reactivity_Hammett + vs ln(kdx/kdH) BCA_DMSO.txt BCA Reactivity_Hammett +
 vs ln(Kx/KH) BCA_Acetone.txt BCA Reactivity_Hammett + vs ln(Kx/KH)
 BCA_DMSO.txt BCA Reactivity_Hammett +_LOG_Relaxation time_2d_DMSO
 Leak_EGMEMA_CF3 vs H vs OMe.txt BCA Reactivity_ka vs kd_Acetone.txt BCA
 Reactivity_ka vs kd_DMSO.txt Biocatalysis_%Organic
 Solvent_TIEDAPEX2_Normalized Conversion.txt Biocatalysis_12 5%
 Acetone_Triplicate_Days_Acetone Material_TIED APEX2_PBS_Material vs
 Control.txt
 Biocatalysis_20250226_TIEDAPEX_INCUBATION_ACETONE_expt_0005_trans.TIFF
 Biocatalysis_20250226_TIEDAPEX_INCUBATION_DMSO_expt_0010_trans.TIFF
 Biocatalysis_20250226_TIEDAPEX_INCUBATION_ETOH_expt_0003_trans.TIFF
 Biocatalysis_20250226_TIEDAPEX_INCUBATION_MEOH_expt_0005_trans.TIFF
 Biocatalysis_20250226_TIEDAPEX_INCUBATION_PBS_expt_0006_trans.TIFF
 Biocatalysis_Baseline Corrected_12 5% Acetone_Triplicate_Days_Acetone
 Material_TIED APEX2_PBS_Material vs Control.txt Biocatalysis_Renewed APEX2
 vs Pristine.txt Biocatalysis_TIEDAPEX2 Material_DMSO_Time Trace_FL.txt
 Biocatalysis_TIEDAPEX2_Normalized Final FL_Organic Solvent.txt
 Biocatalysis_TIEDAPEX2_Organic Solvent Incubation Overnight.txt
 Biocatalysis_Triplicate_Acetone Material_vs Pristine Spores.txt
 Biocatalysis_Triplicate_Days_Acetone Material_TIED APEX2_PBS_Material vs
 Control.txt Biocatalysis_Triplicate_Days_OD600_Acetone Material_TIED
 APEX2_PBS.txt Disassembly__ACETONE_8h_trans.tiff
 Disassembly_ACETONE_0.5h_trans.tiff Disassembly_ACETONE_0.5htrans.tiff
 Disassembly_ACETONE_8h_0002_trans.tiff Disassembly_ACETONE_8h_trans.tiff
 Disassembly_CF3_37C_1000RPM_0.2mM_7days_0015.TIFF
 Disassembly_CF3_37C_1000RPM_0.2mM_7days_0017.TIFF
 Disassembly_CF3_37C_1000RPM_0.2mM_7days_0027.TIFF
 Disassembly_CF3_50C_1000RPM_0.2mM_3days_0000.TIFF
 Disassembly_CF3_50C_1000RPM_0.2mM_3days_0003.TIFF
 Disassembly_CF3_50C_1000RPM_0.2mM_3days_0011.TIFF
 Disassembly_CF3_60C_1000RPM_0.2mM_1day_0012.TIFF
 Disassembly_CF3_60C_1000RPM_0.2mM_1day_0015.TIFF
 Disassembly_CF3_60C_1000RPM_0.2mM_1day_0022.TIFF
 Disassembly_CF3_70C_1000RPM_0.2mM_1day_0003.TIFF
 Disassembly_CF3_70C_1000RPM_0.2mM_1day_0004.TIFF
 Disassembly_CF3_70C_1000RPM_0.2mM_1day_0008.TIFF
 Disassembly_CF3_80C_1000RPM_0.2mM_1day.tif
 Disassembly_DMSO_0.5h_0002_trans.tiff
 Disassembly_DMSO_0.5h_0003_trans.tiff
 Disassembly_DMSO_0.5h_0004_trans.tiff Disassembly_DMSO_8h_0001_trans.tiff
 Disassembly_DMSO_8h_0003_trans.tiff Disassembly_DMSO_8h_0004_trans.tiff
 Disassembly_PY79_ACETONE_0.5h_trans.tiff Disassembly_ZOOM_CF3
 DISASSEMBLY_80C_1000RPM_0.2mM_1day_0013-1.tif Disassembly_ZOOM_CF3
 DISASSEMBLY_80C_1000RPM_0.2mM_1day.tif GPC_P1.txt GPC_P2.txt GPC_P3.txt
 GPC_P4.txt GPC_P5.txt GPC_P6.txt GPC_P7.txt GPC_P8.txt GPC_P9.txt
 GPC_P10.txt GPC_P11.txt Material
 Microscopy_Optical__EG90CF3_NOSPORE_0003.tiff Material
 Microscopy_Optical__EG90CF3+SPORES_0001.tiff Material
 Microscopy_Optical__EG90CF3+SPORES_0007.tiff Material
 Microscopy_Optical__EG90CF3+SPORES_0009.tiff Material
 Microscopy_Optical__EG90CF3+SPORES_0015.tiff Material
 Microscopy_Optical__EG90H_0414_NOSPORES_0001.tiff Material
 Microscopy_Optical__EG90H_0414_spores_0003.tiff Material
 Microscopy_Optical__EG90H_0414_spores_0009.tiff Material
 Microscopy_Optical__EG90H+SPORES_0013.tiff Material
 Microscopy_Optical__EG90H+SPORES_0014.tiff Material
 Microscopy_Optical__EG90OMe_0324_NOSPORES_0000.tiff Material
 Microscopy_Optical__EG90OME+SPORES_0013.tiff Material
 Microscopy_Optical__EG90OME+SPORES_0014.tiff Material
 Microscopy_Optical__EG90OME+SPORES_0017.tiff Material
 Microscopy_Optical__EG90OME+SPORES_0018.tiff Material
 Microscopy_Optical__EG100_NOSPORE_0002.tiff Material
 Microscopy_Optical__EG100_WT_0001_trans.tiff Material
 Microscopy_Optical__EG100_WT_0009_trans.tiff Material
 Microscopy_Optical__EG100+SPORES_0013.tiff Material
 Microscopy_Optical__EG100+SPORES_0014.tiff Material
 Microscopy_SEM__EG90_CF3_RAFT_Spores.tif Material
 Microscopy_SEM__EG90_CF3_Spore.tif Material
 Microscopy_SEM__EG90_H_Spores.tif Material
 Microscopy_SEM__EG90_OMe_Spores.tif Material
 Microscopy_SEM_EG100_Spores_001.tif NMR_BCA-CF3+1-octanethiol.txt
 NMR_BCA-H+1-octanethiol.txt NMR_BCA-OMe+1-octanethiol.txt Rheology_2d_DMSO
 Leak_EGMEMA_CF3 vs H vs OMe.txt Rheology_Relaxation time_2d_DMSO
 Leak_EGMEMA_CF3 vs H vs OMe.txt Solvent Removal_DMSO
 evaporation_Dogbone_CF3-M-EG-H.txt Solvent Removal_EtOH
 evaporation_Dogbone_CF3-M-EG-H.txt Spore Labeling_1mg/mL&200mM_NMR
 Detection of Spore Surface tM Bond (CF3).txt Spore
 Labeling_1mg/mL&200mM_NMR Detection of Spore Surface tM Bond (H).txt
 Spore Labeling_1mg/mL&200mM_NMR Detection of Spore Surface tM Bond
 (OMe).txt Spore Labeling_680_DMSO_20241003_Spore
 Labeling_Maleimide_TCEP.txt Spore
 Labeling_1015_NBD-OH_EndPoint_Fluorescence vs. Concentration_Standard
 Curve.txt Spore Labeling_1127_SporeLabeling_Triplciate_NFL_120uM.txt Spore
 Labeling_1127_SporeLabeling_Washes_120uM_Triplicates.txt Spore
 Labeling_2024_1003_sCy5_WT_AMINE_0004_cy5.TIFF Spore
 Labeling_2024_1003_sCy5_WT_AMINE_0004_trans.TIFF Spore
 Labeling_2024_1003_sCy5_WT_MALEIMIDE_0002_cy5.TIFF Spore
 Labeling_2024_1003_sCy5_WT_MALEIMIDE_0002_trans.TIFF Spore
 Labeling_2024_1003_sCy5_WT-red_AMINE_0004_cy5.TIFF Spore
 Labeling_2024_1003_sCy5_WT-red_AMINE_0004_trans.TIFF Spore
 Labeling_2024_1003_sCy5_WT-red_MALEIMIDE_0004_cy5.TIFF Spore
 Labeling_2024_1003_sCy5_WT-red_MALEIMIDE_0004_trans.TIFF Spore
 Labeling_2024_1011_WT_M90-NBD-CF3_0005_gfp.TIFF Spore
 Labeling_2024_1011_WT_M90-NBD-CF3_0005_trans.TIFF Spore
 Labeling_2024_1011_WT_M90-NBD-CF3_0022_gfp.TIFF Spore
 Labeling_2024_1011_WT_M90-NBD-CF3_0022_trans.TIFF Spore
 Labeling_2024_1011_WT_M90-NBD-CF3_0024_gfp.TIFF Spore
 Labeling_2024_1011_WT_M90-NBD-CF3_0024_trans.TIFF Spore
 Labeling_2024_1011_WT_M90-NBD-CF3_0028_gfp.TIFF Spore
 Labeling_2024_1011_WT_M90-NBD-CF3_0028_trans.TIFF Spore
 Labeling_2024_1011_WT_M90-NBD-CF3_0030_gfp.TIFF Spore
 Labeling_2024_1011_WT_M90-NBD-CF3_0030_trans.TIFF Spore
 Labeling_2024_1011_WT_M90-NBD-CF3_0034_gfp.TIFF Spore
 Labeling_2024_1011_WT_M90-NBD-CF3_0034_trans.TIFF Spore
 Labeling_2024_1011_WT_M90-NBD-OMe_0017_gfp.TIFF Spore
 Labeling_2024_1011_WT_M90-NBD-OMe_0017_trans.TIFF Spore
 Labeling_2024_1011_WT_M90-NBD-OMe_0023_gfp.TIFF Spore
 Labeling_2024_1011_WT_M90-NBD-OMe_0023_trans.TIFF Spore
 Labeling_2024_1011_WT_M90-NBD-OMe_0028_gfp.TIFF Spore
 Labeling_2024_1011_WT_M90-NBD-OMe_0028_trans.TIFF Spore
 Labeling_2024_1011_WT_M90-NBD-OMe_0035_gfp.TIFF Spore
 Labeling_2024_1011_WT_M90-NBD-OMe_0035_trans 2.TIFF Spore
 Labeling_2024_1011_WT_M90-NBD-OMe_0044_gfp.TIFF Spore
 Labeling_2024_1011_WT_M90-NBD-OMe_0044_trans.TIFF Spore
 Labeling_2024_1127_120uM_5wash_NBD-CF3_0004_gfp.TIFF Spore
 Labeling_2024_1127_120uM_5wash_NBD-CF3_0004_trans.TIFF Spore
 Labeling_2024_1127_120uM_5wash_NBD-H_0002_gfp.TIFF Spore
 Labeling_2024_1127_120uM_5wash_NBD-H_0002_trans.TIFF Spore
 Labeling_2024_1127_120uM_5wash_NBD-OH_0002_gfp.TIFF Spore
 Labeling_2024_1127_120uM_5wash_NBD-OH_0002_trans.TIFF Spore
 Labeling_2024_1127_120uM_5wash_NBD-OMe_0000_gfp.TIFF Spore
 Labeling_2024_1127_120uM_5wash_NBD-OMe_0000_trans.TIFF Spore
 Labeling_20250509_MaleimideCappedSpores_0009_cy5.tiff Spore
 Labeling_20250509_MaleimideCappedSpores_0009_trans.tiff Spore
 Labeling_Full Spectrum_DMSO_20241011_M90_CF3_OMe.txt Spore
 Labeling_NFL_540_DMSO_20241011_M90_CF3_OMe.txt Spore Labeling_Stock
 Solution Fluorescence_100x dilution.txt Spore
 Viability_Germination_EG90_CF3.txt Stress Strain Curve_EG90_CF3_1.txt
 Stress Strain Curve_EG90_CF3_2.txt Stress Strain Curve_EG90_CF3_3.txt
 Stress Strain Curve_EG90_CF3_halfspores_1.txt Stress Strain
 Curve_EG90_CF3_halfspores_2.txt Stress Strain
 Curve_EG90_CF3_halfspores_3.txt Stress Strain
 Curve_EG90_CF3_nospores_1.txt Stress Strain Curve_EG90_CF3_nospores_2.txt
 Stress Strain Curve_EG90_CF3_nospores_3.txt Stress Strain
 Curve_EG90_CF3_RAFT_0 29CTA_2.txt Stress Strain Curve_EG90_CF3_RAFT_0
 29CTA_3.txt Stress Strain Curve_EG90_CF3_RAFT_0 29CTA.txt Stress Strain
 Curve_EG90_H_1.txt Stress Strain Curve_EG90_H_2.txt Stress Strain
 Curve_EG90_H_3.txt Stress Strain Curve_EG90_H_nospores_1.txt Stress Strain
 Curve_EG90_H_nospores_2.txt Stress Strain Curve_EG90_H_nospores_3.txt
 Stress Strain Curve_EG90_OMe_0414_2.txt Stress Strain
 Curve_EG90_OMe_0414_3.txt Stress Strain Curve_EG90_OMe_0414.txt Stress
 Strain Curve_EG90_OMe_nospore_0414_2.txt Stress Strain
 Curve_EG90_OMe_nospore_0414.txt Stress Strain Curve_EG95_CF3_2.txt Stress
 Strain Curve_EG95_CF3_3.txt Stress Strain Curve_EG95_CF3.txt Stress Strain
 Curve_EG100_spores_1.txt Stress Strain Curve_EG100_spores_2.txt Stress
 Strain Curve_EG100_spores_3.txt ## Access information Other publicly
 accessible locations of the data: *
 [https://pubs.acs.org/doi/10.1021/jacs.5c13976](https://pubs.acs.org/doi/10.1021/jacs.5c13976) Data was derived from the following sources: *