Toehold switch-enabled translation regulation verified in Nucleus Cytosol
Abstract¶
These results are part of preliminary testing for the pH-responsive DevCell module conducted during the DevCell Kickoff workshop. Using Nucleus Cytosol, the toehold switch enabled translation regulation of pHtdGFP in the presence of trigger ssDNA.
Introduction¶
As part of the development for the pH-responsive DevCell module for the Chicago node, the Liu lab conducted a brief test to validate the toehold switch system in Nucleus Cytosol. The specific toehold switch used in this test is published in Hwang et al. (2025), and is originally developed for PUREfrex2.0. This preliminary test was conducted at neutral pH, and pH-responsive protein expression was not tested.
Results¶
This preliminary test demonstrated that, using Nucleus Cytosol, the toehold-pHtdGFP linear DNA template was able to produce toehold pHtdGFP RNA and induce pHtdGFP expression gated by the presence of the trigger ssDNA. This preliminary test followed standard protocol for bulk reactions using Nucleus Cytosol (pOpen-deGFP plasmid concentration is 3 nM), and the toehold-pHtdGFP linear DNA template and trigger ssDNA concentrations are 2 nM and 4.8 nM, respectively, following those used in Hwang et al. (2025). For each condition, three identical 10-μL reactions were incubated for 6 hours at 37 °C in a plate reader. The composition for each reaction is provided in Table 2.
Table 1:Experiment conditions
| Name | Description |
|---|---|
| Cytosol Positive Control | Control Cytosol reaction expressing deGFP using pOpen-deGFP construct |
| toehold-pHtdGFP + trigger ssDNA | DNA template producing toehold-pHtdGFP RNA mixed with trigger ssDNA |
| toehold-pHtdGFP | DNA template producing toehold-pHtdGFP RNA without trigger ssDNA |
Table 2:Reaction composition
| Component | Stock Concentration | Final concentration | Cytosol Positive Control [µL] | toehold-pHtdGFP + trigger ssDNA [µL] | toehold-pHtdGFP [µL] |
|---|---|---|---|---|---|
| SMix* | 3.33x | 1x | 10.5 | 10.5 | 10.5 |
| PMix | 15 mg/mL | 1.80 mg/mL | 4.2 | 4.2 | 4.2 |
| Ribosomes | 10 µM | 1.8 µM | 6.3 | 6.3 | 6.3 |
| tRNA | 35 mg/mL | 3.5 mg/mL | 3.5 | 3.5 | 3.5 |
| pOpen-deGFP DNA template | 124 nM | 3 nM | 0.85 | 0 | 0 |
| toehold-pHtdGFP DNA | 40 nM | 2 nM | 0 | 1.75 | 1.75 |
| trigger ssDNA | 100 nM | 4.8 nM | 0 | 1.68 | 0 |
| RNAse Inhibitor | 40000 U/mL | 2000 U/mL | 1.75 | 1.75 | 1.75 |
| Water | 7.9 | 5.32 | 7 | ||
| Total mastermix volume [µL] | 35 | 35 | 35 | ||
| Reaction Volume × No. of replicates | 10 µL × 3 | 10 µL × 3 | 10 µL × 3 |
*SMix contains Mg-acetate and provides 8 mM final Mg-acetate concentration in the reaction.
Kinetics of cell-free expression over time quantified via GFP fluorescence intensity.
Endpoint GFP fluorescence intensity after a 6-hr incubation at 37 °C.
Conclusion and Future Steps¶
This preliminary test shows that the toehold switch system is compatible with Nucleus Cytosol. Further testing of Nucleus Cytosol at acidic pH levels (~pH 6-6.5) would be needed to fully validate the compatibility of Nucleus Cytosol for the proposed pH-responsive DevCell module.
- Hwang, S.-W., Li, Y., Green, A. A., & Liu, A. P. (2025). pH-Responsive Synthetic Cells for Programmable in Situ Protein Synthesis and Release. openRxiv. 10.1101/2025.11.16.688650