Overview¶
The Cx43 Module expresses connexin 43 (Cx43), a mammalian gap junction protein, that self-assembles into hexameric hemichannels (connexons) that spontaneously integrate into the membrane of synthetic cells and permit the passage of molecules up to ~1 kDa (Sihorwala et al., 2023).
Schematic representation of connexin and its relationship to a connexon: (left) Connexins are membrane-spanning proteins whose N- and C-termini are located in the cytoplasm; (right) connexons are complexes of six connexins. Figure by Totland et al., 2023 used under CC-BY-4.0 / cropped from original.
Reference Composition¶
| Construct | Description | Length (bp) | Supply route | File |
|---|---|---|---|---|
pOpen-pT7-Cx43 | Expresses wild-type Cx43 under T7 promoter in pOpen backbone | 3320 | Circular plasmid | pOpen-Cx43.gb |
pOpen-pT7-Cx43-eGFP | Expresses Cx43-eGFP fusion protein under T7 promoter in pOpen backbone | 3996 | Circular plasmid | pOpen-Cx43-eGFP.gb |
The Insertion Assay reaction in NEB PURExpress, 20 µL.
| Component | Volume (µL) |
|---|---|
| NEB PURExpress Solution A | 8 |
| NEB PURExpress Solution B | 6 |
| RNase inhibitor | 1 |
pOpen-pT7-Cx43-eGFP (100 ng/µL) | 2 |
| (Optional) OptiPrep | 0.67 |
| Nuclease-free water | 2.33 |
Base Membrane: POPC/Chol at its default composition.
| Component | Target (mmol/mol) |
|---|---|
| POPC | 699.3 |
| Cholesterol | 299.7 |
| (Optional) Fluorescent lipid label | 1 |
Expected Behavior¶
Cells¶
Insertion Assay¶
The Cx43 Module is expected to localize to the membrane of the synthetic cells that express it. Synthetic cells encapsulating NEB PURExpress and pOpen-pT7-Cx43-eGFP, incubated at 37 °C for 6 h, show green fluorescent rings around the synthetic cells, which confirm membrane localization of Cx43-eGFP. Control synthetic cells lacking the Cx43 plasmid show no rings. The readout is fluorescence microscopy on a Squid+ (Cephla) with an Olympus UPLXAPO40X 40x objective, at 80 ms exposure in the 561 channel and 200 ms in the 488 channel (Hsu, 2025).
The −Cx43 control omits the plasmid and adds 2 µL nuclease-free water.
Ribosomes, polymerase and plasmid stay inside the synthetic cell (Sihorwala et al., 2023; Hsu, 2025).
Fluorescence micrograph of synthetic cells expressing Cx43-eGFP. Green fluorescent rings surrounding the synthetic cells indicate successful membrane localization of Cx43-eGFP hemichannels. 488 nm (green, Cx43-eGFP) and 561 nm (red, membrane label) channels overlaid.
Fluorescence micrograph of control synthetic cells without Cx43 plasmid. No green fluorescent rings are observed. Faint green signal within the synthetic cells is encapsulated PURE.
Leakage Assay¶
The Cx43 Module is expected to let encapsulated dye leave the synthetic cells that express it. Synthetic cells co-encapsulating NEB PURExpress, pOpen-pT7-Cx43, and Alexa Fluor 647 dye, incubated at 37 °C for 6 h and imaged by confocal microscopy every 10 min, lose encapsulated fluorescence over time. Control synthetic cells without the Cx43 plasmid retain it. The readout is confocal microscopy on an Operetta at 40x, at 80 ms exposure in the 561 channel and 20 ms in the 647 channel (Hsu, 2025).
| Component | +Cx43 (µL) | -Cx43 (µL) |
|---|---|---|
| NEB PURExpress Solution A | 8 | 8 |
| NEB PURExpress Solution B | 6 | 6 |
| RNase inhibitor | 1 | 1 |
pOpen-pT7-Cx43 (100 ng/µL) | 2 | 0 |
| Alexa Fluor 647 (1 mM) | 0.1 | 0.1 |
| (Optional) OptiPrep | 0.67 | 0.67 |
| Nuclease-free water | 2.23 | 4.23 |
Plot of background-subtracted Alexa Fluor 647 fluorescence intensity over 6 h at 37 °C. Synthetic cells containing Cx43 show a progressive decrease in encapsulated dye fluorescence relative to controls, consistent with pore-mediated dye leakage.
Confocal time series of synthetic cells expressing Cx43 and encapsulating Alexa Fluor 647 over 6 h at 37 °C (images every 10 min, starting 40 min after preparation). Progressive loss of fluorescence is observed as dye leaks through Cx43 channels. Scale bar: 500 µm.
Confocal time series of control synthetic cells encapsulating Alexa Fluor 647 over 6 h at 37 °C. Dye fluorescence is maintained throughout, confirming that leakage requires Cx43 expression. Scale bar: 500 µm.
Confocal image of synthetic cells expressing Cx43 at the start point, 40 min after preparation. Some synthetic cells already show reduced fluorescence, consistent with early dye leakage at room temperature. Scale bar: 500 µm.
Confocal endpoint image (6 h 40 min) of synthetic cells expressing Cx43. A higher proportion of non-fluorescent synthetic cells is observed relative to controls. Scale bar: 500 µm.
Confocal image of control synthetic cells at the start point, 40 min after preparation. The synthetic cells remain fluorescent, confirming dye retention in the absence of Cx43. Scale bar: 500 µm.
Confocal endpoint image (6 h 40 min) of control synthetic cells. Most synthetic cells remain fluorescent throughout the experiment. Scale bar: 500 µm.
Requirements¶
Requires a membrane (e.g. Base Membrane: POPC/Chol).
Requires pT7 transcription and translation, when expressing from DNA (e.g. Base Cytosol).
Requires that molecules passing through the pore be about 1 kDa or smaller (Sihorwala et al., 2023; Hsu, 2025).
Requires, when the interior reaction must keep running after the pore is open, that the outer solution hold every species the pore passes that the interior needs, at working concentration: SMix at 1x for a Base Cytosol interior, or NEB Solution A at 1x (the stock is 2.5x) for an NEB PURExpress interior (Sihorwala et al., 2023; Hsu, 2025).
Processes¶
Encapsulation: Phase Transfer — prepares synthetic cells by encapsulating an inner solution in a membrane.
Credits¶
Module contributed by Ahmed Sihorwala (Belardi Lab, UT Austin). Validation data by Yen-Yu Hsu (b.next).
Supported by¶
DevNotes that support this specification include:
- Sihorwala, A. Z., Lin, A. J., Stachowiak, J. C., & Belardi, B. (2023). Light-Activated Assembly of Connexon Nanopores in Synthetic Cells. Journal of the American Chemical Society, 145(6), 3561–3568. 10.1021/jacs.2c12491
- Totland, M. Z., Omori, Y., Sørensen, V., Kryeziu, K., Aasen, T., Brech, A., & Leithe, E. (2023). Endocytic trafficking of connexins in cancer pathogenesis. Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease, 1869(7), 166812. 10.1016/j.bbadis.2023.166812
- Hsu, Y.-Y. (2025). Cx43 Cell: DNA Validation. 10.63765/xvxu3274