Cx43 Cell

Overview

The Connexin 43 Cell expresses the mammalian channel protein, connexin 43 (Cx43). Cx43 is a four-pass transmembrane protein that self-associates to form hexameric nanopores called Cx43 connexons. These nanopores act as conduits that facilitate exchange of different molecules across the Cell’s membrane. The Connexin 43 Cell is largely based on a paper by Ahmed Z. Sihorwala, Jeanne C. Stachowiak, and Brian Belardi: Light-activated assembly of connexons in synthetic cells.

More specifically, the Connexin 43 Cell encapsulates a plasmid DNA encoding for Cx43-GFP (green fluorescent protein) along with the PURE cell-free expression system. Following PURE expression, Cx43 is able to spontaneously insert into the Cell membrane, and subsequently form connexons. Successful integration of connexons in the Cell membrane leads to release of fluorescent small molecules, thus connecting the Cell lumen to its external environment.

Design

The Connexin 43 Cell expresses Cx43-GFP under the control of a T7 promoter. Cx43 expression leads to the formation of membrane nanopores, called connexons. These connexons enable release of a fluorescent small molecule, Alexa Fluor 647, from the Cell to its external environment.

Protocol

Modules

This component does not function as a stand alone component in PURE, it requires the membrane environment

DNA components

pT7-Cx43-eGFP - express Cx43 eGFP fusion protein in the pRSET plasmid

prset_cx43-egfp_predicted.gb11.1KB

Key Materials

Name	Product	Manufacturer	Part #	Price	Link
Alexa Fluor 647	Alex Fluor 647 NHS Ester	Thermo Scientific	A20006	$429.00	[link]
Atto 390	DOPE-Atto390	ATTO TEC	AD 390-161	$445.00	[link]

Protocol

To create Lipid-Oil Mixture (500uL):

Add 400 uL Silicone Oil (SO) and 70 uL Mineral Oil (MO) to large glass vial, vortex to mix.

Add 0.2 mg POPC and 0.286 ug Atto390-DOPE to small glass vial. Evaporate with nitrogen to create lipid film. Use glass syringes to handle lipids, which are aliquoted in chloroform. These need to be handled and rinsed carefully. Lipids are stored in the -80 fridge.

Resuspend lipid film in 30 uL decane, vortex

Add 30uL of lipid-decane solution to SO / MO mixture, vortex

In an Eppendorf tube, add 90 uL of HEPES buffered saline (5mM HEPES, 30mM NaCl) to the bottom. Gently add 20 uL of lipid-oil solution over the aqueous solution and let the interface stabilize for 30 min.

Inner aqueous solution:

On ice, prepare PURExpress reaction:

Component	Volume [uL]
NEB PURExpress Solution A	5
NEB PURExpress Solution B	3.75
Cx43-GFP in pRSET Plasmid (total amount 70 ng)	0.25
Opti-PREP (for final dilution = 10% OptiPREP)	1.25
Alexa Fluor 647 (start with 100uM AF647)	1.25
“Top it off with” 25mM HEPES; 150mM NaCl	1.25
Total	12.75

The total volume is 12.5 uL, but only 5 uL is needed for encapsulation.

Create Emulsion:

In a large glass vial, mix 80 uL of lipid-oil mixture with 5 uL inner aqueous solution. Vortex to emulsify.

Create Vesicle

Gently add the emulsion to the Eppendorf tube.

Centrifuge at 2400xg for 10 min at 4C.

Puncture side of tube near the bottom using 26G needle and eject vesicle solution into fresh tube. Ejected solution contains vesicles.

Incubate the vesicles at 37C for 2 hours. Cover the vesicles with foil.

Performance assays

Insertion assay. Observe liposomes under microscope. Ref paper uses 60x confocal and obtained time series by taking different preps at 10, 30, 60, 120, and 180 min; alternatively can use a single sample with on-stage incubation. Note: expression plateaued at 60 min.

Leakage assay. Observe liposomes under microscope. Ref paper uses 60x confocal and obtained time series by taking different preps at 10, 30, 60, 120, and 180 min; alternatively can use a single sample with on-stage incubation. Report percentage of vesicles with dye leakage. Expect 20% leakage in Negative Control and 60% leakage in Sample. Most leakage occurred within 30 minutes. Can also quantify leakage by measuring supernatant of pelleted vesicles, a 2-fold difference was observed.

Note: that leakage can be inhibited by presence of 2 mM Ca++ in outer solution.

Performance data

performance data is available in the original publication [link]

Resources and References

Sihorwala, A. Z., Lin, A. J., Stachowiak, J. C. & Belardi, B. Light-Activated Assembly of Connexon Nanopores in Synthetic Cells. Journal of the American Chemical Society vol. 145 3561–3568 (2023) [link]

Credits

Ahmed Z. Sihorwala (UT Austin)
Brian Belardi (UT Austin)
Jeanne C. Stachowiak (UT Austin)