Cx43 Cell | Notion

Overview

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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

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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

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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.gb

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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.