Construction of Out-of-Equilibrium Metabolic Networks in Nano- and Micrometer-Sized Vesicles

Summary

We present a protocol for reconstituting membrane proteins and encapsulating enzymes and other water-soluble components in lipid vesicles of sub-micrometer and micrometer size.

Abstract

We present a method to incorporate into vesicles complex protein networks, involving integral membrane proteins, enzymes, and fluorescence-based sensors, using purified components. This method is relevant for the design and construction of bioreactors and the study of complex out-of-equilibrium metabolic reaction networks. We start by reconstituting (multiple) membrane proteins into large unilamellar vesicles (LUVs) according to a previously developed protocol. We then encapsulate a mixture of purified enzymes, metabolites, and fluorescence-based sensors (fluorescent proteins or dyes) via freeze-thaw-extrusion and remove non-incorporated components by centrifugation and/or size-exclusion chromatography. The performance of the metabolic networks is measured in real time by monitoring the ATP/ADP ratio, metabolite concentration, internal pH, or other parameters by fluorescence readout. Our membrane protein-containing vesicles of 100-400 nm diameter can be converted into giant-unilamellar vesicles (GUVs), using existing but optimized procedures. The approach enables the inclusion of soluble components (enzymes, metabolites, sensors) into micrometer-size vesicles, thus upscaling the volume of the bioreactors by orders of magnitude. The metabolic network containing GUVs are trapped in microfluidic devices for analysis by optical microscopy.

Introduction

The field of bottom-up synthetic biology focuses on constructing (minimal) cells^1,2 and metabolic bioreactors for biotechnological^3,4 or biomedical purposes^5,6,7,8. The construction of synthetic cells provides a unique platform that allows researchers to study (membrane) proteins in well-defined conditions mimicking those of native environments, enabling the discovery of emergent properties and concealed biochemical ....

Protocol

1. General preparation

1. Chemicals
   1. Dissolve lipids (in powder form) to 25 mg/mL in CHCl₃ for making preformed liposomes.
      NOTE: It is preferable to prepare fresh lipid stocks, but the stock solutions can also be stored at -20 °C for a few weeks. Working with lipids in powder form is more accurate than using lipids already solubilized in CHCl₃. CHCl₃ should be handled using glass pipettes and/or syringes and stored in glass containers as C.......

Discussion

We present a protocol for the synthesis of (membrane) protein containing sub-micrometer size lipid vesicles (proteoLUVs), and the conversion of proteoLUVs into giant-unilamellar vesicles (proteoGUVs). The protocol should be applicable for the reconstitution of other membrane proteins^13,19,30,40 and the encapsulation of metabolic networks other than the L-arginine breakdown and glycerol 3-phosph.......

Materials

Name	Company	Catalog Number	Comments
Agarose	Sigma Aldrich	A9414-25g
Amicon cut-off filter	Sigma Aldrich	Milipore centrifugal filter units Amicon Ultra
BioBeads	BioRad	152-3920
CHCl3	Macron Fine Chemicals	MFCD00000826
D(+)-Glucose	Formedium	-
D(+)-Sucrose	Formedium	-
DDM	Glycon	D97002 -C
Diethyl Ether	Biosolve	52805
DMSO	Sigma-Aldrich	276855-100ml
DOPC	Avanti	850375P-1g
DOPE	Avanti	850725P-1g
DOPG	Avanti	840475P-1g
DTT	Formedium	DTT005
EtOH	J.T.Baker Avantor	MFCD00003568
Extruder	Avestin Inc	LF-1
Fluorimeter	Jasco	Spectrofluorometer FP-8300
Glycerol	BOOM	51171608
Gravity flow column	Bio-Rad	732-1010
Hamilton syringe 100 µL	Hamilton	7656-01
Hamilton syringe 1000 µL	Hamilton	81320
Handheld LCP dispenser	Art Robbins Instruments	620-411-00
Handheld Sonicator	Hielscher Ultrasound Technology	UP50H
HCl	BOOM	x76021889.1000
Imidazole	Roth	X998.4-250g
K2HPO4	Supelco	1.05099.1000
KCl	BOOM	76028270.1
KH2PO4	Supelco	1.04873.1000
Kimwipe	Kimtech Science	7552
Large Falcon tube centrifuge	Eppendorf	Centrifuge 5810 R
L-Arginine	Sigma-Aldrich	A5006-100G
Light microscope	Leica	DM LS2
L-Ornithine	Roth	T204.1
LSM Laser Scanning Confocal Microscope	Zeiss	LSM 710 ConfoCor 3
MgCl2	Sigma-Aldrich	M2670-1KG
Microfluidic chip	Homemade	PDMS based	DOI: https://doi.org/10.1039/C8LC01275J
Na-ADP	Sigma-Aldrich	A2754-1G
NaCl	Supelco	1.06404.1000
Nanodrop Spectrometer	Isogen Life Science	ND-1000 spectrophotometer NanoDrop
NaOH	Supelco	1.06498.1000
Needles for GUVs	Henke-Ject	14-14575	27 G x 3/4'' 0.4 x 20 mm
Needles for microfluidics	Henke-Ject	14-15538	18 G x 1 1/2'' 1.2 x 40 mm
Ni2+ Sepharose	Cytiva	17526802
Nigericin	Sigma-Aldrich	N7143-5MG
Nutator	VWR	83007-210
Osmolality meter	Gonotec Salmenkipp	Osmomat 3000 basic freezing point osmometer
Plasmacleaner	Plasma Etch	PE-Avenger
Polycarbonate filter	Cytiva Whatman	Nuclepor Track-Etch Membrane Product: 10417104	0.4 µm
Polycarbonate ultracentrifuge tube	Beckman Coulter	355647
Pyranine	Acros Organics	H1529-1G
Quartz cuvette (black)	Hellma Analytics	108B-10-40
Sephadex G-75 resin	GE Healthcare	17-0050-01
Sonicator	Sonics Sonics & Materials INC	Sonics vibra cell
Syringe filter	Sarstedt	Filtropur S plus 0.2	0.2 µm
Syringe pump	Harvard Apparatus	A-42467
Tabletop centrifuge	Eppendorf	centrifuge 5418
Teflon spacer	Homemade	Teflon based	45 x 26 x 1.5 or 45 x 26 x 3 or 20 x 20 x 3 mm
Tris	PanReac AppliChem	A1086.1000
Triton X-100	Sigma Aldrich	T8787-100 ml
Ultracentrifuge	Beckman Coulter	Optima Max-E
UV lamp	Spectroline	ENB-280C/FE
UV/VIS Spectrometer	Jasco	V730 spectrophotometer
Valinomycin	Sigma-Aldrich	V0627-10MG
Widefield fluorescence microscope	Zeiss	AxioObserver
β-Casein	Sigma Aldrich	C5890-500g